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ANALYSIS OF THE Trypanosoma brucei GENOME AND IDENTIFICATION AND CHARACTERIZATION OF A GENE FAMILY ENCODING
PUTATIVE EF-HAND CALCIUM-BINDING PROTEINS
DISSERTATION
Presented to the Graduate Council of the
University of North Texas in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
James H. DeFord, B.S., M.S.
Denton, Texas
May, 1998
DeFord, James, H. Analysis Of The Trypanosoma brucei Genome And
Identification And Characterization Of A Gene Family Encoding Putative
EF-Hand Calcium-Binding Proteins. Doctor of Philosophy (Biology), May, 1998,
171 pp., 4 tables, 17 figures, bibliography, 159 titles.
The flagellum of Trypanosoma brucei contains a family of antigenically
related EF-hand calcium-binding proteins which are called the calflagins.
Genomic Southern blots indicated that multiple copies of calflagin genes occur
in T brucei. All of the copies were contained in a single 23 kb Xho\-Xho\
fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded
calflagin sequences. Two new members of the calflagin family were found from
genomic clone sequences. The deduced amino acid sequences of the
genomic clones showed the calflagin genes were arranged tandemly along the
genomic fragments and were similar to previously described calflagins. The
calflagin genes were related by two unrelated 3' flanking sequences. An open
reading frame that was unrelated to any calflagin was found at the 5' end of the
2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three
EF-hand calcium-binding motifs and one degenerate EF-hand motif. In
general, variability among the T. brucei calflagins is greater than related
proteins in T. lewisii and T. cruzi. This variability results from amino acid
substitutions at the amino and carboxy termini, and duplication of internal
segments.
Trypanosoma DNA was sequenced by M13 shotgun cloning techniques
to expedite the discovery of novel targets. 455 sequences were generated
representing approximate 1% of the T. Brucei genome. The sequences were
evaluated for putative genes and non coding elements against the nr (non-
redundant) and GSS databases at the National Center for Biotechnology
Information using BLAST family software. 84 sequences are reported with
significant homology to database sequences.
Several sequences were found to be of particular interest and are
discussed separately. Putative sequences for: a putative Pyruvate: ferredoxin
oxidoreductase (dbGSS no. B13566); a putative Fe-superoxide dismutase
(dbGSS no. B13733); and a non-coding sequence (dbGSS no. B13593) with
homology to a region 3' to human apoptosis genes are reported.
In conclusion the identification of novel targets via shotgun cloning
methods is a viable method that is augmented with each submission to the
database.
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mu /fa.
ANALYSIS OF THE Trypanosoma brucei GENOME AND IDENTIFICATION AND CHARACTERIZATION OF A GENE FAMILY ENCODING
PUTATIVE EF-HAND CALCIUM-BINDING PROTEINS
DISSERTATION
Presented to the Graduate Council of the
University of North Texas in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
James H. DeFord, B.S., M.S.
Denton, Texas
May, 1998
ACKNOWLEDGEMENTS
I would like to express my thanks and gratitude to my major professor, Dr.
Robert Benjamin for his support and advice in both my research and career
decisions. I would also like to express special thanks to the following persons
and institutions for their help and assistance in this project. To Dr. Larry Ruben
at Southern Methodist University for his expert advice, guidance, and use of his
lab. To Dr. Glen Evans at the McDermott Center for Human Growth and
Development at the University of Texas Southwestern Medical Center at Dallas
for the opportunity to learn use some of the most sophisticated DNA equipment
in the world. And to Michelle for her untiring help and belief in both the project
and myself.
TABLE OF CONTENTS
List Of Tables vi
List Of Figures vii
Abreviations ix
1. Introduction 1.1 African trypanosomes. 1 1.2 South american trypanosomes. 4 1.3 Rationale for research expediency/epidemiology. 6 1.4 Calcium. 7 1.5 Calcium binding proteins. 11 1.6 Calf lag ins. 16 1.7 Calcium/calcium binding protein interactions on trypanosomal
metabolism and development. 19 1.8 Calcium: effects on trypanosome killing and treatment. 27 1.9 A shotgun approach to identification of novel targets. 30
2. Materials and Methods 2.1 Amplification and isolation of genomic fragments. 33 2.2 Isolation of plasmids (analytical scale). 35 2.3 Agarose mini gel analysis. 36 2.4 Gel photography. 37 2.5 Digesting dna with restriction endonucleases. 38 2.6 Large scale isolation of recombinant plasmids. 38 2.7 Preparative scale digestion of cloned dna fragments. 42 2.8 Preparative scale isolation of cloned dna fragments detected by
uv shadowing techniques. 42 2.9 Electroelution and dna recovery from excised from gels. A1 2.10 Screening isolated dna fragments for restriction sites. 50 2.11 Subcloning into m13mp and pucvectors. 52 2.12 TSS competant cells. 53 2.13 Transformation of dh5 alpha F'with recombinant m13mp 18/19. 54 2.14 Transformation of dh5 alpha F or F cells with recombinant
plasmid. 54 2.15 Preparation ofm13mp single stranded template for sequencing. 55 2.16 Nucleotide sequencing double-stranded and single-stranded
templates by the dideoxyribonucleotide method. 57 2.17 Preparation and electrophoresis of polyacrylamide sequencing
gels. 59 2.18 Autoradiography and sequence analysis. 65
2.19 Detection of genomic sequences using radio labeling techniques: southern blotting. 65
2.20 Radio labeled probes: end labeled; random primed. 67 2.21 Hybidizations. 67 2.22 M13 library construction: isolation , size fractionation and
preparation of genomic dna. 6 8 2.23 M13 library construction: vector preparation and ligation. 70 2.24 M13 library construction: transformation of competant cells. 71 2.25 M13 library sequencing and analysis. 72 2.26 Cosmid library construction: vector preparation. 73 2.27 Cosmid library construction: isolation and preparation of
genomic dna. 74 2.28 Cosmid library construction: ligation of genomic dna tovector. 75 2.29 Pakaging, titering, and plating the cosmid library. 78 2.30 Cosmid library finger printing. 79
3. Results 3.1 Detection and isolation of genomic clones. 80 3.2 Restriction site analysis and subcloning of genomic clones. 85 3.3 Sequence analysis of genomic clones pB1.7 and pB2.5. 85 3.4 Comparisons of coding and flanking sequences to other calflagin
clones. 96 3.5 Analysis of a cloned T. brucei repeated element. 104 3.6. Detection of novel T.brucei sequences using M13 shotgun
sequencing. 107 3.7 M13 Sequences of particular interest. 113 3.8 Cosmid library analysis. 114
4. Discussion 132
References 142
LIST OF TABLES
1. Labeling Mix Table. 60
2. Sequenase Dilution Table. 60
3. Sequencing Reaction Table. 61
4. Trypanosoma brucei: Shotgun Genomic Sequence Survey. 108
List Of Figures
IA. Electrophresis stand and buffer pump assembly for vertical agarose gel electrophoresis. 45
IB. Assembled cassette for vertical acrylamide gel electrophoresis. 45
2. Principle of UV shadowing for the visualization of DNA
bands in agarose gels. 48
3A. Sau3A I partial digestions of genomic T. brucei DNA 76
3B. Preparation of scos seq 3 cosmid vector 76
3C. Conversion of ligation products to high molecular weight recombinant concatamer DNA 76
4. Southern blot of T. brucei DNA probed with 32P-labeled
cDNA, pTB44A. 81
5A. Recombinant plasmid pB2.5. 83
5B. Recombinant plasmid 1.7. 83
6. Restriction digested genomic clones pB1.7 and pB2.5. 86
7. Restriction map and sequencing strategy of the genomic clonepB1.7. 88
8. Restriction map and sequencing strategy of the genomic clone pB2.5. 90
9. The complete nucleotide sequence of the genomic clone pB1.7. 92
10. The complete nucleotide sequence of the genomic clone pB2.5. 94
11. Common features of calflagin clones pB2.5, pB1.7, pBTB44A, and pTB17. 98
12A. The sequence and the translation of the carboxyl end of and ORF in the genomic clone Tb-24. 100
12B. A Kyte and Doolittle hydropathy plot of the 5' open reading frame in TB-24. 100
13. Comparisons of calflagin protein sequences from T. brucei,
T. cruzi, and T. lewisi. 102
14. Analysis of a Trypanosoma brucei repeated element. 105
15A. Identification of genes involved in regulation of oxidative stress inT. brucei. 115
15B. A comparison between a translated region of T. brucei and Fe-superoxide dismutase from T. cruzi. 115
16. Comparison between coding and non-coding regions of T. brucei and a human apoptosis gene sequence. 117
17. Analysis of random cosmid clones from T. brucei. 119
LIST OF ABREVIATIONS
BAP bacterial alkaline phosphatase BLAST basic local alignment search tool bp base pairs CaBP calcium binding proteins cDNA copy DNA CIAP calf intestinal alkaline phosphatase DMSO dimethyl sulfoxide dpm disintegrations per minute DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EST expressed sequence tags EtBr ethidium bromide ETOH ethanol g gravity GSS genome sequence survey k thousand kb kilobases IPTG isopropyl-1-thio-B-D-galactopyranoside kDa kilodalton LB Luria-Bertani mb megabases mcs mutiple cloning site ml milliliter mm millimeter mM millimolar MW molecular weight NCBI National Center for Biotechnology Information nm nanometer nr non-redundant data base (NCBI) PFO pyruvate: ferredoxin oxidoreductase ORF open reading frame rpm revolutions per minute RT room temperature SDS sodium dodecyl sulfate TEMED NNN"N"-tetrametylethylene-diamine TLF trypanosome lytic factor VSG variable surface glycoprotein X-gal 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside
1. Introduction
1.1 African trypanosomes.
African trypanosomes are protozoan parasites that infect a variety of animals.
Within the family Trypanosomatidae are a number of genera. Two of these,
Leishmania and Trypanosoma, present major health and economic problems
for humans and their livestock. Members of the genus Trypanosoma cause two
distinct disease processes limited to, but not restricted to, two geographical
areas. One of these processes known as "sleeping sickness" is found in vast
areas of central Africa. The other is known as Chagas disease and is found in
Central and South America.
The economic impact of African trypanosomiasis (the disease) is mainly
caused by three organisms that are essentially indistinguishable from each
other serologically. The organisms, T. brucei brucei,T. brucei gambiense, and
T. brucei rhodesiense, are separated by their clinical features and geographical
locations. T. rhodesiense is the cause of East African or Rhodesian sleeping
sickness, and is the most virulent of the three affecting both humans and
animals. T. gambiense affects only humans and is found in Central and West
Africa. T. brucei is endemic to all Africa except North Africa. It is apparently non-
infective for humans and its mortality on native game is low. However, it is
devastating to domestic cattle and other economically important animals.
Several species of Glossina (tsetse flies) are vectors of human and
animal trypanosomiasis. Both sexes of the flies transmit the disease and are
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infective for life (2-3 months). The tsetse fly becomes infected when taking a
blood meal from an infected host. There are a wide variety of reservoir hosts for
the T. rhodesiense and T. brucei forms. Trypomastigotes (blood forms) are
ingested by the fly and transformation to the procyclic trypanomastigote form
takes place in the midgut of the fly. These forms continue to divide for ten days
before migrating to the salivary glands of the fly and transforming into
epimastigotes. The epimastigotes are transformed into metatrypanosomes,
which is the infective stage for humans and animals. While the minimum
infective dose of trypanosomes is 300-500, the tsetse fly is capable of injecting
40,000 or more (1). The metatrypanosomes will transform into trypomastigotes
in the subcutaneous tissues, and a primary chancre will mark the site of
inoculation. Dissemination into the blood and lymph of the new host completes
the life cycle. Other modes of transmission include eating infected raw meat and
mechanical transmission by biting flies.
The pathology is related to fluctuating parasitemias due to antigenic
variation of the trypanosomes. Two classes of antibody are directed against
either cellular antigens (IgG) or surface Ags (IgM). Within a given population of
trypanosomes in an infected host, a subpopulation escapes detection by host
antibody. The undetected subpopulation of trypanosomes are variants that
have different antigenic surface glycoproteins (VSGs). As the original
population is destroyed, a new subpopulation begins to multiply until it to is in
turn detected and destroyed by host immune response. As before, another
subpopulation arises taking the place of the population before it.
In studies involving experimental infections, one hundred
immunologically distinct variants were found (2). T. rhodesiense infections
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produce acute sleeping sickness with parasitemias as high as 100 K
trypanosomes per milliliter. These peak parasitemias can last 2-3 days,
occurring at irregular intervals from 2-10 days. T. gambiense infections produce
much lower parasitemias, resulting in a more chronic form of infection.
After inoculation, the cardinal signs of inflammation occur at the bite site.
On or about the sixth day, the trypanosomal chancre starts to develop. If the
chancre is recognized for what it is and treatment is begun, recovery is usually
without incident. Suramin is the drug of choice. If treatment is delayed, flu-like
symptoms develop. The appearance of enlarged lymph nodes, particularly the
posterior cervical nodes (Winterbottom's sign), and spleen enlargement are
characteristic but variable. Symptoms including, headache, stiff neck, insomnia,
and depression are manifestations of central nervous system (CNS)
involvement. Treatment at this stage is more difficult in that only
chemotherapeutic agents that can cross the blood brain barrier are effective.
Melarsoprol in combination with Suramin can be effective but is highly toxic. As
the CNS involvement progresses, a generalized meningoencephalitis with
cerebral edema develops. CNS function deteriorates resulting in abnormal
reflexes, esthesia, ataxia, psychosis, and eventually convulsions and coma.
The heart and lungs are affected by a general pattern of edema which in
combination with deteriorating respiratory reflexes results in pneumonia.
Pneumonia, sepsis, or both are the leading causes of death in most cases. If
untreated, death can occur in a few weeks to nine months for T. rhodesiense
and months to years for T. gambiense. Drug prophylaxis is limited with a risk of
cryptic infection and side effects. Development of a vaccine has been
hampered by the previously described antigenic variation of the parasite (1).
1.2 South American trypanosomes.
Trypanosoma cruzi is essentially an intracellular parasite transmitted by vectors
in the order Hemiptera, family Reduviidae, subfamily Triatominae (triatomids) of
which there are nine genera. These are also more commonly known as
conenose bugs, wheel bugs, and kissing bugs.
Infection is usually transmitted at night when these bugs
characteristically feed. The bug bites near the mouth or eyes of a sleeping
victim (hence the name kissing bug), and defecates as it takes a blood meal.
The feces contain the parasites that must be rubbed into the bite site by the
victim if infection is to occur. The bite itself is not painful, but begins to itch after
a short time, thus promoting the infective process.
Infections also occur without contact with the bug. In areas where
thatched roofs exist, bugs live in the roofs and defecate upon sleeping people
who then become infected by rubbing feces into mucus membranes. In rural
areas where people are close to their livestock, the risk of infection increases
because of the many types of reservoir hosts (e.g. dogs, cats, rats, bats, sloths,
and other primates). The trypomastigote form of the parasite is the infective
form for mammals, entering the bite site or mucus membranes and infecting a
wide variety of host cells. Upon entering the cell, the parasites transform into the
non-flagellated amastigote form and begin to divide. After several divisions,
they transform back into trypomastigotes, rupture the cell, and distribute
throughout the host. They infect a variety of tissues including: central nervous
system, gastrointestinal, and cardiac tissue.
Triatomids become infected when they take a blood meal from an
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infected host, and remain infective for life. The trypomastigotes migrate to the
midgut of the bug, transform into epimastigotes, and divide, producing
thousands of organisms which transform into metacyclic trypomastigotes. The
trypomastigotes migrate to the hindgut where they are expelled with the feces.
Edema occurring at the bite site is known as a primary chagoma or
Romana's sign. It is usually located on the face and contains intracellular
amastigotes. This can be diagnostic, especially in young children who are
frequent targets of infection. An incubation period of 4-5 days is followed by the
acute stage which is often asymptomatic and frequently misdiagnosed. It is
characterized by the spread of parasites and accompanied by an intense
inflammatory reaction lasting up to four months. Those individuals that survive
the acute stage enter the chronic stage which results in the slow destruction of
invaded cells. This can lead to heart enlargement with possible ventricular
aneurysm and or megacolon and megaesophogus.
Chronic Chagas disease is quite variable with some persons remaining
asymptomatic their entire lives. Others remain healthy for years before
developing symptoms of chronic Chagas (1). Commonly, infected persons
have abnormal electrocardiograms. Diagnosis in the acute stage is made by
finding trypomastigotes in the blood. In the chronic stage diagnosis is made by
finding amastigotes in a muscle biopsy. Serological test are also available but
are variable during the acute stage. Treatment is difficult. Nifurtimox can help
during the acute stage but it is not as effective during the chronic stage and has
serious side effects. There is also some evidence that the trypanosomes have
become resistant.
1.3 Rationale for research expediency/epidemiology.
Trypanosomes cause death and disease across vast areas of sub-Saharan
Africa and Central and South America. In endemic areas the loss of human life,
productivity, and livestock cannot be over stated.
African trypanosomiasis (the disease), also known as sleeping sickness,
is endemic to an area approximately the size of the United States. This area is
virtually free of human beings and their livestock. The area is limited
geographically by the distribution of the Tsetse fly, the vector of African
trypanosomiasis. Mortality rates climb dramatically when people and their
livestock migrate into these areas for political or economic reasons. Currently, it
is estimated that 50 million people in 36 sub-Saharan countries are at risk with
50,000 new cases reported annually (3). This is considered to be grossly
underestimated due to logistical problems in diagnosis. The disease is
invariably fatal if not diagnosed and aggressively treated in the early stages.
South American trypanosomiasis, known as Chagas' disease, is
endemic to areas as far south as Argentina and as far north as the southern
border states of the U.S. In endemic areas, approximately 20% of the
population is at risk, resulting in 7 million infected persons (4). Some estimates
indicate the number of infected persons at between 10 and 12 million
individuals (5). Ten percent of infected persons die in the acute phase or
develop serious myocardiopathy in the chronic stage of the disease (5). While
vector-transmitted Chagas' disease largely occurs below the Tropic of Cancer,
sporadic cases have been reported in the U.S. with increasing frequency.
There are at least two reasons for this:
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1 .Infected vectors and nondomestic reservoir host appear to be
abundant in North America, though the mode of transmission and epidemiology
does not often involve humans.
2. Political and social unrest in endemic areas have increased the
number of infected people in the U.S. resulting in cases of Chagas' disease
transmitted by blood transfusion (6).
Louis V. Kirchoff has estimated that 100,000 immigrants in the U.S. may
be chronically infected with the vast majority asymptomatic (7). In a study of
205 immigrants from El Salvador and Nicaragua living in Washington, DC, Five
percent were found to harbor the parasite. Data from South America indicate
that approximately 25% of recipients receiving blood from infected persons
could also become infected. Blood recipients can be protected from infection
by treatment of the donated blood with gentian violet (1:4000 dilution), which
kills the trypomastigotes.
These two forms of trypanosomes are spreading geographically with
morbidity and mortality rates expected to climb by millions in the next few years.
To combat this, vector control programs, early diagnosis, and treatment have
shown some limited success. There has also been a renewed interest in
identifying novel aspects of the biology, biochemistry, and molecular biology of
trypanosomes that might be exploited to develop new targets for vaccines or
chemotherapy (3).
1.4 Calcium.
We all began life with a wave of calcium sweeping through the cytoplasm of the
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egg from which we came at the moment of fertilization. Calcium is no less
important to all living things where it initiates the advent of many physiological
processes under strict homeostatic control. Techniques, such as video-imaging
of flourescent indicator dyes at the level of a single cell, has revealed that the
stimulus-induced intracellular calcium signal is remarkably organized both in
time (calcium spikes) and space (calcium waves), suggesting that the method of
delivery of the calcium pulse into the cell is of primary importance in eliciting a
physiological response (8).
In most eukaryotic cells calcium functions as an intracellular signaling
molecule, a so called "second messenger". The first messenger being
hydrophillic molecules communicating at the surface of the cell. These first
messenger molecules interact with cell surface receptors to produce a variety of
cellular activities. These activities are stimulated either by extracellular
molecules interacting with membrane bound enzymes or by interacting with
gated ion channels. In either event the concentration of intracellular mediators
changes which results in a signal that initiates an intracellular response.
Adenylate cyclase is an example of a membrane bound protein that is
activated by extracellular molecules. It is not activated directly by extracellular
molecules but by other activated proteins. When a signaling molecule (first
messenger) binds to the plasma membrane receptor it activates another
membrane bound protein, G protein . The activated G protein activates
adenylate cyclase. One First messenger receptor protein can activate many G
proteins thereby greatly amplifying the adenylate cyclase response. Adenylate
cyclase is composed of two subunits. One has catalytic activity which converts
ATP to cyclic-AMP (cAMP), the other is a regulatory protein that activates the
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catalytic unit when it binds GTP and inactivates it when it hydrolyzes GTP.
Cyclic-AMP in turn acts as a second messenger to activate cAMP-dependent
protein kinases. These enzymes are tetramers which are composed of two
regulatory and two catalytic subunits. The cAMP dependent kinases and their
associated phosphorylases modulate many physiological and metabolic
activities. Some of these activities are calcium dependent (discussed later) and
known to be initiated by: many types of hormones, neurotransmitters, seratonin
and histamine, certain prostaglandins, and other extracellular first messengers.
An alternative to modulating intracellular mediators by activating cell
surface proteins like adenylate cyclase, is for extracellular molecules to activate
cell surface proteins which interact with gated ion channels. The alteration of
intracellular mediator concentration produces transient action potentials, as
seen in electrically active cells or large influxes of ions that mediate intracellular
responses. Action potentials generated in neural and muscle cells are not
accompanied by large changes in intracellular ion concentration, nor is the
initial influx an intracellular signal. The intracellular signal occurs at the nerve
terminal where calcium ion channels are opened. Calcium then acts as a
second messenger to elicit an exocytotic release of neurotransmitters. When
greater influxes of calcium enter the cell, an intracellular response can be
initiated. An enormous calcium ion gradient is available across the plasma
membrane of cells to mediate a variety of intracellular activities. Where as the
concentration of internal and external calcium is more or less equal, eg.,
approximately 10'3 M, the concentration of free cytosolic calcium is 10 "7 M or
less, so that most of the internal calcium is bound to other molecules or
sequestered in cellular organelles. This gradient must be maintained if calcium
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is to serve as an intracellular signal.
Membrane bound ATPases use ATP hydrolysis energy to pump calcium
out of the cell or into cellular organelles. These calcium pumps are sometimes
coupled to the movement of other ions like sodium to maintain homeostatic
gradients and membrane potentials. Endoplasmic reticulum (ER) and
particularly sarcoplasmic reticulum of muscle cells remove large amounts of
calcium from the cytosol. Mitochondria maintain high levels of lumenal calcium
by removing it from the cytosol in a process that is driven by inner mitochondrial
membrane potentials. Calcium is freely permeable to the outer mitochondrial
membrane. This process directs the positively charged calcium ions through
gates where the lumenal negative charges are generated during the electron
transfer steps of oxidative phosphorylation. Calcium, pumped out of cells by
ATPases, and in some cases coupled to reactions that pump other ions into the
cell while pumping calcium out.
When extracellular molecules stimulate second messenger activity by
opening voltage regulated gates or by activating certain membrane bound
enzymes, the cytosolic concentration of intracellular mediators change. For
calcium generated activities this means a cytosolic rise from 10-7 M to 10-5 M.
The proteins that respond must be able to do so at these levels since cytosolic
calcium concentrations rarely get above 10-5 M. Calcium binding proteins
(CaBPs) like calmodulin and others, not only sequester calcium at low levels,
but they are highly specific. They can distinguish calcium ions from other
similar ions such as magnesium even though magnesium is generally
maintained at cytozolic levels of 10-3 M, and can be as much as a thousand
fold higher than Calcium levels.
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1.5 Calcium binding proteins.
CaBPs are proteins that undergo a conformational change when they bind
calcium. These changes mediate cellular functions through their actions on
other proteins. Troponin is one of the most understood CaBPs. It functions as a
complex of three proteins (T, I, and C) that have a close association with actin
and myosin myofilaments, particularly actin. When troponin C binds calcium it
undergoes a conformational change which allows myosin cross-bridges to bind
with actin subunits. It does this by altering the position of tropomyosin on the
actin filament as part of a classic myofibril contraction. While troponin C is the
best understood CaBP, it appears to have evolved from another ubiquitous
CaBP, calmodulin. Calmodulin has been found in all plant and animal cells
that have been examined indicating that it is highly conserved throughout
evolution (9,10,11,12,13). It has been implicated in regulating a wide variety of
cellular activities (14). It is the fourth member of a family of calcium-binding
proteins to be characterized by crystallography, the others are: parvalbumin
(15), intestinal CaBP (16), and Troponin - C (17,18).
Calmodulin is a small dumbbell shaped protein of 148 amino acid
residues (Mol. wt. 16.8K) with two calcium binding domains (4 total) at each
globular end of the dumbbell. The globular ends are completely separated by
an amphipathic 8 turn alpha helix. The binding of calcium by calmodulin
causes a conformational change in the molecule allowing the proteins to
interact with the alpha helix, although it exhibits significant alpha-helicity even
in the absence of calcium binding (19,20,21,22). Calmodulin has no enzyme
activity in itself, but regulates the activity of other enzymes in response to
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cytosolic calcium levels. The calcium binding domains in this family of CaBPs
were described first by Kretinger, et al. (23) and is the basis for the family's
characteristics. Calmodulin and other members of this family, including
trypanosomal calflagins are calcimedins. Calcimedins are molecules that
reversibly expose hydrophobic residues in the presence of Calcium. Three
families have been proposed to compose the Calcimedins they are:
(1) Annexins - A group of related proteins that are involved in regulating
calcium channels and calcium dependant endo- and exocytotic processes.
They have tandem arrays of 70 residues repeated 4 or 8 times interspersed
with a 17 residue consensus sequence "the endonexin" within each repeat.
(2) Protein Kinase C Isoenzymes - A group of calcium dependent enzymes
which can phosphorylate a number of proteins with different cellular functions.
These enzymes are interesting in that they are activated cooperatively by the
products of a triphosphorylated inositol phosopholipid (PIP2). This molecule is
cleaved into inositol triphosphate, a molecule that transiently releases calcium
from its various compartments and diacylglycerol, which transiently activates
the calcium dependent protein kinase C isoenzymes. In a similar process to
the cyclic AMP pathway mentioned earlier, an activated receptor is thought to
activate a G protein (Gp) which in turn activates an enzyme (phosphoinositide-
specific phospholipase C) that cleaves PIP2. The protein Gp has been shown to
be activated by many different membrane receptors.
(3) E F-hand calcium-binding proteins. Kretsinger etal. described a helix loop
helix calcium-binding motif which was called an E F-hand because it resembles
a hand, where the thumb and index finger are extended to represent the two
alpha helices, and the rest of the fingers closed to form the Calcium binding
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loop (24).
Calmodulin is an EF-hand CABP that contains four calcium-binding
domains composed of seven helices (7-19, 29-39, 46-55, 65-92,102-112,119-
128,138-148) with a total helix content of 63% (25,26,27). Helix IV is the
backbone of the molecule, the interactive site for calmodulin regulated proteins,
and the shared helix between Calcium binding domains 2 and 3.
COOH-
NH-Ac
Ca++
Of interest is that Calmodulin target proteins share very little binding domain
sequence homology (28). It has been suggested that the backbone alpha helix
is more flexible than previously thought (29,30). Evidence using small angle x-
ray scattering (SAXS) techniques and nuclear magnetic resonance Imaging
(NMR) shows that it is possible to cross-link N and C terminal domains, and that
the central backbone is extremely flexible in aqueous solution. While the
flexibility of calmodulin is somewhat reduced by calcium binding, it seems to
enhance its affinity for target proteins (31). The flexibility of calmodulin domains
is thought to promote the "Best fit" around the various targets. The domains
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have been described as like a flexible tether (32). When the central linking
region (helix IV) of Calmodulin bends to accommodate the target, it loses some
of its helical structure in the process. The two domains then embrace the target
peptide to stabilize the complex. This is in contrast to relatively recent theory
that allows for the target proteins to embrace the central alpha helix of
calmodulin. Hydrogen bonding between the central helix and target peptides is
minimal (only one) and the complexes are largely held together via side chain
interactions. Another unusual feature of the Calmodulin target interaction is that
the target itself assumes an alpha helix conformation (28). This is a very rare
type of interaction and is the reason why target proteins can have very little
binding domain homology. While it will be sometime before intracellular
interactions between Calcium, calmodulin, and their target proteins are fully
understood, we can take what we do know about this ubiquitous calcimedin
and extend and apply that knowledge to other members of the E F-hand family.
In trypanosomes, calcium second messengers involving cellular
functions other than motility, are associated with their flagellum. The flagellum
is a complex structure that contains desmosome like structures for adhesion, an
axoneme for flagellar motility, a structural paraxial rod, and a flagellar pocket
where particulate and soluble substances enter the trypanosome (33,34).
Endocytosis of substances at the flagellar pocket are required for normal growth
and may be regulated by calcium. Coppens etal. (35) reported on low density
lipoprotein (LDL) receptors in T. brucei bloodstream forms. They had
previously shown thatT. brucei takes in host LDL through its flagellar pocket
(36). This presumably occurs as a requirement for membrane synthesis
(37,38). Where as cholesterol is the major sterol of trypanosome blood form
15
membranes, it has been reported that they are unable to synthesize it de novo
(37,39). Coppens et al. (35) report that LDL is required for growth in T. brucei.
They demonstrated that growth can be inhibited by low levels of available LDL
or by antibodies against LDL receptors. Growth can be restored in both cases
by addition of excess LDL. Endocytosis of LDL was characterized in part as
being almost completely dependent on calcium. The pocket may also be
involved in host recognition (36). Vesicals within the flagellar pockets are
similar to clatharin-coated vesicles of other eukaryotic cells. A molecule having
a molecular weight similar to the heavy chain of clatharin was observed (40)
but, was not serologically cross reactive with mammalian clatharin. The
vesicles bud from the flagellar pocket membrane and discharge their contents
into an intracellar tubular system which connects with structures similar to the
multivesicular bodies and lysosomes of mammalian cells (34,41). Within the
flagellar pockets are located a variety of receptors through which extracellular
molecules can stimulate intracellular signals. Calmodulin has been localized to
the flagella of trypanosomes and is no doubt involved in various cellular
processes including regulation of metabolism, growth, and development as it is
in mammalian cells. These receptor mediated processes might provide an
excellent target for a vaccines or chemotherapeutic agents if calmodulin were
not so highly conserved (even between mammals and trypanosomes).
In mammalian hosts African trypanosomes possess a continuous
variable surface glycoprotein coat (VSG) that can be altered antigenically and
biochemically to evade host immune responses (42,43,44). These VSG's have
in the past, been targets of much vaccine research. It now looks like
trypanosome VSG's are not going to be useful as vaccine targets based on
16
their antigenic diversity (45). New targets for vaccines and chemotherapeutic
agents are being sought. The endocytotic interface between host and
trypanosome has been suggested as a new avenue of vaccine attack (41). The
clatharin-like protein that is not cross reactive with mammalian clatharin might
be a possible site for a vaccine. Another group of proteins for possible vaccine
or chemotherapeutic agent research, has the potential at least of hitting a
number of regulatory targets. These are a group of E F-hand Calcimedins
found thus far, exclusively in trypanosomes. They are the Calflagins (46) and
like calmodulin, have been localized to the flagella (7,47). The calflagins may
also be involved in cellular activities such as growth, metabolism, and
development, similar to calmodulin. If this is the case, then they might make
good targets for vaccines or chemotherapeutic drugs, since they are not cross
reactive with mammalian calcimedins (7, 47, 48).
1.6 Calflagins.
Calflagins were first reported in 1985 by Lizardi, et. al and Gozalez, ef a/.(48)
who were searching for a vaccine to Trypanosoma cruzi by cloning genes that
coded for major surface antigens. A positive clone (IF8) was isolated by
screening expression libraries using IgG from sera of chronic, as well as, acute
chagas patients. IF8 was found to contain a single open reading frame which
was used for a homology search of protein sequences. Remarkably good
homologies were found with a number of E F-hand containing calcium binding
proteins such as parvalbumin, troponin C, calmodulin, and catalytic myosin light
chain. Hybridization studies of nick-translated IF8 probed to T. cruzi DNA
17
showed that it belonged to a group of tandemly arranged genes, and that there
was at least 30 copies of the basic 940 bp repeat unit. They showed that the
gene is transcribed in a polycistronic manner, and that the resultant mRNA is as
abundant as B -tubulin mRNA (about 5% of the total poly A mRNA). The
relative abundance of this calcium binding protein has stimulated further
interest in possible regulatory roles similar to those of calmodulin.
The T. cruzi calflagins were further characterized by Engman, et. al. in
(7) and later by Ouaissi et.al. (49). They were also selecting T. cruzi calflagin
cDNA clones with sera from infected animals. They expressed calflagin cDNA
in E. coli and showed that it could be detected repeatedly with different sera
and different expression libraries. The protein was localized to the
trypanosome flagella using mouse antisera, and it was shown that recombinant
as well as native proteins bound calcium. They demonstrated that the protein
has a low calcium binding capacity, <2 mol. Ca++/mol. protein, and a high
calcium binding affinity Kd <50u M Ca++ which is consistant with other E F-
hand calcium binding proteins. This suggests that the protein might be involved
in trypanosome motility due to its location and the role calcium plays in the
motility of other organisms. They proposed the name FCaBP for flagellar
calcium binding protein.
Lee, et. al. (50), reported that while screening African trypanosome cDNA
clones for evolutionarily conserved genes that encode cell surface proteins,
they found a cDNA clone (Tb17) with an open reading frame that shared
significant homology to the E F-hand calcium-binding proteins of T. cruzi
previously identified by Lazardi and Engman. When this clone was probed
back to genomic DNA several other clones encoding related proteins were
18
found, isolated and later sequenced (51)
In 1992, Wu, et. al. (47) reported a similar 44kDa protein from T. brucei
and described a family of flagellar E F-hand calcium binding proteins they call
the predominant calcimedins of T. brucei. Of interest is the differences between
the family of calcimedins described by Wu, et. al. (51) in T. brucei, and the
tandemly repeated genes described by Lazardi, et. al.(48), and Engman, et.
al.(7), in7. cruzi. Wu, et. al.(51), point out that the differences in gene
organization between T. brucei and T. cruzi indicate that at least some of the
calflagins might function in rolls other than motility. They characterized the
organization of these genes in T. brucei as comprising a family of proteins
consisting of a 44KDa protein (Tb-44) and a 23-26 KDa cluster that probably
includes the gene Tb-17 described by Lee et al (50). They isolated the protein
Tb-44 to the trypanosome flagella by immunofluorescence and further
characterized the proteins of Tb-44 and 23-26 KDa cluster. Partial sequence
analysis and calcium-dependent phenyl-Sepharose binding experiments
(defines calcimedins as binding to specific hydrophobic matrices in a calcium
dependent manner), demonstrated that the proteins were calcimedins of the
E F-hand type. Antibody cross-reactivity and immunoblot analysis indicate that
these proteins probably comprise a family of related proteins and that they were
not members of the annexin family.
The genomic organization of the calflagin family of proteins is currently
under investigation, and it has been shown by Wu,etai. (51) that multiple
calflagin family members occur in T. brucei where as only a single member
occurs in T. cruzi. Evaluation of the calflagin gene organization in
T. Brucei suggests evolutionary origins of calflagin variants. Of interest are
19
variants not found in T. cruzi, suggesting that at least some calflagin family
members are not required for motility, but serve some other functions. Currently
however, none of calflagin members has a known function.
1.7 Calcium/calcium binding protein interactions on trypanosomal metabolism
and development.
Evidence that Calcium is interactive with proteins like calmodulin and the
calflagins in a way that regulates trypanosomes metabolically or
developmentally has remained elusive though indirect evidence is continuing
to be compiled. Much of the evidence is gleaned from other protozoa and
mammalian investigations. This can involve some sweeping assumptions. For
instance, metazoan organisms evolved from protozoa and retained much of
their calcium metabolism. There can be further risk in comparing other
protozoa with trypanosomes since trypanosomes seem to have more than their
share of unique features. None the less, circumstantial and direct evidence is
continuing to be sought for the role calcium plays in the development and
metabolism of trypanosomes and the family Trypanosomatidae in general.
It has been shown that the cellular complement of calcium binding
proteins varies during the trypanosome life cycle (52, 53), and that calmodulin
does not represent the majority of calcium binding activity (54, 55, 56). This
suggests that trypanosomes respond to calcium signals differently during their
life cycle. Several calcium dependent proteins have shown stage specific
activation by calcium.
Paindavoine et al. (57) report on a group of cyclase glycoproteins in
20
T. brucei that are calcium dependent and developmental^ regulated. They
showed that four transcripts for adenylate and guanylate cyclases have been
found in the bloodstream forms of T. brucei. These transcripts have partial
sequence homologies to known eukaryotic adenylate and guanylate cyclases
(57,58). One of these genes ESAG 4 (expression site associated gene 4) was
found to be developmental^ regulated and transcribed only in the blood stream
form, while the other three transcripts were also found in the procyclic (insect)
form. ESAGS are a well documented group of genes (23,24,44,58,59,60,61).
ESAGs are transcribed on large multicistronic transcripts along with the
telomeric VSG (variable surface glycoprotein) gene expression site. Differential
expression of ESAGs in trypanosomes was also demonstrated by Gram and
Barry (62) who showed that ESAGs could be expressed with VSG in slender
bloodstream forms or with out VSG expression in procyclic forms. Mathews
etal. (63) showed that ESAGs are not present in the telomeric expression site
during the metacyclic trypanosome stage. Paindavoine et al. (57) showed
ESAG 4 and related glycoproteins of 150kDa appear to be trans membrane
proteins localized exclusively to the flagellum. They showed that when ESAG 4
and another related glycoprotein (GRESAG 4.1) complement a Saccharomyces
cervisiae mutant for adenylate cyclase, only ESAG 4 was activated by calcium,
the others being inhibited by it.
Another group of calcium dependent proteins found to be
developmental^ regulated in trypanosomes are protein kinase C isoenzymes.
As mentioned earlier, these are calcimedins that are calcium dependent and
known to phosphorylate a number of proteins with different functions in
mammalian cells. Keith et al. (64) report on protein kinase activities in T. brucei.
21
They identified protein kinase C enzymatic activity and isolated it to either
slender blood forms or procyclic insect forms. Using DEAE cellulose they
separated proteins based on their ability to phosphorylate histone in the
presence of calcium and diacylglycerol. Peaks of activity differed between
lysates of T. brucei bloodstream and procyclic forms. Isoelectric focusing and in
situ protein kinase assays further characterized the two groups as having
different properties. The authors conclude that the bloodstream kinases are
either more numerous or more stable than the procyclic forms. One of the
bloodstream kinases was remarkable in that it shared many characteristics with
mammalian protein kinase C. The authors report that this protein kinase C was
shown by Western blot analysis to contain an epitope that is recognized by
monoclonal antibody raised against mammalian protein kinase C. They
conclude that the bloodstream form of T. brucei has a protein kinase C
homologue which is not found in procyclic forms.
Parsons et at. (65) report on the role of protein kinases in organisms that
diverged early in the eukaryotic lineage. They report that primitive eukaryotes
possess a large complement of protein kinases, and that they play a pivotal role
in proliferation and differentiation in protozoa. They examined the activity of
renaturable protein kinases during the life cycle of T. brucei. They
demonstrated that six of eight renaturable protein kinases were regulated
during the trypanosome life cycle. Based on these finding and a previous work
where the authors demonstrated that tyrosine phosphorylation is stage specific
in T. brucei(66) they conclude that protein phosphorylation networks are
important in regulating the cyclical development of T. brucei. Tyrosine
phosphorylation is a key regulator of cell proliferation and development in
22
higher eukaryotes (66). In further support of this hypothesis they site the
observation that a strain of T. brucei defective for the slender to stumpy
transformation had substantially decreased activity in two of the stage specific
renaturable protein kinases. It was suggested by these authors that a proper
study of specific protein kinases could be done through genomic manipulation,
and that the protein kinases described in their paper would be targets of
particular interest once the relevant genes were cloned.
Some of the best evidence that calcium/calcium-binding protein
interactions are involved in the development and regulation of trypanosomes is
where calcium dependent gene products are associated with a certain
developmental stages or located on a transcriptional units with other regulatory
genes. Wong et al. (46) investigated transcription of housekeeping genes in T.
brucei. They characterized the expression of two ubiquitin genes up stream of a
EF-hand superfamily member gene and a calmodulin gene cluster. Ubiquitin is
part of the proteolytic machinery responsible for selective protein degradation in
eukaryotes. It is easy to speculate that genes transcribed with the ubiquitin
genes might also have some house keeping function. Wong etal. (46) reported
that the genes are linked and transcribed polycistronically. They suggest that
all four genes are under the control of a single distant upstream promoter. They
showed that RNA molecules that span the intergenic regions can be detected
by PCR. They conclude that there is coordinated transcription of three different
genes, each of which encode a protein with a potential regulatory function. The
coordinated production of ubiquitin with two calcium binding proteins implicates
calcium as being involved in the regulation of trypanosome metabolism. The
authors suggests that regulation of transcription initiation plays an important
23
role in the coordinated expression of housekeeping genes in trypanosomes.
As evidence that calcium plays an important role in the regulation of
trypanosome development and metabolism continues to be compiled, it
becomes as important to consider calcium homeostasis in trypanosomes as it is
to consider calcium dependent proteins. In general calcium homeostasis is
regulated by the concerted action of the plasma membrane, endoplasmic
reticulum, and mitochondria. As mentioned earlier, low levels of free ionic
calcium are maintained in the cytosol (10-5 M) in eukaryotic cells. These
delicately balanced calcium levels are critical to the physiology of the cell and
are maintained by high affinity ATPases (67). It has been shown that members
of the family Trypanosomatidae are able to maintain very low ionic calcium
concentrations (50 nM) (68,69,70,71). Information on how this is accomplished
is sketchy at best, but it has been suggested (72,73) that high affinity calcium
activated ATPases could have a role in regulating intracellular calcium levels
and calcium transport in these protozoa. High affinity ATPases have been
implicated in the transport of calcium across plasma membranes and
maintenance of cytosolic calcium levels in of some parasitic kinetoplastids (L
braziliensis, L. donovani, T. cruzi, and T. rhodesiense).
Benaim and Romero (69) demonstrated the presence of a calcium pump
in plasma membrane vesicles of L braziliensis promastigotes. This pump is
capable of maintaining low levels of cytoplasmic calcium. They describe a high
affinity calcium ATPase (Km 0.7uM) that saturates at 5 uM. They report that
maximal calcium-ATPase activity is attained when equimolar amounts of
magnesium-ATP are present in the medium. This suggests to them that the
magnesium-ATP is the substrate for the calcium-ATPase. This ATPase was
24
stimulated by calmodulin (70-80% with 5ug/ml) and inhibited by trifluoperazine.
Their work is the first to describe such a pump in L braziliensis. They showed
that the vesicles accumulated calcium against a gradient and released it after
addition of a calcium inonophore (A23187). Calcium uptake and transport were
completely inhibited by vanadate (20uM). They conclude that kinetic
parameters of the enzyme are compatible with long term maintenance of
intracellular free calcium levels in these parasites.
Ghosh et al. (72) describe a calcium dependent ATPase in
L. donovani. It appears to be involved in calcium transport and maintenance of
cytoplasmic calcium levels. The enzyme is composed of two subunits, MW
51,000 and 57,000 and has a total molecular weight of 215,000, indicating a
tetrameric structure. The authors report that this enzyme is significantly different
from mammalian types and it is apparently some what different from the
ATPases reported by Benaim etai (68,69,74) in L braziliensis and T.cruzi.
The ATPase described by Benaim and Romero (69) maximized enzyme activity
with equimolar amounts of ATP-Mg, this enzyme reported by Ghosh etai (72) is
inhibited by magnesium at higher concentrations. Ghosh etai. (72) generally
describe this enzyme as a high affinity ATPase with an extremely low Km
(35nM) which is further lowered (10-15nM) in the presence of calmodulin. The
enzyme is sensitive to vanadate and to a lessor degree trifluoperazine.
Labeling studies of the enzyme and membrane suggest that it is an integral
membrane protein that has its catalytic site on the inner face. The authors
envision that the enzyme is a pump involved in the extrusion of calcium, but
they maintain that this idea will remain speculative until the enzyme is
integrated on liposomes and ATP-driven calcium movement is detected. Their
25
report and others is strongly suggestive to the authors of a role for calcium in
the physiology of these parasites. They conclude that the localization,
orientation, and high affinity of the calmodulin sensitive calcium ATPase is
suggestive that calcium movement plays some role in the life cycle of
Ldonovani.
Similar ATPase investigations have been reported in trypanosomes.
Benaim et al. (74) also reported on a calmodulin activated ATPase in
T. cruzi. This ATPase is reported to be involved in calcium transport by plasma
membrane vesicles. Unlike other similarly described parasitic protozoan
ATPases where magnesium was inhibitory (72,73) or stimulatory, but not
required (69) the calcium-ATPase described here has an absolute requirement
for magnesium. An investigation of calcium transport by plasma membrane
vesicles using Arsenazo III as a calcium indicator showed that vesicles
accumulated calcium only when magnesium was present. Calcium was
released by the vesicles in response to the calcium ionophore A23187. The
enzyme was completely inhibited by vanadate at low levels (10uM). A Km of
0.3 uM for free calcium indicated a high affinity enzyme, but activity and
transport were further stimulated by calmodulin from endogenous and
mammalian sources. Trifluoperazine and calmidazolium inhibited calmodulin-
stimulated ATPase activity. The authors conclude that their observations
support the existence of a maganesium-depenent calcium pump in these
parasites.
Lastly but probably the first to report on ATPases in parasites of the
family Trypanosomatidae was John McLaughlin (73). He described a calcium-
ATPase in the blood stream forms of T. rhodesiense. The ATPase besides
26
having no magnesium requirement was inhibited by submicromolar levels of
magnesium. Cell fractionation studies localized the ATPase to the surface
membrane. An enriched surface membrane fraction was shown to have
ATPase activity using very low levels of free calcium (200 nM). He investigated
a range of ATPase modulators and found only vanadate had any effect (47%
inhibition). Enzyme activity was unaffected by calmodulin or calmodulin
antagonist (Chlorpromazine and trifluoperazine). He concluded that the kinetic
properties of the calcium-ATPase are compatible with a role in calcium
regulation in the bloodstream forms of T. rhodesiense.
It is interesting to speculate as to why some of these ATPases require, or
are stimulated by, magnesium and others are inhibited by it. One clear
separation of the two groups are the author's descriptions of the ATPase
location in the cell. Beniam et al. (68,69,70) described ATPases from plasma
vesicles of two different organisms, L braziliensis promastigotes, and T. cruzi
epimastigotes. These ATPases required, or were stimulated by, magnesium. In
contrast Ghosh et al. (72) and McLaughlin (73) described ATPases on surface
membranes of L donovani promastigotes and T. rhodesiense bloodstream
forms respectively. As other ATPases are discovered in similar protozoan
parasites it will be interesting to associate their magnesium requirements with
the description of their ATPase location. Of the four T. rhodesiense was the
only organism whose ATPase was not stimulated by calmodulin. Some
eukaryotic ATPases have been found to be evolutionarily related. Calcium
ATPases in the sarcoplasmic reticulum of muscle cells are one of the best
understood calcium pumps. It was found via cloning and sequencing
techniques that these pumps are evolutionarily related to the large catalytic
27
subunit of the Sodium-potasium-ATPase (pump). Similar molecular studies of
these organisms might add to their related evolutionary history as well as
contributing to their biology.
1.8 Calcium: effects on trypanosome killing and treatment.
In ways not fully understood calcium/calciun binding protein interactions
appears to be very important to the metabolism, physiology, and development
of parasitic protozoans in the family Trypanosomatidae. It may also be
important to the treatment of these infections by targeting or suplementing
various components of calcium metabolism.
Since 1902 normal human serum has been shown to have cytotoxic
effects on T. brucei (75). The trypanocidal activity was further characterized by
D'hondt et al. (76) who reported that calcium played a role in the trypanocidal
activity of HDL as an essential cofactor. They showed that normal serum, when
dialyzed, lost trypanocidal activity, but regained it after the addition of calcium.
Calcium was also found to stimulate lysis of nondialysed serum. Magnesium
was reported to have a negligible effect. Calcium could not restore heat
inactivated serum (45 mln. @ 60 °C) indicating that calcium is dependent on
some other factor for this activity. The authors presented evidence that alpha-2-
M (a protein ligand for calcium) might be involved as a calcium carrier for the
activity since serum treated with antialpha-2-M showed no trypanocidal activity.
Several D-sugars were also shown to inhibit trypanocidal activity when added
to the medium. It was thought that they might compete for a trypanocidal factor's
binding site. Glycerol was shown to have the opposite effect even in resistant
28
clones of T. rhodesiense. Rifkin (77,78) showed that high density lipoprotein
(HDL) in the serum of humans and baboons was cytotoxic to T. brucei. Rifkin
found that rabbit and rat serum had no cytotoxic effects. It was shown that HDL
induced trypanocidal activity, could be lost by digestion with phospholipase.
This was an important finding in that it showed the importance of phospholipids
in the biological activity of HDL Trypanocidal activity by HDL could also be
inhibited by adding rabbit or rat HDL to the medium. Purified phospholipids or
phosphatidylinositol could also inhibit trypanocidal activity when added to the
medium, with the latter being the most effective. Trypanocidal HDL was
characterized as having low phosphoinositol content and high
phosphatidlycholine content. As recently as July 1994 a mechanism for the
trypanocidal activity of HDL was proposed by Hager et al. (79). This work was
based on the previous work by Rifkin referenced earlier and Hajduk (Hajduk
et a/. 1989, Hajduk etal. 1992). The mechanism was reported to be based on a
minor (<1%) subclass of human and presumably baboon serum HDL called
trypanosome lytic factor (TLF). Briefly the mechanism as reported involves TLF
binding at high affinity to the flagellar pocket of the trypanosome. TLF becomes
associated with a coated pit (CP) (34) and internalized in coated vesicles (CV).
These small vesicles were thought to contain an inactive form of TLF since it
has no effect on the plasma membrane. TLF is found to be active in a
lysosomal compartment of the cell. Studies where the pH was raised indicate
that TLF must pass though an acidic environment in order to become active.
Large lysosome-like vesicals are shown to contain TLF before lysis. The
mechanism involved in the disruption of these large vesicles is still speculative,
however studies involving leupeptin (a thioprotease inhibitor) inhibited lysis by
29
69% but did not block endocytosis of TLF. The authors speculate that an
alteration in the pH environment could activate the enzyme. Alternatively TLF is
composed of two linked apolipoprotein oligomers (L-l, L-lll), which maybe
activated by modification to the protein or lipid component. The authors
conclude that TLF is a unique natural killing factor in that it not only has to be
internalized, but also requires a putative activation step. Hajduk's group has
recently showed (82) that TLF is composed of two apolipoproteins
(haptoglobin-related protein, paraoxonase-arylesterase) that exhibit peroxidaes
activity. They contend that TLF is endocytosed bound to hemoglobin and
targeted to the lysosome where it reacts with H2O2 to cause peroxidation of the
lysosomal memebrane and ultimately the death of the cell. Wheather or not
calcium or calcium/calcium binding protein interactions are envolved in TLF
killing as reported by D'hondt etal. (76) or if they are envolved with internalizing
HDL as it has with LDL (described earlier) remains unclear, but it is cause for
speculation and further studies in this area.
In the area of treatment calcium has been shown to enhance drug
therapy. The synergistic effects of calcium on trypanocidal compounds were
described by Clarkson and Amole (83). They reported that the synergistic
effects of serum on the drug combination of salicylhydroxamic acid (SHAM) and
glycerol were due to diffusible calcium ions. They were able to remove the
synergistic activity by dialysis or by adding chelating agents (EGTA, EDTA) to
the serum. They further showed that a buffer containing calcium could mimic
the synergistic activity of the serum. Neither magnesium or zinc showed any
synergistic activity. Trypanosome killing by melarsoprol was also enhanced by
calcium even more than it enhanced the SHAM plus glycerol killing. The
30
authors suggested that a mechanism may involve activation of phospholipases
by an increase in intracellular calcium which would result in lysophospholipids
causing membrane damage and cell lysis. Calcium has been shown to
influence other antiprotozoan compounds as well. In Plasmodia antimalarial
compounds maybe related to their interactions with the parasites calmodulin
(84), and in leishmania phenothiazine antileishmanials are thought to inhibit
calmodulin activity in pathogenic and vector forms (85).
Calcium whether acting alone or in concert with calcium binding proteins
appears to play a pivotal role in the biology, evolution, and control of
protozoans in the Trypanosomatidae family. To further the understanding of
the role calcium plays in these organisms, the molecular genetic analysis of a
group of flagellar calcium binding proteins of the EF-hand type was undertaken
in T. brucei. These proteins called "calflagins" (51) are thought to be
representative of the type of targets that may ultimately control these organisms.
1.9 Shotgun approach to identification of novel targets.
In recent years the indentification of novel targets and genes of a variety of
organisms has come from the random (shotgun) sequencing of genomic and
cDNA fragments (86,87,88,89). Seeking genes in this way not only contributes
to our knowledge of an organism but also contributes to the database, which is
proportional to the chance that a given shotgun sequence will contribute to a
larger contiguous sequence and ultimately the entire genome. This scenario
plays well to the strategy for understanding trypanosome biology because of
the high density of genes on the larger chromosomes and polycistronic
31
transcripts which code related genes of various cellular functions. Furthermore
difficulties in applying standard molecular techniques to trypanosomes and
their relatively small genome of 4 x 107 bp (90) makes them an attractive
candidate for whole genome sequencing. Trypanosomes are ancient
organisms that present unusual features of genomic organization not found in
other eukaryotes. In
T. brucei the nuclear organization consists of a diploid genome of seven pairs
of non-condensing homologous chromosomes (0.3 - 5.0 mb) and
approximately 100 minichromosomes (50 -150 kb) of unknown ploidy (91).
The chromosomes can not be stained but have been characterized by pulse-
field gradient-gel electrophoresis. It was shown by this method that karyotypes
can vary dramatically even within strains of a given species (92,93,94,95,96).
The minichromosomes contain few if any genes and are composed mainly of
tandemly arranged 177 bp repeats (97). Approximately 15% of the
trypanosome genome is of a repeated nature due to the minichromosomes and
telemeric repeats. Trypanosomes disperse their genetic material clonally but
sexual recombination has been reported, including: recombination of haploid
nuclei (98); haploid and diploid nuclei (99); and diploid nuclei (100). A unique
mitochondrial structure called the kenetoplast, is found in trypanosomes which
contains unusual DNA in the form of concatonated mini and maxi circles. Most
of the mitochondrial transcripts are incomplete, but are reconciled by small RNA
molecules (guide RNA) that act as templates in a process called "RNA editing"
(101). Other unique features related to the molecular biology of trypanosomes
include: Polycistronic transcription (102) and transplicing of precursor RNAs
(103,104).
32
In recent years, random (shotgun) sequencing has been applied to
Trypanosoma brucei (105,106) and entered into the EST (expressed sequence
tag) and the GSS (genome sequence survey) databases of the National Center
for Biotechnology Information (NCBI). In searching these sequences, many
interesting and potentially advantagous genes can be found by comparing
them to other related sequences using the BLAST software. Most EST and
GSS sequences in the database are derived from a variety of shotgun cloning
techniques. Recently, El-Sayed and Donelson (105,106) compared the
efficiency of sequencing random genomic fragments with random cDNAs of T.
(brucei) rhodesiense. They concluded that sequencing random DNA
fragments is as efficient in identifying novel genes as sequencing cDNA clones
due to the high density of Trypanosome genes.
In this report a method of M13 shotgun cloning was used to generate 455
Trypanosoma brucei genomic sequences totaling more than 300,000 bases.
These sequences were evaluated for putative genes and non-coding elements
against the nr (non-redundant) and GSS databases at the National Center for
Biothechnology Information using BLAST family software.
Many hopeful inroads into the biology of trypanosomes lie with the
discovery of new genes that may code unique targets for chemotherapeutic
agents or vaccines. Molecular genetic analysis of T. brucei will ultimately
reveal sequences involved in the development, survival, and pathogenicity of
these parasites and may be extended to include other similar parasites. This
study begins with the genomic organization of the Calflagin gene family and
was expanded to encompass a basic analysis of the entire T. brucei genome.
2. Materials and Methods
2.1. Amplification and isolation of genomic fragments.
Two clones Tb-1.8 (1.8 Kb) and Tb-24 (2.5 Kb) were previously
subcloned by Lee (50) and were made available in an ampicillin resistant
plasmid vector pl)C18 . The fragments were ligated into the BamH I multiple
cloning site of the vector. Nanogram quantities of Tb-1.8 and Tb-24 were
amplified by transformation of competent E.coli cells. Competent cells were
prepared by growing overnight cultures of E. coli strains DH5 alpha and/or DH5
alpha F* in YT (8 g Difco™ Bactotryptone, 5 g yeast extract, 5 g NaCI) or LB (10g
Difco™ Bactotryptone, 5 g yeast extract, 10 g NaCI) culture media. Inoculums
were taken from either frozen log phase cultures (50 ul) or colonies from fresh
agar plates. Inoculated broths were incubated in a New Brunswick™
shaker/incubator at 37° at 250 rpm. 500 ul of the overnight culture was then
added to 50 ml of YT or LB culture media in a 250 ml flask and returned to the
shaker incubator. The optical density (OD) was monitored at 550 nm with a
Beckman™ DU40 spectrophotometer until the culture reached an optimal
absorbance of 0.45. The culture was then divided in half and placed into two
30 ml Corex™ tubes and placed on ice for 10-20 min. The culture was then
centrifuged for 10 minutes at 2200 rpm (1000 x g) at 4 °C. The supernatant was
discarded and the pellet was resuspended in 2.5 ml sterile fresh (within 2
weeks) TSS solution (0.85 g Difco™ Bactotryptone, 0.43 g yeast extract, 0.85 g
NaCI, 5.0 ml 5% DMSO, 10 g of 10% PEG 8000 MW, 50mM MgCl2 (pH 6.5),
34
and ddH20 up to 100 ml). The competent cells were place on ice until ready for
transformation. Optimally they were used within 6 hours. Cells were
transformed by placing nanogram quantities of vector plasmid (10-25 ul) in the
bottom of a microfuge tube. To this was added 200 ul of DH5 alpha competent
cells. The tubes were then placed on ice for 10 minutes. The cells were then
heat shocked in a water bath at 42 °C for 2 minutes. One ml. of YT or LB media
was added to the tubes and they were placed in a shaker/incubator at 37 °C for
one hour at 250 rpm . Transformed cells were screened on ampicillin plates
using blue/white selection (107). Ampicillin plates (8.0 g Difco™ tryptone, 5.0 g
yeast extract, 5.0 g NaCI, 15.0 g agar, 50 ug/ml ampicillin, and ddH20 up to one
liter) were prepared for visualizing transformed cells by spreading 10 ul of 100
mM IPTG (isopropyl-b-D-thiogalactopyranoside) and 50 ul of 2% X-gal (5-
bromo-4-chloro-3-indoyl-b-D-galatoside) (25mg in 1.25 ml dimethylformamide)
over the surface of the plates and allowing the solutions to soak in for a few
minutes. Optimum plating densities were determined by plating volumes of 10,
50, and 100 ul of transformed cell culture. The transformed cells were spread
onto the surface of the ampicillin plates and allowed to soak in for a few minutes
before incubation. Controls included: Competent cells without plasmid
(nontransformed) on selective media to test antibiotic killing and competent
cells transformed with varying amounts of plasmid (.01-1.0 ug) on ampicillin
plates without X-gal or IPTG to test transforming efficiency and the level of
competence. Blue colonies (carry plasmids with no DNA inserted into the
multiple cloning site (MCS)) were noted on non-control plates as a measure of
ligation efficiency. White colonies (carry plasmids with DNA inserted into the
MCS, thus inactivating the lac Z gene) were inoculated into 5 ml tubes of YT or
35
LB broth containing 50 ug/ml ampicillin and incubated over night in a shaker/
incubator at 250 rpm and 37 °C.
2.2 Isolation of plasmids (analytical scale).
Positive cells (white colonies) were cultured overnight, harvested, and
analytical scale plasmid DNA isolation performed by standard alkaline lysis
"rapid prep" procedures as outlined in the monograph by Maniatis et al. (107),
which is a modification of a procedure described by Birnboim and Doly (108)
and Ish-Horowicz and Burke (109). 3.0 ml of the overnight culture was
centrifuged at 10,000 x g for one minute. The remaining 2 ml of culture was
stored at 4 °C or combined with 80% glycerol and stored at -70°C. The
supernatant was discarded and the pellet was resuspended by vortexing in ice
cold "Solution A" (50.0 mM glucose, 10.0 mM EDTA
(ethylenediaminetetraacetate, disodium form), 25.0 mM Tris-HCI pH 8.0, and
6.0 mg/ml lysozyme (added just prior to use). This solution was allowed to set
at room temperature for 5 minutes. Next was added 200 ul of freshly prepared
"Solution B" (0.2 N NaOH, 1.0% SDS (sodium dodecyl sulfate)). The tube was
then inverted gently two or three times and placed on ice for 5 minutes. To this
solution was added 150 ul of ice-cold potassium acetate, pH 4.8. The solution
was mixed by inverting two or three times and stored on ice for five minutes.
The solution was then centrifuged for 5 min at 10,000 x g and the supernatant
transferred to a new tube. An equal volume (450 ul) of phenol:chloroform was
added to the supernatant and thoroughly vortexed. The solution was then
centrifuged at 10,000 x g for 2 minutes and the supernatant was transferred to a
36
fresh tube (phenol layer discarded). Two volumes of ice-cold 100% ethanol
was added to the supernatant and mixed by vortexing. The samples were then
placed in an iced ethanol bath for 5 minutes, then centrifuged at 10,000 x g for
10 minutes at 4°C. The supernatant was then discarded and the pellet was
washed with 1 ml of ice cold 70% ethanol by gently inverting the tube once or
twice. The samples were then centrifuged once again at 10,000 x g for 10
minutes at 4°C. The supernatant was removed from the samples and the
pellets were further dried in a vacuum desiccator for approximately 5 minutes.
Care was taken not to over dry the samples which would have made
resuspending them more time consuming. The dry samples were resuspended
in 30 ul of TE buffer (10.0 mM Tris base pH 8,1.0 mM EDTA) and stored at
-20°C. DNA samples were analyzed by agarose minigel electrophoresis for the
presence and size of plasmid DNA.
2.3 Agarose mini gel analysis.
1 % agarose gels were cast by melting one gram agarose/100 ml 1 X
TBE (10.8 g tris base, 5.5 g boric acid, 4 ml 0.5 M EDTA, and ddH20 up to 1
liter) in a microwave oven. The agarose solution was weighed prior to heating
and rehydrated afterwards. The hot agarose solution was poured into open
ended plexiglass trays with the ends sealed with masking tape. A well form
was then placed square to and near one end of the tray and the agarose was
allowed so solidify at room temperature. In preparation for electrophoresis of
"rapid prep" plasmid DNA, the gel was placed in an electrophoresis tank
(masking tape removed) and covered (1-2 mm) with 1 X TBE solution. Typically
37
a convenient volume (25 ul) of a sample was prepared by adding 3 ul of rapid
prep DNA to 5 ul Loading Buffer (25 % glycerol, 0.5% SDS, 0.1% bromophenol
blue leading dye, 0.1 % xylene cyanol lagging dye, 50.0 mM EDTA) and 17 ul
of ddH20. The samples plus a size marker (usually Hind III digested lambda
DNA) were placed into the agarose slots and electrophoresed at 80 volts for
one hour. To visualize the plasmid DNA the gel was stained for 30 minutes with
an ethidium bromide (EtBr) solution (1 mg / liter ddH20) and destained for 30
minutes with ddH20. Ethidium bromide fluoresces orange in UV light. The EtBr
intercalates between DNA bases. Caution: EtBr is considered a potent
mutagen and extreme care is taken when working with this substance including
the use of gloves and protective Benchcoat™. "Rapid prep" samples showing
bands of a size consistent with the plasmid plus the insert were noted for further
treatment and / or photographed.
2.4 Gel photography.
A UV radiation source (Fotodyne™ transilluminator) was used in
conjunction with a Polaroid™ MP-4 camera system. Gels were placed on the
transilluminator and visualized with UV. The gels were then centered and
brought into focus with the camera. The camera was then loaded with
Polaroid™film Type 55 Positive/Negative (ISO 50). A red filter was placed over
the aperture, and the aperture setting was typically adjusted to 4.5. Higher
aperture settings were used with faint bands. At an aperture of 4.5 the film was
exposed for 1 minute and developed according to package instructions.
38
Negatives were treated by soaking in 9% sodium sulfite and kept as part of the
data.
2.5 Digesting DNA with restriction endonucleases.
The inserts were removed from the plasmid DNA by BamH I restriction
endonuclease, and samples were analyzed by agarose mini gel
electrophoresis. 1- 5 ug of plasmid DNA was typically digested in a convenient
volume (typically 20 ul for mini gel analysis) with no more than 10%/unit volume
restriction endonuclease and the appropriate 10X reaction buffer. One unit of
enzyme was used to digest 1 ug DNA for 1 hr. Protocols were essentially the
same for other low molecular weight DNA fragments. Twice the amount of
enzyme was used to digest genomic DNA. Digested samples were analyzed
by mini gel electrophoresis and positive clones showed banding patterns
consistent with the size of the plasmid and the insert. These clones were noted
on the selective antibiotic plate from which they came in preparation of
obtaining large quantities of the insert for sequencing and other analysis.
2.6 Large scale isolation of recombinant plasm ids.
Large scale ultra pure isolation of recombinant plasmids was performed
on positive isolates. Up to two liters of each E. coli clone was prepared for
isolation by inoculating a 25-50 ml LB broth culture and incubating overnight in
a shaking (250 rpm) incubator at 37 °C. 10-50 ml of the overnight culture was
used to inoculate 1 or more 2800 ml Fernbach™ flasks containing one liter LB
39
broth each. Some cultures containing plasmid vectors whose copy numbers
were less than that of pUC vectors were monitored at 550 nm until they reached
0.8-1.0 (log growth phase) at which time 2 ml of 20 ug/ml chloramphenicol in
95% ethanol was added to increase plasmid yields. Cultures were incubated
overnight in a shaking (250 rpm) incubator at 37 °C. The cells were collected
by centrifugation in a precooled (4 °C) Sorvall™ centrifuge with a GS3 rotor.
Approximately 500 ml/bottle was centrifuged at 6000 rpm for 6 minutes. The
cells were resuspended in 20 ml 0.15 M NaCI and placed in 45 ml Oakridge™
style tubes. Each tube contained the pellet equivalent of 750-1000 ml of
culture. These tubes were centrifuged in a precooled (4 °C) Sorvall™
centrifuge with a SA600 rotor at 6000 rpm for 5 minutes. From this point on all
work was done on ice in a cold room at 4 °C. The pellet of each tube was
thoroughly resuspended in 10 ml 50 mM Tris, 25 % sucrose, pH 8.0. Two ml of
5mg/ml lysozyme was added to each tube and mixed by gently inverting the
tubes 2-3 times. The tubes were then placed on ice for 5 minutes. The addition
of 4 ml 0.25 M Na2 EDTA, pH 8.0, followed and the tubes were again mixed by
inverting and allowed to sit on ice for 5 minutes. Five milliters of 5 M NaCI and
2 ml of room temperature 10% SDS was then added and the tubes mixed by
gentle inversion after each addition. The tubes were then balanced and left on
ice in the cold room for 90 minutes. The tubes were then centrifuged at 16,350
rpm for 45 minutes in a SA600 rotor. The supernatant was then poured into a
graduated cylinder and one volume of isopropanol was added to it. This
solution was then poured into a GSA centrifuge tube and placed in a dry ice
ethanol bath for twenty minutes or left over night at -70 °C. The frozen contents
of the GSA centrifuge tubes were then thawed in a tap water bath. The tubes
40
were then centrifuged in a GSA rotor for 20 minutes at 8000 rpm. The pellet
was resuspended in 15 ml/liter of culture TE (10mM Tris, 1mM EDTA, pH 8.0) by
slowly stirring on a stirring plate. Care was taken to avoid foaming of the
solution. A concentration of 20 ug/ml DNase free RNase was added to the
solution. Larger pieces of pellet were broken up by repeated pipetting.
Approximate yields of plasmid were determined by mini gel analysis.
Undissolved material was removed by centrifugation at 4 °C in a ™Sorvall
centrifuge using a SA600 rotor at 10,000 rpm for 10 minutes. To the
supernatant was added 5.3 g CsCI/5 ml TE. The solution was mixed gently to
avoiding the introduction of bubbles. Four hundred microliters of 10 mg/ml EtBr
(ethidium bromide) was added per 8 ml of solution under reduced lighting to
prevent DNA cleavage. The plasmid preparation was then introduced into Til
270 ultracentrifuge tubes, one per liter of culture. The tubes were balanced to
within 0.03 g by adding a balancing solution of either TE/CsCI (heavier) or
mineral oil (lighter). Great care was taken to insure that the tubes were full to
the top to prevent the tubes from collapsing during centrifugation. The tubes
were crimped shut with Ultracrimp™ caps and crimping device. The tubes were
centrifuged in a Dupont™ OTD65 ultracentrifuge for 40 hours at 36,000 rpm.
The tubes were then removed from the rotor and clamped into a ring stand.
Bands were visualized with a hand-held UV source. Care was taken to work in
subdued light to prevent photo-activated cleavage of the DNA by EtBr.
Protein
• • <4— Chromosomal DNA Plasmid
J ^ ~ \ RNA
41
Protective eye ware was also used through out the experiment where UV light
was used. EtBr stained plasmid DNA bands were extracted from the centrifuge
tubes while the tubes were clamped in a ring stand. A 20 gage needle was
inserted in the top of the tube near the cap to act as a vent. Another 20 gage
needle attached to a 3 ml syringe was inserted directly into the lower portion of
the plasmid containing band and the contents withdrawn. In some cases the
bands containing the plasmid DNA were subjected to another 40 hour
centrifugation as previously described to further remove contaminating
materials. After centrifugation, the plasmid DNA was placed into 15 ml conical
tubes and extracted with water-saturated isobutanol in subdued light to remove
the EtBr. The EtBr was removed with the top isobutanol phase (water-saturated
isobutanol is the upper phase of the mix). Several extractions were necessary
to remove all of the EtBr as monitored by a visually clear isobutanol layer.
When the isobutanol layer was clear two volumes of ddH20 and 9 volumes of
100% ethanol were added to the plasmid preparation and the solution was
thoroughly vortexed . The solution was then poured into 30 ml Corex™ tubes
and centrifuged at 4 °C in a Sorvall™ centrifuge at 8000 rpm for 10 minutes.
After centrifugation the supernatant was poured off completely and the pellet
was redissolved in 300 ul of 0.3 M sodium acetate and transferred to a 1.5 ml
microfuge tube. The DNA was extracted with 1 ml of 100% ice cold ethanol and
placed in a dry ice ethanol bath for two minutes. The DNA was then pelleted by
centrifugation (10,000 x g for 10 min. ® 4 °C). The supernatant was then
removed and the pellet was washed with 1 ml 70% ethanol by inverting the
tube one time (care was taken not to disturb the pellet while adding the
ethanol). The DNA was collected by centrifugation (10,000 x g for 5 minutes at
42
4 °C). The samples were then placed in a vacuum desiccator for 2 minutes to
remove residual ethanol. Care was taken not to completely dry the pellet in
order to facilitate later resuspension. The pellet was resuspended in 1 ml of TE
buffer or ddH20/liter of culture (0.5 ml if the pellet was divided into two tubes).
The absorbance was scanned from 220 nm to 320 nm (Beckman™ DU-40
spectrophotometer) to give an estimate of the yield and purity.
2.7 Preparative scale digestion of cloned DNA fragments.
100-200 ug of recombinant plasmid DNA (100-200 ul) was prepared for
fragment isolation. The plasmid DNA was digested in 1 ml microfuge tubes with
restriction enzymes. Both plasmids containing T. brucei calflagin sequences
were digested with Bam HI restriction enzyme. The digestion mixture was
completed in a volume of 250 ul and contained 25 ul of 10 X reaction buffer
(New England Biolabs) and 250 units of restriction enzyme (New England
Biolabs). Digestions were allowed to proceed for at least 3 hours at an optimal
temperature for the enzyme (37 °C for BamH I). The digested plasmid DNA was
purified (buffer salts removed) by ethanol precipitation and analyzed by mini gel
electrophoresis (see Agarose mini gel analysis).
2.8 Preparative scale isolation of cloned DNA fragments detected by UV
shadowing techniques.
The vector was separated from genomic DNA fragments by vertical
agarose gel electrophoresis. The technique required the assembly of an
43
agarose gel cassette. Glass plates (20 x 20 cm and 20 x 22 cm) were
thoroughly cleansed with Windex ® and then wiped with non-denatured 95%
ethanol. The plates were then wiped with a solution of 1%
dimethyldichlorosilane under a fume hood and allowed to dry for a few minutes.
The plates were then baked briefly in a drying oven at a warm setting. Prior to
assembly of the cassette, the plates were inspected visually at low angle and
wiped with a clean dry Kimwipe® to remove any dust particles. The cassette
was assembled by placing the two siliconized surfaces together, and placing
between them two Delrin® 20 x 1.0 x 0.15 cm spacers flush with the edge along
the long axis of the plates. A cellulose sponge (1.0 cm x 18.5 cm) saturated n
TBE buffer was placed between the two plates perpendicular to the long axis
and flush with the edge of the plates leaving the 22 cm plate to overhang at the
opposite end. The plates were then clamped firmly together with three medium
sized binding clips. Care was taken to insure that there was no space between
the Delrin® spacers and the sponge. Excess buffer from the sponge was
poured out from between the plates. The cassette was then placed into the
lower buffer well of a vertical agarose gel stand (which circulates the buffer from
the lower to the upper buffer wells) and clamped into place with the
overhanging 22 cm plate facing outward (Fig. 1 A and B). Care was taken to
insure the cassette was in a true vertical position. Two neoprene spacers (1 cm
x 2 cm x 0.6 cm) were placed between the 22cm plate and the gel stand forming
the upper buffer chamber. A small amount of vacuum grease was placed
between the top of the Delrin spacers and the neoprene spacers. A well
forming comb was inserted into the cassette such that the top of the slots of the
comb were above the level of the 20 x 20 cm plate. The size of the comb was
44
chosen according to the amount of DNA to be loaded, generally 500 ug DNA to
a 7-8 cm well. 1X TBE electrophoresis buffer was placed in the lower buffer
well up to the top of the sponge. The gel was cast while the cassette was in the
gel stand. 10-12 ml of melted agarose was pipetted into the cassette down one
edge with care being taken to avoid the introduction of bubbles. The level of
TBE in the lower buffer chamber was then raised to the level of the agarose, but
not exceeding it, forming a plug in the bottom of the cassette. After the agarose
had solidified the remainder of the cassette was filled with molten agarose. The
gel was allowed to solidify completely and the well forming comb was carefully
removed. Both the upper and lower buffer chambers were filled to capacity and
the circulating pump turned on to test for leaks. The DNA was then loaded into
the wells with agarose loading buffer (see Mini Gel Analysis). Care was taken to
insure that the samples were distributed evenly across the wells. The gel was
run at 80 volts for 4-5 hrs depending on the size of the fragment that was to be
isolated. The bands were detected by UV shadowing techniques. After the
DNA had electrophoresed for the desired length of time the gel was transferred
45
7 1 _ X L i l i r L i J T
poer r„, Buffer Chamber Z
Agarose Gel
. Sample Wells
•Delrin Spacer
Lover Buffer Chember
-Binding Clip
PUMP
i m r o f c w
^ / / / / / / / / / ^ ^ ^ ^ ^ ^
#WAA{ IAAAA0/ mwm.
front view side view
B 3/4 Inch Offset - [
0.5 Inchx 1.5 mm Delrin™ Spacer
Cellulose Sponge
Delrin Comb
Medium Clip
46
Figure 1 A Electrophresis stand and buffer pump assembly for
vertical agarose gel electrophoresis.
Figure 1 B Assembled cassette for vertical acrylamide gel electrophoresis.
47
to a thin layer chromatography (TLC) plate impregnated with an ultraviolet
indicator. A shortwave (260 nm) hand held UV lamp was held over the gel.
The plate fluoresced except where the DNA was present to absorb the UV light
which presented a shadow or darkened area on the gel. (Fig. 2). Those bands
corresponding to known desired fragment sizes were excised from the gel and
prepared for electroelutlon.
2.9 Electroelution and DNA recovery from excised from gels.
The excised bands were placed into cut lengths of dialysis tubing
according to the size of the gel slice. A dialysis bag clip was placed at one end
of the bag and the bag was filled with a quantity of 0.25X TBE buffer. Another
clip was placed at the other end of the bag and care was taken to remove all the
bubbles. The dialysis bag with the gel slice inside was then placed horizontally
in an electrophoresis chamber and covered with .25X TBE buffer. The level of
buffer was adjusted so it just covered the bag. Electroelution proceeded at 80
volts for 2-4 hours depending on the size of the fragment. When the
electroelution was complete the current was reversed for approximately one
half a minute to dislodge the DNA from the side of the dialysis tubing. The
DNA/TBE buffer was removed with a siliconized Pasteur pipette. The gel was
washed in the tubing with an additional volume of 0.25X TBE. Electroeluted
samples were extracted twice with one volume each TE saturated Phenol. The
aqueous DNA solution was then extracted with one volume of ether. The upper
ether layer was removed and the samples were allowed to sit under a fume
48
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49
Figure 2. Principle of UV shadowing for the visualization of DNA bands in
agarose gels.
50
hood while residual ether volitalized for a few minutes. The samples were
centrifuged at 10,000 x g to remove any residual agarose and the supernatant
was transferred to a fresh tube. The samples were then placed in a vacuum
desiccator to reduce the volume to a more workable level (250 ul). The DNA
was then precipitated by adding one tenth volume (approximately 25 ul) of 3 M
sodium acetate and 3 volumes of 100% EtOH. Each sample was then vortexed
and placed in a dry ice ethanol bath for five minutes. The DNA was then
pelleted by centrifugation at 10,000 x g for 10 minutes. The supernatant was
removed and the pelleted DNA sample was gently washed (inverted once) with
1 ml of 70% ethanol and recentrifuged for an additional 5 minutes at 10,000 x g.
The supernatant was again removed and the pelleted DNA sample was placed
in a vacuum desiccator for five minutes to remove residual EtOH and H2O. The
DNA pellet was then resuspended in 25 ul of TE buffer by vortexing, and
alternately heating (65°C) and freezing the sample. A small fraction of the
sample (1.5 ul) was prepared for mini gel analysis (see Agarose Mini Gel
Analysis) to verify DNA fragments. The analysis was carried out with a tf/'nd III
cut lambda ladder for size reference. Once the fragments were verified they
were screened for restriction sites that would allow for subcloning.
2.10 Screening isolated DNA fragments for restriction sites.
Isolated DNA fragments were cut with various restriction enzymes
(described earlier) and electrophoresed on agarose gels (see sections 2.3 and
2.5) or 6% non-denaturing polyacrylamide gels (described below). Non-
denaturing polyacrylamide gels were used to identify small DNA fragments
51
(100-500 bp), which are beneficial in subcloning with some vectors. Digested
DNA fragments (1-2 ug) were combined with 2.5 ul of 5X nondenaturing
polyacrylamide gel electrophoresis loading buffer (0.25% bromophenol blue,
0.25% xylene cyanol, 0.5% SDS (sodium dodecyl sulfate), 25.0% glycerol) and
heated to 65 °C for five minutes. Samples not used immediately were stored at
-20 °C. A 20 x 20 x 0.15 cm cassette was assembled to contain the gel during
electrophoresis. The nondenaturing polyacrylamide gel solution was made by
mixing the following: 12.0 ml acrylamide stock solution (40.0 g electrophoretic
grade acrylamide, 1.0 g bisacrylamide, ddHteO to a final volume of 100 ml); 8.0
ml 10X nondenaturing TBE buffer; 60.0 ml ddH20; and 0.1 g ammonium
persulfate. The solution was filtered into a 250 ml vacuum flask and then
degassed until no bubbles were visible. Note: It is important to remove radical
scavengers like O2 because they will inhibit the polymerization process. After
degassing, 20 ul of TEMED (N,N,N\N\-Tetramethylethylene-diamine) was
added to the solution with gentle swirling of the flask. The solution was then
poured into the cassette prior to polymerization. Once the gel had polymerized
it was pre-electrophoresed for one hour at 300 volts. Samples and reference
markers (pBR322 plasmid cut with Hae III, Hpa II, or Hinf I restriction enzymes)
were loaded on to the gel with a 25 ul Hamilton® microsyringe. The gel was
electrophoresed at 250 volts until the bromophenol blue dye (leading dye)
migrated to about 80% of the gel's length. On completion of the electrophoresis
the gel was stained with EtBr for 5-10 minutes and then destained for 10
minutes. Some gels were photographed for later reference (see Gel
Photography).
52
2.11 Subcloning into M13mp and pUC vectors.
After the samples were screened for restriction sites using a variety of
restriction enzymes, the fragments were analyzed for further subcloning (110)
and sequencing. The restriction fragments for subcloning were ligated into
M13mp18/19 virus vectors or pUC 18/19 plasmid vectors. The variants of each
vector (18/19) have multiple cloning sites in opposite orientations permitting
sequencing of both DNA strands. Insertion of foreign DNA into the multiple
cloning site of either vector causes the inactivation of the amino-terminal
fragment of the beta-galactosidase gene which can be detected visually (white
plaques or colonies) when the presence of a synthetic inducer, isopropylthio-B-
D-galactoside (IPTG) and a substrate, 5-bromo-4-chloro-3-indoyl-l3-D-
galactoside (X-gal) is present in the media. Vectors were prepared by digesting
with restriction endonucleases complementary to the target DNA fragments.
Vectors were digested such that DNA fragments could be subcloned in either
direction or cloned unidirectionally. The vectors were further treated with
bacterial or calf intestinal alkaline phosphatase (BAP, CIAP) which removes
the 5' phosphate and prevents closing of the restriction digested ends. The
phosphatase treatment involved combining 100 ng of linearized vector with 100
units of bacterial alkaline phosphatase enzyme in a volume of 50 ul diluted with
TE buffer. The digested and phosphatase treated vector was then extracted
with phenol and precipitated with ethanol to remove enzyme and buffer salts.
The target DNA and the vector were then brought together in a ligation mixture
at a ratio of 1:1 - 3:1 (although other ratios were used to maximize
53
transformants). The ligation was contained in a volume of 10-20 ul and
contained: approximately 100 ng of vector DNA; 100 ng of target DNA; 5X DNA
ligase reaction buffer (250.0 mM Tris-HCL (pH 7.6), 25%(w/v) PEG 8000, 50.0
mM MgCl2, 5.0 mM rATP, 5.0 mM DTT (dithiotheitol)), T4 DNA ligase (0.1 BRL
unit for sticky ends, 1.0 unit for blunt ends), and sterile water up to volume. The
mixture was incubated at room temperature for 4 hours. The reaction was
stopped with 1/5 volume 0.25 M EDTA and sterile water up to a final volume of
30 ul.
2.12 TSS competent cells.
DH5 alpha F or P cells were inoculated into a 5 ml. culture of YT or LB
broth and grown overnight at 37 °C and 250 rpm in a New Brunswick™
shaker/incubator. 500 ul of the overnight culture was used in inoculate 50 ml
YT or LB broth in a 250 ml flask. The culture was returned to the shaker
/incubator and monitored until the absorbance 550 nm reached 0.45. The
culture was then divided into two sterile tubes and placed in an ice bath for 10-
20 minutes. The culture was then centrifuged at 1000 x g for 10 minutes at 4
°C. The supernatant was then discarded and the pellet resuspended in 2.5 ml
of sterile (filtered or autoclaved) TSS (0.85 g Bacto™ tryptone, 0.43 g yeast
extract, 0.85 g NaCI, 5.0 ml DMSO, 10.0 g PEG 8000, 50 mM MgCl2 (pH 6.5),
ddH20 to 100 ml). Each tube was then placed on ice until ready for use or
stored at 4°C for up to two weeks.
54
2.13 Transformation of DH5 alpha F with recombinant M13mp 18/19.
Recombinant M13mp RF forms were used to transform DH5 alpha F'
cells by the TSS competent cell method. Transformation was visualized as a
plaque in a lawn of cells. Plaques containing recombinant phage appear as
clear while plaques without recombinant phage appear blue. 300 ul of TSS
competent cells (described above) were added to a 1.5 ml microfuge tube with
approximately 10 ul ligation mixture (25-50 ng recombinant DNA). This mixture
was placed on ice for 30 minutes and then heat shocked for 2 minutes at 42 °C.
The cells were then added to a tube containing: 3 ml YT soft agar (10 g Bacto™
peptone, 8 g NaCI, 6 g Bacto™ agar/ liter) stored at 42 °C; 50 ul 2% X-gal, 10 ul
100mM IPTG, and 200 ul of lawn cells. The entire contents was swirled briefly
and poured on to a YT agar plate. The contents was spread evenly over the
plate by tilting the plate as necessary. The top agar was allowed to solidify at
room temperature and then placed at 37°C overnight.
2.14 Transformation of DH5 alpha F or F cells with recombinant plasmid.
1.0 - 0.01 ug of recombinant plasmid in a volume of 10 - 25 ul was
placed into the bottom of a 1.5 ml microfuge tube with 200 ul TSS competent
cells (described above) and placed on ice for 10 minutes. The cells were then
heat shocked in a 42 °C water bath for 2 minutes. 1 ml of sterile YT or LB broth
was then added to each tube and incubated at 37°C in a shaker/incubator at
55
250 rpm for one hour. During the incubation period LB/ampicillin plates were
prepared by spreading 10 ul of 100 mM IPTG and 50 ul 2% X-gal on to each
plate with a sterile hockey stick. At the end of the incubation period transformed
cells were spread onto the plates in varying amounts (10, 50,100 ul) and
incubated overnight at 37 °C. Plasmid was isolated by "rapid prep" protocols
(see Isolation of plasmid 2.2)
2.15 Preparation of M13mp single stranded template for sequencing.
The plaques containing the recombinant phage (clear) were removed
from the plate with sterile Pasteur pipette by driving the end of the pipette into
the plaque and pulling out the plaque along with a plug of agar. The plugs
were placed in 5 ml of YT media along with 100 ul of overnight DH5 alpha F
culture. The culture with the plaques were shaken at 250 rpm for 5 hours at 37
°C. After the incubation period 3.6 ml of phage infected culture was transferred
to 5 ml polypropylene tubes and centrifuged for 8 minutes at 1000 x g in a
microcentrifuge. The supernatants were transferred to new sterile 5 ml
polypropylene tubes where they could be stored at 4 °C, or used. The pellet
contained Replicative Form DNA (RF DNA) and could be saved for a small
scale preparation of the clone. To proceed with the isolation of the single
stranded template for sequencing, the supernatants were centrifuged again in
the same manor as before and transferred to sterile tubes to ensure that all
bacterial debris was removed. To the clean supernatants was added 900 ul of
a solution containing 20% PEG 8000 (polyethylene glycol) and 2.5 M NaCI.
The tubes were covered with Parafilm® and thoroughly vortexed. The tubes
56
were then set aside at room temperature for 15 minutes. Following the
incubation the parafilm was removed and the tubes were centrifuged for 10
minutes at 10,000 x g to pellet the phage particles. The supernatant was
removed completely with a drawn out pasteur pipette and the phage pellets
resuspended in 100 ul of TES buffer (20 mM Tris-HCI, pH 7.5,10 mM NaCI, 0.1
mM Na2 EDTA) by thoroughly vortexing. The solution was then transferred to
1.5 microfuge tube containing 100 ul of TE-saturated phenol and vortexed 20
seconds. The solution was then centrifuged for 2 min at 1000 x g followed by
the removal of 80 ul of the phenol layer. The solution was then vortexed for 20
seconds with 100 ul of chloroform and again centrifuged for 2 minutes to
separate the organic and aqueous phases. 80 ul of the upper aqueous phase
was then transferred to sterile 1.5 ml microfuge tubes. To these tubes was
added 9 ul of 3 M sodium acetate (300 mM final concentration) followed by
vortexing and 200 ul of 100% EtOH followed by more vortexing. The tubes
were then place in a dry ice ethanol bath, or -70 °C freezer to precipitate the
DNA. The DNA was then pelleted by centrifugation at 1000 x g for 10 minutes.
The 100% ethanol was removed and the pellet was then washed with 70%
ethanol by inverting the tube once and then recentrifuging the pellet for 5
minutes at 1000 x g. The Ethanol was again removed and the pellet was dried
in a vacuum desiccator for 2 minutes to remove any residual ethanol and H2O.
The viral DNA pellet was resuspended in 30 ul of TE buffer by alternately
vortexing, centrifuging, and placing the tube in a 65 °C water bath until the
pellet was thoroughly dissolved. Three microliters of the sample was analyzed
by mini gel electrophoresis (see 2.3). Recombinant phage with at least 300 bp
of insert will have reduced mobility compared to nonrecombinant M13mp DNA.
57
(108,109,110)
2.16 Nucleotide sequencing of double-stranded and single-stranded templates
by the dideoxyribonucleotide method.
DNA sequencing reactions were performed on genomic sub-clones as
described in the Sequenase® version 2.0 DNA sequencing kit obtained from
US Biochemical (111,112,113). Double-stranded templates (plasmid) were not
initially treated the same way as single-stranded templates. From the primer
annealing step on, however, the sequencing reaction was the same for both
templates.
To prepare for double stranded sequencing, approximately 4 ug of clean
plasmid DNA was dissolved in 30 ul of sterile water. The DNA was denatured
with 3 ul of 2 N NaOH at room temperature for five minutes. The samples were
then ethanol precipitated by vortexing with 120 ul 100% ethanol followed by the
addition of 5 ul of 3 M sodium acetate (pH 5.0) and more vortexing. The DNA
was pelleted at 1000 x g for 10 minutes at 4 °C. The supernatant was carefully
removed and the pellet was washed with 70% ethanol by inverting the tubes
once and then repelleting the DNA the same as before. The supernatant was
again removed and the pellet was dried in a Speed Vac™ for 5 minutes. The
denatured DNA pellet was then resuspended in 8 ul of sequencing water,
primer solution, and 2 ul of reaction buffer, all supplied in the sequencing kit.
The primer was annealed to the template by placing the DNA primer mixture in
a 37 °C water bath for 45 minutes.
58
To prepare for single-stranded templates, a primer solution (all
components supplied in the kit) was typically made for up to 12 samples (13 ul
of primer, 26 ul reaction buffer, 26 ul sterile water, total 65 ul). Five microliters of
the mixture was then added to 5 ul of the single-stranded template preparation
and placed in a 65 °C water bath for 2 minutes. The samples were then
removed and the primer was allowed to anneal to the template by placing the
tubes in a tray of 65 °C water, which was then allowed to cool slowly (30-40
minutes) at room temperature. At this point the procedures for both double-
stranded and single-stranded sequencing were the same. While the templates
were annealing a series of termination tubes were set up. One set consisted of
four different colored .5 ml microfuge tubes with 2.5 ul of one kind of termination
mix per color (ddGTP, ddATP, ddTTP, ddCTP). The Labeling Solution was
also prepared at this time. The solution consisted of 0.1 M DTT, distilled water,
and labeling mix, all supplied in the sequenase kit, and 3E>S-ATP, supplied
separately, for a total volume of 45.5 ul/12 samples. The Sequenase™ enzyme
was diluted with dilution buffer with or without pyrophosphatase according to
the number of samples (26.4 ul for 12 samples). Both sequenase and labeling
mixes were adjusted according to the number of samples to be sequenced
(Table 1. A and B). After the annealing, 3.5 ul of the 35S-ATP labeling mix was
added to each of the annealed template samples. For each reaction 2 ul of the
diluted sequenase was added to the annealed templates and allowed to
incubate at room temperature for 4 minutes. While the incubation was taking
place, the termination tubes (G, A, T, C) were placed in a 37 °C water bath. On
completion of the 4 minute incubation, 3.5 ul of the enzyme reaction mixture
was transferred to each tube in a set of termination tubes and mixed by
59
pipetting up and down a few times. The termination tubes were then returned to
the water bath and incubated for an additional 20 minutes. At the end of the 20
minute incubation period the reaction was stopped by adding 4 ul of formamide
stop solution to each of the G, A, T, C tubes. The tubes were then stored at -20
°C until ready for use. The samples were heat denatured in a 90 °C water bath
for 2 minutes and then plunged into an ice bath prior to loading onto
sequencing gels. Multiple samples, up to 12, were "staged" during the reaction
procedure according to a time table (Table 2.).
Many of the genomic subclones were further subcloned to obtain
fragments more suitable for sequencing or to obtain sequence from the
opposite strand. Another sequencing strategy was to employ the use of
oligonucleotide primers in the sequencing reactions (Biosynthesis, Lewisville,
Tx) to extend the sequence from larger subclones where restriction sites were
not present for subcloning.
2.17 Preparation and electrophoresis of polyacrylamide sequencing gels.
Sequenase reaction products were analyzed by electrophoresis on 6%
denaturing polyacrylamide gels. One hundred milliters of 6 % polyacrylamide
gel solution (42.4 g urea, 20 ml 30 % acrylamide/1 % bisacrylamide, 10 ml 10X
TBE buffer, ddH20 up to 100 ml) was prepared in a 250 ml beaker by placing
the solution on a stirring plate and covering the beaker with Parafilm® until the
urea was dissolved (20 minutes) during which time the cassette was usually
assembled.
60
Table 1.
LABELING MIX TABLE
Samples I 2 3 4 5 6 7 8 9 10 11
DTT 1.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
Label Mix 0.4 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8
H2O 4.6 4.8 6.4 8.0 9.6 11.2 12.8 14.4 16.0 17.6 19.2
35S 0.5 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Table 2.
SEOUENASE™ DILUTION TART F
Samples 1 2 2 4 5 6 7 8 q 10 n
Enzyme
Dilution 3.5 5.3 7.0 6.5 8.1 9.7 11.3 13.0 14.6 16.2 19.5
Buffer
Pryophos- 0.5 0.5 0.5 0.63 0.75 0.9 1.0 1.13 1.25 1.4 1.5
phatase Sequenase 0.5 0.7 1.0 1.3 1.5 1.8 2.0 2.3 2.5 2.8 3.0
61
Table 3.
SEQUENCING REACTION TABLE
TIME ACTION
-1 Place all termination tubes for templates 1-4 @ 37 °C 0 Add 2ul diluted sequence to template #1, mix, place @ RT 1 Add 2ul diluted sequence to template #2, mix, place @ RT 2 Add 2ul diluted sequence to template #3, mix, place @ RT 3 Add 2ul diluted sequence to template #4, mix, place @ RT 4 Dispence 3.5ul of template #1 to each GATC tubes @ 37°C 5 Dispence 3.5ul of template #2 to each GATC tubes @ 37°C 6 Dispence 3.5ul of template #3 to each GATC tubes @ 37°C 7 Dispence 3.5ul of template #4 to each GATC tubes @ 37°C 8 Dead time/catch up
9 Place all termination tubes for templates 1-4 @ 37 °C 10 Add 2ul diluted sequence to template #5, mix, place @ RT 11 Add 2ul diluted sequence to template #6, mix, place @ RT 12 Add 2ul diluted sequence to template #7, mix, place @ RT 13 Add 2ul diluted sequence to template #8, mix, place @ RT 14 Dispence 3.5ul of template #5 to each GATC tubes @ 37°C 15 Dispence 3.5ul of template #6 to each GATC tubes @ 37°C 16 Dispence 3.5ul of template #7 to each GATC tubes @ 37°C 17 Dispence 3.5ul of template #8 to each GATC tubes @ 37°C 18 Dead time/catch up
19 Place all termination tubes for templates 1-4 @ 37 °C 20 Add 2ul diluted sequence to template #9, mix, place @ RT 21 Add 2ul diluted sequence to template #10, mix, place @ RT 22 Add 2ul diluted sequence to template #11, mix, place @ RT 23 Add 2ul diluted sequence to template #12, mix, place @ RT 24 Dispence 3.5ul of template #9 to each GATC tubes @ 37°C 25 Dispence 3.5ul of template #10 to each GATC tubes @ 37°C 26 Dispence 3.5ul of template #11 to each GATC tubes @ 37°C 27 Dispence 3.5ul of template #12 to each GATC tubes @ 37°C 28 Add 4ul of stop solution to all template 1-4 tubes 37 Add 4ul of stop solution to all template 5-8 tubes 46 Add 4ul of stop solution to all template 9-12 tubes
62
plates are thoroughly cleaned. Cleaning was carried out first with Windex® and
then with 95% ethanol. The inside of the cassette plates were then siliconized
by wiping them down with 5% dichloromethylsilane dissolved in heptane under
a fume hood and allowed to dry (5 minutes). The plates were occasionally
subjected to a drying oven and "baked" at 200 °C for 10 minutes. The plates
were then returned to the rubber stoppers siliconized side up and inspected at
low angle for dust particles, which were removed with a Kimwipe®. The two
plates were then placed one on top of the other, siliconized sides towards each
other and offset lengthwise by 13 mm (0.5 inch). Two Delrin® spacers (51 cm
X 13 mm X 0.25 mm) were placed between the plates and flush with the
lengthwise edges. A third spacer (45 cm X 1.3 cm X 0.25 mm) was placed
between the plates perpendicular to the first two and flush at one end (this will
eventually be the top of the cassette). The cassette was then clamped together
with large binding clamps (three to a side). The spacer junctions were
inspected for potential leaks.
Gel casting: After the urea was dissolved the polyacrylamide solution
was filtered through a Buchner funnel containing a piece of Whatman™ No.1
qualitative filter paper or equivalent and collected in a 250 ml Erlenmeyer
vacuum filtration flask. The Buchner funnel was then removed and 0.12 g of
solid ammonium persulfate was added to the filtered solution. The solution was
then degassed under vacuum for 3-5 minutes with gentle swirling to remove all
the bubbles and gasses. The solution remained under vacuum while a funnel
was created at the open end of the cassette with warmed plastocene®.
Immediately before adding the acrylamide solution to the cassette 25 ul of
TEMED was added to the solution and swirled gently with out introducing
63
bubbles to the solution. The acrylamide solution was then added to the
cassette by carefully pouring the solution down one side of the cassette with the
aide of the plastocene® funnel. When the cassette was 3/4 full it was placed
horizontally on four stoppers and the acrylamide solution was allowed to flow
out the end of the cassette. A small spacer (20 x 13 X 0.25 mm) was then
placed between the plates at the open end of the cassette and the acrylamide
was allow at least one hour to solidify. If the gel was not to be used right away
the open end of the cassette was covered with plastic wrap to prevent drying.
The gels were used within 24 hrs.
Preelectrophoresis and loading of acrylamide gels: The plastic wrap and
short spacer were removed from one end of the cassette and the binding clips
and long spacer were removed from the other end. The cassette was then
placed into a sequencing electrophoresis gel stand such that the bottom of the
cassette (while the gel was being poured) was at the top of the stand and such
that the more elevated of the offset plates was facing away from the stand. All
sequencing gel electrophoresis was carried out in a heated box constructed
with wood and plexiglass. The temperature was elevated to between 30-35 °C
with a small space heater. End spacers were placed vertically between the
plates extending the existing vertical spacers to the top of the glass. Neoprene
spacers took up the space between the top of the offset plate and the stand.
The cassette was then clamped into the stand with large binders such that the
plates completed the formation of the upper buffer chamber and with the bottom
of the cassette in the lower buffer chamber. Both chambers were then filled with
1X TBE sequencing buffer and leaks that may occur in the upper chamber were
addressed using vacuum grease. Air bubbles were removed from the exposed
64
areas of the gel in the upper and lower chambers with a bent Pasteur® pipette.
The electrical leads were connected to a power supply and the gel was
preelectrophoresed at 1700-2300 volts (50 amps constant power) for one hour.
The glass plates were monitored for excessive heat (>55 °C) and the voltage
was adjusted accordingly. When the preelectrophoresis was complete the
power was turned off and the upper and lower buffer chambers were again
cleared of bubbles and any urea that might have diffused into the buffer above
the gel. Prior to loading, the samples were placed in a water bath at
90 °C for two minutes and then placed into an ice bath until loading. Shark's
tooth well-forming combs were inserted between the plates and spaced such
that the gel would be oriented from left to right. Two microliters of each sample
(G,A,T,C) were drawn into a Hamilton® microliter syringe with a 32 gage needle
and placed between the spaces of the shark's tooth combs. Care was taken not
to introduce bubbles into the chamber. The syringe was rinsed in ddH20 after
each loading. At least one space was kept between sequencing reactions.
Generally 12 sequencing reaction were run on a single gel. Electrophoresis
continued at approximately 2300 volts or 50 constant amps according to the
migration of the xylene cyanol component of the sequencing reaction mixture
(cm). Gel runs of 35, 70, and 120 centimeters allowed reads approaching 500
bases. When the electrophoresis was completed the power was turned off and
the cassette was removed from the stand and placed flat on a bench top. The
plates were then separated by inserting a single edge razor blade between the
two plates and carefully separating them. The plates were separated such that
the gel was left intact on one of the plates. If the gel appeared to be stuck to
both plates, techniques such as gently blowing on the gel or the delicate use of
65
a pasture pipette was employed to preserve as much of the gel as possible.
Once the plates were separated a 17 x 14 inch piece of 3 MM Whatman®
paper was placed on the gel even with the bottom edge. The over hanging gel
was then trimmed away with a razor blade. The paper was then carefully lifted
away from the plate with the gel stuck to it and placed gel side up on the bench.
A piece of plastic wrap was then stretched over the gel and paper and the
excess trimmed away. The gel sandwiched between plastic wrap and watman
paper was then dried on a slab drier at high heat for one hour. After drying the
plastic wrap was removed and the gel and paper were briefly (1- 2 minutes)
submerged in a solution of 12% methanol 10% acetic acid to remove excess
urea. The gel was again covered with plastic wrap and dried on a slab drier at
high heat for two hours.
2.18 Autoradiography and sequence analysis.
Dried gels were placed into film cassettes (plastic wrap removed) gel
side up. A piece of Kodak XAR-5 autoradiographic film was placed on top of
the gel in a dark room. The film was exposed from one to several days
depending on the dpm counts emitting from the gel. Nucleotide sequence
analysis was determined for both genomic fragments using the Macintosh®
DNA analysis software program DNAsis® (Hitachi).
2.19 Detection of genomic sequences using radio labeling techniques:
Southern blotting.
66
Labeled DNA fragments or oligonucleotide probes were employed to
screen genomic digest and various libraries using methods originally described
by Southern (114). DNA from genomic digests or cloned fragments were
separated by gel electrophoresis. Some gels were stained with EtBr and
photographed. Gels were then placed in a denaturing solution (0.5 M NaOH,
0.5 M NaCI, for 30 minutes. During gel denaturation 12 cm X 22 cm Biodyne®
nylon membranes (Gibco BRL Life Technologies Inc.) were prewetted in ddH20
then soaked in the denaturing solution described above. Two thin (.25 inch
each) sponges were also presoaked in denaturing solution. The DNA was
transferred from the gel to the nylon membrane by capillary action. The transfer
was conducted in a shallow cafeteria type tray and assembled in the following
manner. The two sponges were placed in the center of the tray, on top of them
were placed four pieces of blotting paper (Schleicher & Schuel), two pieces of
GB002 and two pieces of GB004. The gel was placed on top of the blotting
paper well side down (the DNA runs closer to the bottom of the gel). A notched
and labeled nylon membrane was placed on the gel taking care to avoid
bubbles between the gel and the membrane. Two more pieces of GB004
blotting paper were placed on the membrane followed by a one inch stack of
paper towels cut to size. The assembly was weighted with a 0.5 inch thick
piece of plexiglass on top. The tray was then filled with denaturing solution to
the level of the first sponge. The paper towels were changed out at 30 minute
intervals (x 2) and then at one hour intervals (x 2). At the end of three hours,
the assembly was carefully dismantled. The membrane was gently washed in
2.5 X SSPE to remove any residual agarose. The membrane was then allowed
to dry overnight or placed in a warm oven for a few hours or UV crosslinked to
67
the filter with a hand held UV source or commercial membrane crosslinker at
300 milijoules of UV.
2.20 Radiolabeled probes: end labeled or random primed.
Small (oligonucleotide) probes used in hybridizations were radio labeled
at their 5' ends by using T4 polynucleotide kinase to transfer the alpha-
phosphate from a [gam ma-32 P] ATP to the free 5' hydroxyl group of the probe
(115). Approximately 0.1 ug of oligonucleotide probe was combined in a
microfuge tube with 2 ul of [g-32P] ATP (3000 Ci/mmol, DuPont), 2.5 ul 10 X
kinase buffer (500mMTris-HCI pH 7.4, 50mM MgCl2, 20mM DTT, and 1.0 mM
spermidine), and 1.5 ul 10U/ul T4 polynucleotide kinase. The contents of the
tube were mixed gently and incubated at 37 °C for 45 minutes. The reaction
was stopped by heating to 65 °C for five minutes. Unincorporated nucleotides
were removed by Nensorb™ 20 nucleic acid purification cartridges (NEN
Research Products Bioresearch Systems, Dupont) or NucTrap® probe
purification columns (Statagene).
For larger probes such as cloned sequences, where fewer moles of
probe required more extensive labeling, random priming was employed using
the Life Technologies® Random Primers DNA Labeling System (Gibco BRL)
according to packaged instructions. Labeled probes were cleansed of
unincorporated nucleotides by the same method used for end labeling (above).
2.21 Hybridizations.
68
Screening denatured DNA fragments crosslinked to nylon membranes
with radio labeled probes was carried out in a hybridization vessel. The
membrane was added to the vessel and prehybridized in 5 ml. of Life
Technologies® DNA Typing Grade Hybridization Buffer (Gibco BRL) for 30 min
at 65 °C. During the prehybridization the probe was denatured by heating at
100 °C for 5 minutes. The probe was then added to the hybridization vessel.
The vessels was resealed and put in motion for 12-16 hours at 65 °C. After
hybridization, the hybridization solution was removed and the membrane was
washed in the hybridization vessel with varying concentrations of SSC (0.1 X -
2X), 0.1% SDS until the dpm signal emitting from the membrane was between
1- 2 k. The membranes were then wrapped in plastic and analyzed by
autoradiography using an intensifying screen at -70 °C.
2.22 M13 library construction: isolation, size fractionation and preparation of
genomic DNA..
Genomic DNA from Trypanosoma brucei brucei antigenic type M110
(116) was used in this study. Genomic DNA was isolated by methods
previously described by Medina-Acosta and Cross (117). Genomic DNA was
digested with either Xba I or Xho I and size selected in a 0.8% agarose gel
between 10-14 Kb.
The DNA was placed into Hospitak "up-mist" medication nebulizers with
200 ul 10x TM buffer (100 mM Tris; 50 mM MgCfe), 1.6 ml 40% glycerol, and
ddH20 up to 2 ml. The nebulizers were actuated on ice with 10 lb of N2 for 1
minute. The chambers were then briefly centrifuged and the samples divided
69
into four 1.5 ml microfuge tubes of approximately 490 ul each. The DNA was
then precipitated with 50 ul of 3 M NaAcOH and 1 ml 100% EtOH. The samples
were resuspended in 30ul H20 per tube and then combined (120 ul total
volume).
The ends of nebulized DNA were repaired (blunted) in a preparation
containing:
30 ul of the nebulized sample
5 ul 10x polynucleotide kinase buffer
5 ul 10 mM rATP, 7.5 ul 2 mM dNTP
5 ul Klenow fragment (2 U/ ul)
3 ul T4 polynucleotide kinase (10U/ul).
The preparation was incubated at room temperature for 10 minutes followed by
incubation at 37° for 50 minutes. Following incubation 13 ul of 5X agarose gel
electrophoresis loading buffer (25% glycerol, 0.5% SDS, 0.1% bromophenol
blue, 0.1% xylene cyanol, 50 mM EDTA) was added in preparation for size
selection. Nebulized and end repaired DNA was size selected in a TAE 0.8%
agarose gel. Samples were electrophoresed at 80V for approximately 1 hour
concurrently with a 1 kb ladder (Gibco BRL). DNA was visualized with ethidium
bromide. The DNA was size selected from approximately 1.5 kb to 3.0 kb by
removing the unwanted lower MW end of the gel, reversing the current, and
running the DNA against a small piece of dialysis membrane embedded in the
gel lane at the 3 kb marker. The DNA was then "pushed" into the membrane by
increasing the voltage to 200V for about one minute. The wet membrane was
then carefully removed from the gel and placed into a 1.5 ml. microfuge tube
with 10 ul of H20 (total volume 15-20 ul). The membrane was crushed into the
70
bottom of the tube with a pipette tip and the tube was place in a 65 °C water
bath for 10 minutes to remove the DNA from the membrane.
2.23 M13 library construction: vector preparation and ligation.
RF M13 vector was prepared for ligation by digesting with SmaI
restriction endonuclease followed by treatment with calf intestinal alkaline
phosphatase (CIAP). The reaction was set up as follows:
5 ug RF M13 vector
5 ul (50 U) Sma I
20 ul 10X buffer
17Q Ml cWH#
200 ul total volume
The digestion was incubated at room temperature for at least one hour.
Fifty units of CIAP was added to the digestion followed by further incubation at
37 °C for one hour. The enzyme activity was stopped by heating 65°C for 10
minutes. The vector was extracted with phenol and precipitated in ethanol. The
vector was resuspended in 100 ul ddH20. Ligations were set up in a total
volume of 10 ul containing:
6 ul (3-4 ug) end repaired and size selected genomic DNA
2 ul (100 ng vector [Sma I/CIAP])
1 ul T4 ligase (400 U/ul)
1 ul T4 ligase buffer
10 ul total volume
71
Ligations were incubated overnight at room temperature. Following incubation
90 ul H2O was added to each ligation in preparation for transformation of
competent cells.
2.24 M13 library construction: transformation of competent cells.
5-10 ul of the ligation mix was used to transform Epicurian Coli ® XL2-
blue MRF' ultracompetent cells (Statagene). Competent cells were thawed on
ice. 100 ul of cells was placed into prechilled Falcon® (12 x 75 mm
polypropylene) tubes on ice. 1.7 ul of 1.4 M beta-mercaptoethanol was added
to the cells in the bottom of the tube. The tubes were shaken on ice every 2
minutes for 20 minutes. The ligation mix was added to the cells and allowed to
sit for 30 minutes with shaking every five minutes. The cells were then heat
shocked at 42°C for 45 seconds then placed back on ice for two minutes. The
cells were then returned to the bench at room temperature for 30 minutes. To
each sample of transformed cells was added:
6 ml top agar
80 ul 2% X-gal
16 ul 100 mM IPTG
200 ul XL2-blue MRF' lawn cells.
Transformants were selected using blue/white screening techniques
(118,119,107) on YT agar plates. Clear recombinant plaques were taken with a
sterile toothpicks and grown up overnight at 37 °C with shaking in 800 ul 4YTG
(10 g tryptone, 17 g yeast extract, 5 g NaCI, 2 g dextrose, ddH20 up to one liter)
with 80 ul of overnight XL 2-blue MRF'cells. Following incubation, the cells
72
were pelleted and 600 ul of the supernatant was transferred to a deep-well
microtiter plate. DNA was precipitated by adding to 180 ul of 20% PEG
8000/2.5 M NaCI to the supernatant and inverting several times. After a ten
minute incubation at room temperature the plates were centrifuged at 3000 rpm
(Beckman GS-6R centrifuge, GH-8 rotor, microtiter carrier) to pellet the DNA.
The plates were then inverted onto paper towels and spun again at 300 rpm for
10 seconds to remove excess PEG. The phage pellets were resuspended in
20-40 ul of Triton-TE extraction buffer (0.5% Triton X-100/1X TE) at 90 °C in
preparation for sequencing. The remaining suspensions (not sequenced) were
stored in 96 well microtiter plates at -20°C.
2.25 M13 library sequencing and analysis.
Enzymatic extension reactions for sequencing were done in Perkin
Elmer thermal cyclers (Cetus Corp.) using AmpliTaq® DNA polymerase and
ABI PRISM™ dye terminator (FS enzyme) or dye primer (CS+ enzyme) Ready
reaction kits following the manufacturer's instructions. Samples were
sequenced by single passage through an automated ABI Prism 377 DNA
sequencer. Sample sequences were filtered for vector and low compositional
complexity sequences (120). Sequences were analyzed with a basic local
algorhythm search tool (BLAST) with and without filters (120,121,122,123,
124, 125) at the National Center for Biotechnology Information (NCBI). The
sequences were "BLASTed" in-batch via E-mail using TBLASTX formatting.
Each sequence was then submitted to the Genome Sequence Survey (GSS)
database. "Nearest Neighbors" to the GSS sequences were reported by NCBI
73
and were analyzed for inclusion in the study. All sequences under
consideration were then reBLASTed for the most updated alignments using
BLASTP or BLASTN formatting at the NCBI website. Sequences were
considered individually for inclusion based on homology, statistical (P) values,
length, and the repetitive nature of the sequences.
2.26 Cosmid library construction: vector preparation.
Cosmid libraries were constructed in a modified Supercos 1 (Statagene)
cosmid vector. The vector was modified (site directed mutagenesis) to hold
approximately 1 kb more insert by removing the amp resistance portion of the
vector and part of the pUC multiple cloning site containing Xho I and Hind III
restriction sites. The vector (scos-seq3) was modified in this way by Dr. Lisa
McDaniel at the University of Texas Southwestern Medical Center for use in the
Human Genome Project and others. 13 ug of the vector was linearized with
Xba I at nucleotide 1435 in the following manner.
10 ul vector (13ug)
10 ul enzyme (100 U)
20 ul buffer (10X)
160 ul ddH2Q
200 total volume
The digestion was carried out at 37° C for 1 hour. The vector was then treated
with CIAP (50 U) for 1 hour at 37° C. The reaction was stopped by phenol
extraction and precipitated with ethanol. The DNA was resuspended in 175 ul
ddH20. The digestion was visualized on a 0.8% agarose gel stained with EtBr
74
(Fig. 3 B2) The cos ends of the vector were exposed by digesting with BamH I
at 37 °C for two hours in the following reaction:
175 ul vector
20 ul buffer
5 ul BamH I (50 U)
200 ul total volume
The vector was then extracted with phenol-chloroform-isoamyl (25:24:1),
extracted with chloroform, precipitated with 100% ethanol, washed with 70%
ethanol, and resuspended in 40 ul of ddH20. Two separate bands were
predicted (1.1 kb and 6.5 kb) and detected on 0.8 % agarose gel (Fig. 3 B4).
2.27 Cosmid library construction: isolation and preparation of genomic DNA..
Genomic DNA from bloodstream form Trypanosoma brucei M110 (116)
was used in this study. Trypanosomes were harvested from rodent blood as
described previously (126). Genomic DNA was obtained by LiCI precipitation
of detergent solubilized trypanosomes (117). The optimal digestion time was
determined for 5 ug of high molecular weight genomic DNA with 0.1 U ofSau
3A I enzyme (Fig. 3A) Digestions were carried out at 37° C in the following
reaction:
10 ul (5 ug) DNA
4 ul 10X buffer
1 ul (.1U) enzyme
25 Ul ddHzQ
40 ul total volume
75
The reaction was stopped in 8 ul aliquots with an equal volume of phenol-
chloroform at time intervals of 1, 2, 4, 8, and 16 minutes. The DNA was then
precipitated in ethanol and resuspended in ddhteO. The results of the partial
digestions were visualized on a 0.3% agarose gel stained with EtBr. The
digestion time containing the largest amount of 40 kb DNA fragments was
determined to be three minutes under the conditions described above. The
above digestion was then repeated with 10 ug DNA, 15 ul ddh^O for three
minutes. The reaction was stopped with a phenol extraction (200 ul phenol-
chloroform) and 160 ul ddhteO. Residual phenol was removed with a
chloroform extraction and the DNA was precipitated with ethanol. The DNA
was then resuspended in 40 ul ddH20 and visualized on 0.3 % agarose gel
with EtBr (Fig. 3A). The partially digested DNA was then treated with CIAP, 26
U at 37° C for one hour, to avoid cloning multiple smaller inserts. The DNA was
again extracted with phenol and chloroform, precipitated in 100% ethanol,
washed in 70% ethanol, and resuspended in 40 ul ddH20.
2.28 Cosmid library construction: ligation of genomic DNA and vector.
Genomic DNA fragments were cloned into the Xba I site of the scos-seq
3 vector with T4 DNA ligase in the following reaction:
8.0 ul genomic DNA (4 ug)
5.0 ul Xba I-CIAP and BamH I digested vector (1.5 ug)
1.5 ul buffer
1,0 ul T4 DNA ligase
15.5 ul total volume
76
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Figure 3. Panel A shows digestion times for 5 ug of High molecular weight
genomic DNA with 0.1 U ofSau 3A I enzyme. The reaction was stopped at time
intervals of 1, 2, 4, 8, and 16 minutes. The optimal digestion time containing the
largest amount of 40 kb DNA fragments was determined to be three minutes.
Molecular weight markers are shown in lanes land 7. The DNA was
visualized on 0.3 % agarose gel stained with EtBr. Panel B shows 13 ug of the
vector linearized with Xba I at nucleotide 1435 (lane 2) and the same vector
DNA digested with Bam H I to expose the cos ends of the vector (lane 4). Two
bands were predicted (1.1 kb and 6.5 kb) and detected on 0.8 % agarose gel. 1
kb size markers are shown in lanes 1 and 3. Panel C shows the conversion of
the ligation components to high MW concatamers was visualized on 0.3%
agarose gel stained with EtBr (lane 2). 1 kb size markers are shown in lane 1.
78
The ligation was carried out at room temperature for 24 hours. The conversion
of the ligation components to high MW concatamers was visualized on 0.3%
agarose gel stained with EtBr (Fig. 3C).
2.29 Packaging, titering, and plating the cosmid library.
The ligated DNA was then packaged into phage particles using
Stratagene's Gigapack® II XL packaging extract (cat. # 200217) according to
the instruction manual. 0.5 ml SM buffer (prepared according to package
instructions) was added to the packaged DNA followed by extraction with 20 ul
chloroform. The supernatant containing the recombinant phage was stored at
4 °C until used. XL 1 Blue MRF' cells (Stratagene) were prepared as host cells
for the phage. A 50 ml culture containing 10 mM MgSCU and 0.2% (w/v)
maltose was prepared from a single colony. The culture was incubated
overnight at 30 °C (prevents over growth) with shaking. The cells were pelleted
at 500 x g for 10 minutes at 4 °C. The cells were resuspended in 10 mM
MgSC>4 to an OD600 of 0.5. 1:10 and 1:50 dilutions of the packaged cosmid
library were prepared in SM buffer. 25 ul of each dilution was combined with
25 ul of the XL1-Blue cells described above in a microfuge tube and incubated
for 30 minutes at room temperature. 200 ul of LB broth was added to the tube
and incubated for 1 hour at 37 °C with gentle shaking every 15 minutes to
initiate kanamycin resistance expression. The cells were then pelleted for one
minute and resuspended in 50 ul of fresh LB broth. The cells were then spread
on to 150 mm LB agar plates containing 50 ug/ml kanamycin using a sterile
hockey stick spreader and incubated over night at 37 °C. Single colonies were
79
taken and inoculated into 12 x 75 mm polypropylene tubes containing 5 ml LB
broth and 50 ug/ml kanamycin. Tubes were incubated at 37 °C with vigorous
shaking overnight. 0.5 ml of each sample was combined with 0.5 ml glycerol
and placed into corresponding wells of four groups of 14, 96 well (200 ul/well)
microtiter plates. One group of plates was stamped onto membranes by a
Biomek™ robot in 4 x 4 arrays (384 samples/7.5 x12 cm membrane). Cosmid
DNA of the remaining 4.5 mis of culture was isolated by alkaline lysis "rapid
prep" (108) (see: Isolation ofplasmids (2.2)).
2.30 Cosmid library finger printing.
"Rapid prep" cosmid DNA was resuspended in 25 ul TE (pH 8.0) with 25
ug/ml heat-treated RNase. Two ul of each sample was digested with EcoR I (20
U) and visualized on EtBr stained 0.8 % agarose gels with a Hind Ill-lambda
ladder. EcoR I restriction patterns of recombinant cosmid DNA was
photographed for later analysis. The remainder of the recombinant DNA was
placed in 96 well microtiter plates and stored at -20 °C.
3. Results
3.1. Detection and isolation of genomic clones.
The predominant calcemedins in T. brucei comprise a family of putative E
F-hand calcium binding proteins. The proteins were reported (47) to consist of a
44 kDa protein and a cluster of 23-26 kDa proteins. E F-hand calcium-binding
proteins of T. Cruzi had been previously reported by Lazardi (48), Gonzalez
(127), Engman (7), and Ouaissi (49). A clone in T. brucei was discovered by
Dr. Lee at Columbia University, New York, New York who reported (50) that
while screening African trypanosome cDNA clones for evolutionarily conserved
genes that encode cell surface proteins, they found a cDNA clone (Tb-17) with
an open reading frame that shared significant homology to previously described
E F-hand calcium-binding proteins of T. cruzi. Dr. Ruben at Southern Methodist
University, Dallas, Texas obtained the clone Tb-17 and used it to screen a
lambda Zap (Stratagene) cDNA library provided by Dr. K. Stuart, Seattle
Biomedical Research Institute, Seattle, Washington. A positive clone, pTB44A,
was considered for further examination and sequencing . A full length pTB44A
was used to probe restriction digested trypanosome DNA, strain M110
(Fig. 4) and several genomic fragments were detected. BamH\ fragments 1.7
and 2.5 were selected for further examination. Dr. Lee also probed BamH I
digested genomic trypanosome DNA strain 427-60 with the TB-17 clone and
obtained similar BamH\ fragments. The two fragments, 1.7 and 2.5, were then
excised from the gel by Dr. Lee and cloned into the BamH\ site of pUC19 (Fig. 5
81
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Figure 4. T. brucei genomic DNA was digested with several restriction enzymes
and electrophoresed on a 1% agarose gel. A Southern blot of the gel was then
probed with a full length 32P labeled cDNA, pTB44A. Autoradiography
detected several homologous genomic fragments. Approximate size markers
are shown in kilobases.
83
Pvull 3.14 Hindlll 2.96 Pstl 2.95 Hindi 2.94 BamHI 2.94
vull Qs,
coRI 0.40 imal 0.42
BamHI 0.42
2.5 kb Genomic Fragment
B coRI 0.40 trial 0.42
BamHI 0.42
1.7 kb Genomic Fragment
BamHI 2.10 Hindi 2.11 Pstl 2.12 Hindlll 2.13
Pvull 2.31
84
Figure 5 A,B. Recombinant plasmids pB2.5 (A) and pB1.7 (B) were the result of
cloning BamH\-BamH\ genomic fragments corresponding to Southern transfer
and autoradiography bands of 1.7 kb and 2.5 kb (see Fig. 4) into the pUC 19
vector.
85
A,B)- Positive size fractionated clones were detected by screening a genomic
BamH\ library with the Tb-17 cDNA probe.
3.2. Restriction site analysis and subcioning of genomic clones.
In order to investigate the genomic organization of these genes the clones were
examined for restriction sites in preparation for subcioning and sequencing.
Restriction site analysis of TB-17 revealed dueling EcoRI sites more or less
central in that clone, clones were therefore digested with BamHI, EcoRI and
BamHI/EcoRI double digestions (Fig. 6). Overlapping subclones were
constructed from both strands of the genomic clones using M13 and pUC
vectors. Where restriction sites were unavailable oligonucleotide primers were
generated from sequence ends to fill in gaps. The sequencing strategy for
pB1.7 is shown (Fig. 7). All subclones for pB1.7 were constructed using M13
bacteriophage vector and sequenced from single stranded templates. pB2.5
subclones were constructed in both M13 and pUC vectors. Further subclones
were also generated from pB2.5 by digesting with two additional restriction
enzymes Hinc II and Pvu II (Fig. 8) to facilitate sequencing.
3.3. Sequence analysis of genomic clones pB1.7and pB2.5.
The sequence generated from subcloned genomic fragments of pB1.7
and pB2.5 was determined by Sanger dideoxynucleotide sequencing using
U.S. Biochemical sequencing kit version 2.0. Overlapping fragments were
86
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Figure 6. 1.0% agarose gel stained with ethidium bromide of restriction enzyme
digested genomic clones pB1.7 and pB2.5. Hind III cut lambda and Hinf I cut
pBR322 are shown as size markers with approximate kilobase sizes are shown
at left.
88
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Figure 7. Restriction map and sequencing strategy of the genomic clone pB1.7.
The darker shaded area labeled Tb-1.7g represents a calflagin open reading
frame. Clone numbers are shown under the arrows. Oligo primers used in
sequencing are shown below.
90
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Figure 8. Restriction map and sequencing strategy of the genomic clone pB2.5.
The darker shaded area labeled Tb-24 represents a calflagin open reading
frame. Clone numbers shown under arrows represent M13 clones and clone
numbers above arrows represent pUC clones. A putative 5' open reading
frame of another unidentified calflagin is shown at the 3' end of the clone. The
3' open reading frame is unidentified. Oligo primers used in sequencing are
shown below.
92
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Figure 9. The complete nucleotide sequence of the genomic clone pB1.7. The
1.7 kb Bam HI-Bam HI genomic fragment was cloned into pUC19 and
sequenced to completion. The ORF for the calflagin Tb-1.7g is indicated by the
single-letter amino acid code.
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Figure 10. The complete nucleotide sequence of the genomic clone pB2.5.
The 2.5 kb Bam Hl-Bam HI genomic fragment was cloned into pUC19 and
sequenced to completion. ORFs for Tb-24 and gene A (the first 18 nucleotides
of a calflagin coding sequence) are indicated by the single-letter amino acid
code. The upstream ORF that encodes a putative non-calflagin protein (bases
1-321) has not been marked.
96
assembled using DNAsis® software to obtain complete sequences of pB1.7
and pB2.5 (Figs. 9 and 10).
3.4. Comparisons of coding and flanking sequences to other calflagin clones.
A schematic diagram of the relationship between conserved coding and
flanking regions of different calflagin clones is shown (Fig. 11). Prominant
features of the genomic clones include an incomplete open reading frame of
321 nucleotides (1-321; Figs. 10,11,12 A, B) at the 5' end of pB2.5. A
hydropathicity plot (Fig. 12 B) indicate that the sequence may code for a
membrane associated protein. The putative translation product of this ORF
represents the carboxyl portion of a protein that is unrelated to the calflagins but
shows significant homology of 10/28 amino acid residues (35%) to a cDNA
sequence reported from Brugia malayi (EMBL acc. no. BM7041). Whether this
ORF is actually transcribed has not yet been determined. Downstream from the
5' ORF is a second ORF (Tb-24) that shares nearly identical 5' and 3' non-
coding flanking regions with TB-44A. It encodes a 24 kDa protein with
extensive homology to the carboxyl half of Tb-44A (Figs. 10,11,13). The 3'
flanking sequences of Tb-24 are identical to the 3' flanking sequences of Tb-
44A (Fig. 11). This conserved downstream segment is followed by 131
nucleotides that are repeats of a region found at the 51 end of Tb-24, and
includes the putative mini-exon acceptor site (Fig. 11). Thirty-six nucleotides
downstream from the putative mini-exon acceptor site, the first 18 nucleotides of
another calflagin gene was observed (gene A, Fig. 11). This calflagin member
97
differs from Tb-24, Tb-17, and Tb-44A in that it has a BamHI site at the 51 end of
the gene.
The sequence of the 1.7 kb BamHI genomic clone begins at precisely the
same location within the calflagin sequence where the 2.5 kb genomic clone
ends (Fig. 9 and 11). This is suggestive that the 2.5 kb and the 1.7 kb genomic
fragments may be linked however, Southern blots (51) and restriction analysis
suggest that more than one calflagin member might contain a 5' BamHI site.
The partial sequence for the calflagin on the 1.7 kb genomic clone was called
Tb-1.7g (Fig. 9,11, and 13). The 3' non-coding flanking sequences of Tb-1.7g
and the cDNA clone Tb-17 were related (Fig.11).
Proteins with homology to the T. brucei calflagins were sought in the
available databases of the NCBI (National Center for Biotechnology
Information). No other organisms were found top have related sequences other
than those within the genus Trypanosoma.. Calflagin sequences from T. brucei,
T. lewisi, and T. cruzi are compared in Fig. 13. Substitutions among the T.
brucei calflagins were most prevalent at the N-terminus. Variability occurred at
the C-terminus, where variations on the hexapeptide sequence GANEGD were
represented once in Tb-24 and Tb-44A and twice in Tb-17 and Tb1.7g. The
most extensive difference occurred between Tb-44A and the remainder of the T.
brucei group as a consequence of an internal repeat in Tb-44A. The T. cruzi
group (1F8, FCaBP, 24 kDa antigen, and FCaBP-PBOL), in contrast, differed
very little (Fig. 13) and the few differences that did occur appeared to be strain
specific. T. cruzi has been shown to produce a single calflagin band from
immunoblots (7,47) and Southern blots show that T. cruzi is more
homogeneous that T. brucei (127). Comparisons of EF-hand
9 8
pB25
pB1.7
B E E B
pTB44A r E
UXSE3 I I E E
I E E E
pTB17 ! EEttE f s I 1
E Bft l S
400 hp
99
Figure 11. Common features of calflagin clones pB2.5, pB1.7, pBTB44A, and
pTB17 are compared. Coding sequences are indicated with a black box.
Common elements in the 5' and 3' non-coding regions are indicated by boxes
that share a common design. The position of restriction endonuclease sites for
EcoRI (R), BamHI (B), Sa/I (S), and Bgl\ (BG) are shown.
100
9
5 1 ATC COT CTC
lie Leu Leu
63
GGC CCG AAA
Gly Pro Lys
117
GTC ATG GTT
Val Met Val
171
ATG AAT CCC
Met. Asn Pro
225
GCA GCA CCA
Ala Ala Pro
279
GGT OTA ATT
Gly Leu lie
18 TTT ATT AftC
27 CTG GOT
36 ATT GTT AST
45 ATT GAA CTC
54 GCT GTT CGG TTG
Arg Leu Phe lie Asn Leu Val H e Val Ser lie Glu Leu Ala Val
72 CAC GCT GTC
81 GTG AGC
90 CTG ATT GGT
99 GCT OTA CCG
108 GCA GCT GCG GTG
Ala Val His Ala Val Val Ser Leu lie Gly Ala Leu Pro Ala Ala
126 CTA CCA GCG
135 TTG CTC
144 ACT ATG CAG
153 GTT GAC CAT
162 GCT GTA TOT ATT
Phe lie Leu Pro Ala Leu Leu Tbx Met Gin Val Asp His Ala Val
180 GAT CGT GTG
189 ACG TTG
198 AAG TAC TOG
207 GGA AGC ATG
216 TTT ACG GAA GAG
Glu Glu Asp Arg Val Thr Leu Lys Tyr Trp Gly Ser Met Phe Thr
234 TCT TGG ACA
243 CGC ATT
252 CGT TGT TAC
261 TTC TAC ATT
270 ATC TTT GTG TTT
Val Phe Ser Trp Thr Arg lie Arg Cys Tyr Phe Tyr lie H e Phe
288 GTA ATG GGC
297 ACC TAC
306 AGC GTC ATT
315 GCT GAA CTC GTC ATG
Val Met Val Met Gly Thr Tyr Ser Val lie Ala Glu Leu
TGA 3'
B Kyte and Doolittle Hydropathicity Plot
Hydrophobic
5 Hydrophilic m
100
101
Figure 12. Panel A shows the sequence and the translation of the carboxyl end
of an ORF in the genomic clone pB2.5. The ORF is located at the 5' end of the
clone (Figs. 10 and 11). Amino acid sequence is given in three letter code
below the sequence. Numbers are in nucleotides. Panel B shows a Kyte and
Doolittle hydropathicity plot revealing the hydrophobic nature of the sequence.
The bottom numbers are in amino acid residues. The hydropathicity is shown
along the left side.
102
_ _ _ _ _ _ _ 60
Tb-44A MGCSASKDTTNSKDGAASKGGKDGKTTADRKVAWERIRCAIPRDKDAESKSRRIELFKRF
lb-17 ***AS**NAS*P**********************************************C*
tb-24 **•***.************...*****«****.*..*..*.**.************•**« Tb-1.7g G**NAS*P*******************************.**************Q* 57
FCaBP/lF8 MOACGSKG*TSDKGIASD****KAK***E******Q****E*T**A*Q* K* 24 kDa Aff MGACGSKG*TSDKGLASD****NAK***E******Q****E*T**A*Q*******K*
FCaBP-PBOL MGACGSKG *TSDKGLASD** * *NAK***E******Q****E*T**A*Q* * * * * * *K*
TL-17 RRSKNS*SSK* * * *AAKTARLRGR3JL*QT* * *ERTD*ARQ* * *D* **K* 49
EF-hand fl
Tb-44A DTNGTGKLSFREVLDGCYSILKLDEFTTHLPDIVORAFDKAKDLGNKVKGVGEEDLVEFL 120
jb.j.7 ********Q***************** ************ ************* *********
Tb-24 ********q*********o***************************************** Tb-1.7g ****** **g*********G*****************************************
FCaBP/lF8 *K*E**.**CYD**HS**LEV*******PRVR**TK******RA**S*LENK*S**F**** 117 24 kDa Ag *K*E****CYD**HS**LEV*******FRVR**TK******RA**S*LENK*S**F**** FCaBP-PBOL *K*E****CYD**YS**l,EV*******SRVR**TK*****SRT**S*LENK*S**F****
Tl-17 *K*NS****YD**YR**IDE********RVR**TK***N****M****EKT*S**F**** 109
EF-hand #2
Tb-44A EFRLMLCYiyDIFELTVMFDTMDKDGSLLIjELQEFKEALPKLKEWGVDITDATTVFNEID 180
Ws-17 *****************i**************j}*************************** ^,.24 ********************************************q***************
Tb-1.7g ************************************************************ FCaBP/lF8 ***********F********EI*AS*NM*VDEE*L*R*V***EA**AKVE*PAAL*K*L* 177 24 kDa Ag ********«**E********EI*AS*NM*VDEE*L*R*V***EA**AKVE*PAAL*K*L* FCaBP-PBOL ***********F********EI*AS*NM*VDEE*F*R*V*«*EA**AKVE*PAAL*K*L*
Tl-17 ***********Y********EI*TS*NM**DDK*****V***EK**LK*EHPEK**KQL* 169
EF-hand *3
Tb-44A
Tb-17g Tb-24
Tb-1.7 FCaBP/lFB 24 KDa Ag
FCaBP-PBOL Tl-17
TNGSGWTFDEFSCWAVTKKLQVCGDPDGEGAAKTTADRKVAWERIRCAIPRDKDAESKS 240
***********************G****D*ENGANEGDGANAODGVPAAEGSA
*************************** *G*ENGANEGN
* * * * * * * * * * * * * * * * * * * * * * * * * * * * 0 * E N G A N E G D < 3 A N A G D G V P A A E G S A
K**T*S******AA**SAV**DAD****NVPES*
K * *T*S * * * * **AA**SAV**ADD** **NVPES*
K**T*S«*****AA * * SAV* * EPTATRTTCRRAREARGRRVTCPRRRQCKHRVRMRRGTC 229
KK**********AA**SA***DAE****K 198
233 218
211
EF-hand #4
Tb-44A RRIELFKQFDTNGTGKLGFREVLDGCYSILKLDEFTTKLPDIVQRAFDKAKDLGNKVKGV 300
FCaBP-PBOL FCWAEHILHFM 240
EF-hand #5
TB-44A GEEDLVEFLEFRLMLCYIYDIFELTVMFDTMDKDGSLLLELQEFKEALKKLKEWGVDITD 360
EF-hand #6
Tb-44A ATTVFNEIDTOGSGWTFDEFSCWAVTKKLQVCGDPDGEENGANEGN 407
103
Figure 13. Comparisons of calflagin protein sequences from T. brucei, T. cruzi,
and T. lewisi. The sequences used are from T. brucei, Tb-44A (accession no.
U06463), Tb-17 (accession no. X53464), Tb-24 (accession no. U06644), Tb-
1.7g (accession no. U05882); from T. cruzi FCaBP/1 F8 (accession no.X02838),
24 kDa antigen (accession no. S43664), FCaBP-PBOL (accession no. L26971);
and from T. lewisi (Lee et al.,1990). Numbers in the right hand margin indicate
the position of amino acids. The amino acids in bold letters indicate the position
of sequences related to the hexapeptide GANEGD. Identical residues are
indicated with an *. Bars indicate the position of EF-hands.
104
Calcium-binding motifs in T. brucei calflagins (Fig. 13) show thatTb-24, Tb-1.7g,
and Tb-17 all have three EF-hand calcium -binding sites while Tb-44A has six
due to the duplication. Most known EF-hand proteins contain multiple copies
(two to eight) of helix loop helix calcium-binding motifs. The EF-hands are
generally arranged as interacting pairs (128). It was shown (51) that in Tb-44A
EF-hands 1 and 4 are unpaired. Instead, the adjacent regions (residues 99-139
and 288-318) have degenerated so that no evidence of calcium co-ordination
could be found. The degenerate calcium co-ordination sites have retained
considerable hydrophobicity, however and may still function to bind calcium. In
that case they may have evolved to interact with target proteins.
3.5. Analysis of a cloned T. brucei repeated element
Degenerate oligo probes acquired from Dr. Larry Ruben at the Southern
Methodist University were end-labeled with 32P and probed against restriction
digests of T. brucei. A weak band was detected from oligo probe #4 on a Hind
III digest at approximately 1 kb. The detected genomic fragment was
electroeluted from the gel and cloned into the Hind III site of pUC 19. Positive
clones were detected by dot-blot analysis with the original probe #4. Plasmids
were isolated by rapid prep procedures and 32P labeled by random priming.
Plasmid probes were then probed to a genomic Southern digested with Hind III
and various other enzymes (Fig. 14 A). Several genomic fragments were
detected in lanes digested with Hind III and double digested with Hind III and
Xho I. Plasmid preparations of the Hind III cut genomic fragment were used in
double stranded sequencing reactions using the Sequenase® version 2.0
105
c o o Q. JC 4Z g 2 g
G A T C
22.6
- %** Wmm
:>Mu
- < ^ '.vv , Sf •\* EmM&m
*>&.> i\:in Kt' •?- rvx ,w .v*
§111
f' WTOi
106
Figure 14. Analysis of a Trypanosoma brucei repeated element. Panel A
shows a Southern blot of T.brucei restriction digests probed with a 32P labeled
Hind III genomic fragment containing a 28 bp repeated element. Approximate
size in kilobases are shown to the left. Panel B shows an autoradiograph of a
portion of the repeated element. The sequence was generated from 35S
labeled sequencing reactions terminating in G.A.T, and C shown at the top. The
gel is read from the bottom to the top.
107
sequencing kit obtained from U S Biochemical. A sample of the sequence
showing the repeated segment is shown (Fig. 14 B). Blast analysis reveal
significant homology of 27/38 bases (71%) with a cDNA clone from Brugia
malayi (EMBL: BAA32121). There were no other matches reported in the
BLAST analysis using standard BLASTN parameters.
3.6. Detection of novel T.brucei sequences using M13 shotgun sequencing.
Genomic DNA was cut with XhoU and Xba\ and fragments from 10-14 kb
were obtained from the two digestions. The DNA from both digestions was then
nebulized together to generate small fragments in the range of 1.5-3 kb,
suitable for cloning into the bacteriophage vector M13. Of the 5,856 M13 clones
stored in 96 well plates, 433 were sequenced from the -21 M13 forward primer
by a single pass through an ABI 377 DNA sequencer. Some sequences were
interrupted by large regions (greater than 100 bases) of unresolved nucleotides
and were treated as two sequences. Therefore, a total of 455 separate
sequences are reported and submitted to the GSS database of the NCBI. An
average read length of 740 nucleotides was obtained for each sequence,
representing approximately 0.84% of the total T. brucei genome. Because M13
sequencing proceeds only in one direction, sequence information was not
obtained from both ends of the insert. The sequences were analyzed using the
BLAST family of software (NCBI) for regions that encoded putative proteins,
structural RNAs, or regulatory elements. 84 sequences with homologies to
sequences already in the database are reported (Table 4).
108
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112
Table 4. illustrates the variety of sequences identified. Internally repeated
motifs were found primarily in structural proteins and proline rich proteins. The
repeats were evident because one or more segments of the query sequence
aligned with multiple regions of the subject sequence (superscript a).
Alternatively, two or more segments of the query sequence overlapped one
another when aligned with the subject sequence (superscript b). Finally, one or
more non-overlapping segments of the query aligned with overlapping regions
of the subject (superscript c).
113
3.7. M13 Sequences of particular interest
Several protein coding regions previously unidentified in T, brucei were
of particular interest. Pyruvate: ferridoxin oxidoreductase (PFO) is one such
structure. The partial amino acid sequence of a trypanosome PFO (dbGSS no.
B13566) is shown with high homology to the PFO from E. histolytica (Fig. 15 A).
A PFO has not yet been described in 7. brucei.
A dismutase capable of destroying superoxide radicals has not been
described previously for T. brucei, although an Fe-superoxide dismutase has
been identified in Crithidia fasciculata and T. cruzi (129,130). Evidence that T.
brucei may contains a Fe-superoxide dismutase (dbGSS no. B13733) with
homology to the enzyme from T. cruzi is shown (Fig. 15 B).
Apoptosis-like events have been described for T. cruzi (131) and
Leishmania (132). Evidence that those events may occur in T. brucei are found
in a clone (dbGSS no. B13593) that contains an ORF and a downstream
untranslated region of 402 bases. The untranslated region has a 95% identity
to human DNA sequences found immediately downstream of the stop codon for
apoptosis inducer Nbk (U49730) (Fig. 16). Two other descriptors contained
similar homology, including a human STS (G26842) and a human Bcl-2
interacting killer (BIK) (U34584) [50,51]. No homology was found between the
coding region for any of the apoptosis genes and the trypanosome sequence.
114
3.8. Cosmid library analysis.
In the pursuance of other novel targets for therapy and of ultimately
sequencing the entire Trypanosome genome a 1X deep cosmid library from T.
brucei strain M110 was analyzed by DNA fingerprinting and cataloged (Fig.17).
1276 random cosmid clones representing approximately 51 million bases were
digested with EcoR I and electrophoresed on 0.8% agarose gels with Hind III
cut lambda size markers. The gels were stained with EtBr and photographed.
Four copies of the library were prepared and stored at -80° C as well as one set
of filters for other analysis and probing.
115
pyruvatesferredoxin oxidoreductase
T.brucei
E.histolytica
3 SNTGGHASKASNLGQVAQFAAAGKNIKKKSLAEIAMSYGYV SNTGG SKA+NLG V +FA++G KK L IAM+YG V
993 SNTGGQKSKATNLGAWKPAS SGCKRPKKDLGAIAMAYGDV
T.brucei
E.histolytica
YVAQVAMGANPAQTLKAIHE 185 YVA +A+GANPAQ KA E YVASIALGANPAQAFKAFKE 1049
B
Fe-superoxide dismutase
T. brucei
T.cruzi
145 GYDGRAAQGLSTVHGRIHYEQDN*GYAN*AIAVAXWGS 32 GYDG AA+GLS +HY++ + GY A A S
11 GYDGLAAKGLSKQQVTLHYDKHHQGYVTKLNAAAQTNS 48
116
Figure 15. Identification of genes involved in regulation of oxidative stress in 7".
brucei. Panel A shows a comparison between a translated region of T. brucei
sequence B13566 and pyruvate: ferredoxin oxidoreductase from E. histolytica
(U30149). Panel B shows a comparison between a translated region of T.
brucei sequence B13733 and Fe-superoxide dismutase from T. cruzi (U39401).
The single letter amino acid code is shown. Identities between the sequences
are indicated by listing the amino acid between the alignments while similarities
are indicated with a plus sign. The asterisk indicates the position of stop
codons. The residue numbers refer to the nucleotide position from the 5' end of
the insert or start methionine of the subject. The region of overlap is short for
the pyruvate: ferredoxin oxidoreductase because the 5' end of clone B13566
encodes the carboxyl terminus of the protein. Conversely, the region of overlap
for the Fe-superoxide dismutase is short because the 3' end of clone B13733
encodes the amino terminus of the protein.
117
T.brucei MTKRSKTKGKKDARKRAEKELQCAEKTEEGTSRGAEKPTSEQPQRRRRR 147 M L E
Human MSEVKPLSRDILMETLLYEQLLEPPTMEVLGMTDSEEDLDPMEDFDSLE 147
T.brucei XRTKRRGKPSEDCGQDLRPPRVREWRPLPRKAHRPILDGPTDPEQGEGR 294 L
Human CMEGSDALALRLACIGDEMDVSLRAPRLAQLSEVAMHSLGLAFIYDQTE 294
T.brucei SNQRVNPS* gaggggctga 331 V S
Human DIRDVLRSFMDGFTTLKENIMRFWRSPNPGSWVSCEQVLLALLLLLALL 441
T.brucei cctgaggccaagggaGCCCCGGCGGTTCAGNGGGGGGNNGGCCCCACCC 380 llllllllll Mil I Mil E 1 I I I I I II I
Human LPLLSGGLHLLLK *gGCCCCGGCGGCTCAGGGCGGGGCTGGCCCCACCC 518
T.brucei CCATGACCATTGCCCTGGAGGTGGCGGCCTGNTGCTGTTATCTTNTTAA 429 11IIII tl I 1111II111111111111111 M I N I M U M Mil
Human CCATGACCACTGCCCTGGAGGTGGCGGCCTGCTGCTGTTATCTTTTTAA 567
T.brucei CTGTTTTCTCATGATGCTTTTTANTATTTAAACCCCGAGATAGTGCTGG 478 lllllllllllllllil Mil 1111111111111111111111111
Human CTGTTTTCTCATGATGCCTTTTTATATTTAAACCCCGAGATAGTGCTGG 616
T.brucei AACACTGCTGAGGTTTAATACTCAGGTTTTTTGTTTTTTTTTTATTCCA 527 I I I II I I I I I I I I I I I M M I I I I I I I I I I I I I I I I I I I I I I I I I II
Human AACACTGCTGAGGTTTTATACCCAGGTTTTTTGTTTTTTTTTTATTCCA 665
T.brucei GTTTTCGTTTTTTCTAAAAGATGAATTCATATGGCTCTGCAATTGTCAC 576 II I I I I I I I I I I I I I I I I II I I I I IiI I I I I I II I I I I I I I II I II I
Human GTTTTCGTTTTTGCTAAAAGATGAATTCCTATGGCTCTGCAATTGTCAC 714
T.brucei CGGTTAACTGTGGCCTGTGCCCAGGAAGAGCCATTCACTCCTGCCCGTG 625 I I I I I II I I I II I IMI I II I I I I I I I I I I I I I I I I I I I I I I II I I II
Human CGGTTAACTGTGGCCTGTGCCCAGGAAGAGCCATTCACTCCTGCCCCTG 763
T.brucei CCCACACGGCAGGTAGCAGGGGGAGTGCTGGTCACACCCATGTGTGATA 674 I I I I I I I I I I II I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I II I
Human CCCACACGGCAGGTAGCAGGGGGAGTGCTGGTCACACCCCTGTGTGATA 812
T.brucei TGTGATGCCCTCGGCAAAGAATCTACTGGAATAGATTCCGAGGAGCAGG 723 I I I I I I II I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I II
Human TGTGATGCCCTCGGCAAAGAATCTACTGGAATAGATTCCGAGGAGCAGG 861
T.brucei AGTGCTCAATAAAATGTTGGTTTCC 748 I I M I I I I M I M I M M I M M M
Human AGTGCTCAATAAAATGTTGGTTTCC 886
118
Figure 16. Comparison between coding and non-coding regions of T. brucei
sequence B13593 and a human apoptosis gene sequence (U49730). Coding
regions are represented with the single letter amino acid code and are
italicized. Shared amino acid residues are shown between the alignments
while identical nucleotides are connected with a line. Stop codons are indicated
by an asterisk. Intragenic non-homologous sequence are in lower case letters.
N's in the DNA sequence are unresolved nucleotides.
119
- 1 -12 -
GEL#1|
-13-36—
J s ' • > ' i * «•- i n
37-48- 49-62
GEL#2|
78-95— GEL#3|
™96*108——
M m ® ^
w i g » * • *
w *
1 2 0
-109-120- -121-132- -133-144—
GEL#4| ^ m i | H If p ^ If m • m ' i I ^ y f If# *m
-145-156-- 157-168- 169-180—
GEL#5|
GEL#6|
181-192- 193-205
-206-217- -218-229- 230-241-
GEL#7
i —0~—242-253- -254-265- X" -266-277-
GEL#8
- «t|
H & m m •
tp m mm m
GEL#9|
121
X
290-301 302-305* j.
1 2 2
-306-317- -318-329— -330-341-
GEL#10
354-365 366-377-
Gel#11
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131
Figure 17. Analysis of random cosmid clones from T. brucei strain M110. Clones are digested with EcoR I and electrophoresed on 0.8% agarose gels with Hind III digested lambda DNA. The gels were stained with EtBr to reveal banding patterns. Clone numbers are shown between lambda markers at the top of the gels.
4. Discussion
This work is a continuation of work begun by Wu et al.,1992. The
sequences, transcription and genomic organization of EF-hand proteins were
obtained using several different trypanosome strains. The original partial
protein sequence (data not shown) of Tb-44 was obtained from strain M110,
while the Tb-44A cDNA was cloned from a library of IsTAFM. Tb-17, Tb-24, and
Tb-1.7g were all obtained from strain 427-60. The M13 library and the cosmid
librarys were constructed from strain M110. Some sequence variability that
was observed may therefore reflect differences between strains.
Several lines of evidence indicate that multiple calflagin family members
occur within a single trypanosome. These include: the detection of multiple
bands by immunoblot analysis of strain M110 (47); the detection of Tb-44-
related sequences in both the IsTAFM cDNA library and M110 cell lines; the
detection of identical restriction fragments when genomic DNA of strains M110
or 427-60 were hybridized with coding regions of calflagins (Fig. 4); and the
cloning of Tb-17, Tb-24 and Tb-1.7g from the same strain. These data are in
contrast to T. cruzi where genomic Southern blots identified a 900 bp direct
repeat upon digestion with BamH\, Hind\\\, Pst\ or Sail (127), and only a single
band on immunoblots (7,47). Overall, the data suggest that multiple functions of
the T. cruzi calflagins are limited.
The different patterns of 3' flanking sequences for the calflagins may help
indicate the evolutionary origins of the calflagin variants. For example, Tb-17
and Tb-1.7g are very similar in their coding sequences. They share the same
133
amino terminus and carboxyl terminal extensions as well as the same 3' non-
coding sequences. Therefore they are probably variants of a common
progenitor (Fig. 11,13). Tb-24 and Tb-44A also share similar coding sequences
including amino and carboxyl termini that are distinct from Tb-17 and Tb-1.7.
They share the same distinct 3' non-coding region, suggesting that Tb-44A
arose from a progenitor to Tb-24. In addition, Tb-44A contained a near perfect
internal repeat of 559 bp, which indicated that further heterogeneity in the
calflagin family arose from gene duplication of internal sequences. The
presence of only 12 nucleotide substitutions between the two halves of Tb-44A
indicates that the duplication event occurred relatively recently in the evolution
of trypanosomes. This suggestion was strongly supported by the observation
that T. cruzi contained a 23 kDa calflagin, but not the 44 kDa calflagin
(133,134). These data indicate that the duplication event occurred after the
lineage for T. brucei and T. cruzi diverged. Two different mRNA species of 1.2
and 1.6 kb were identified that encoded the calflagins. The larger mRNA likely
results from the transcription products of Tb-44A, Tb-17, and Tb-1.7g. Despite
the large size differences between the coding regions for Tb-44A and Tb-17,
each cDNA was approximately 1.5 kb. Tb-1.7g has downstream sequences
that are related to Tb-17, suggesting the use of a common polyadenylation site
and common sized transcript. By contrast, the short 3' non-coding intergenic
region of Tb-24 suggests that the smaller 1.2 kb mRNA is encoded by this gene.
The presence of multiple EF-hand calcium-binding proteins in cilia and
flagella may be a general phenomenon. The flagellum of Chlamydomonas
contains calmodulin, and a separate EF-hand protein (135,136,137). Cilia from
Tetrahymena contain at least 3 other EF-hand proteins besides calmodulin
134
(138). All of the EF-hand proteins of cilia and flagella are structurally divergent,
and may have evolved to perform special functions. The function of
trypanosome calflagins is under investigation. The flagellar location of these
calcium-binding proteins suggests a role in the motility of trypanosomatids.
However, the requirement of multiple calcium-binding proteins including
calflagins and calmodulin for motility is not known. The situation in T. cruzi
provides evidence that multiple calflagins, especially the larger Tb-44A is not
an absolute requirement for motility. Both immunoblot analyses (7,47), and
molecular cloning (127) demonstrate that the T. cruzi flagellum functions as a
motile apparatus in the absence of Tb-44A. An alternative possibility is that
some members of the calflagin family mediate signal/recognition functions of
the trypanosome flagellum. Calcium-sensitive adenylate cyclase (139), host-
parasite junctional complexes (140), or the cellular interactions required for
genetic exchange (141) are potential targets for calflagin regulation. Gene
cloning of the trypanosome calflagins will facilitate these functional analyses.
The repeated nature of the calflagins reflects the organization of the
trypanosome genome. Approximately 15% of the trypanosome genome is of a
repeated nature. Tandemly arranged 177 bp repeats of the minichromosomes,
telomeric repeats, and several families of retroposon repeated elements make
up the bulk of non-coding repeated DNA. Coding sequences lack introns,
occur at high density and are often duplicated in tandem arrays. This level of
repetition presents special problems in applying classical molecular techniques
to gene analysis. Restriction analysis of the calflagin gene locus using
Southern blotting for example was inconclusive due to similarities between
135
coding and non-coding sequences. The current hypothesis is that at least one
and possibly two other calflagin genes may exist at the locus (data not shown).
Strategies to resolve the calflagin locus are currently being considered for
investigation. In highly repeated genomes, false bands may be observed when
labeled probes bind non-specifically to DNA dense areas within a Southern
blot with certain restriction digests. False bands may also occur when probes
bind weakly to repeated elements resulting in a signal that can be amplified
many times over. Such was the case where a degenerate oligo probe for a T.
brucei VSG was used to probe a genomic Hind III digest (data not shown). A
weak band was observed and a positive Hind III fragment was cloned into a
pUC plasmid vector and sequenced (Fig. 14A). The sequence revealed a 28
bp repeated element. Reevaluation of a heavily EtBr stained gel revealed an
abnormal distribution of Hind III fragments. A dense area of fragments was
observed in the area from which the positive fragment was cloned suggesting
non-specific binding of the probe to that area. The clone was then probed back
to a Hind III digest of genomic DNA where several bands were observed
(FIG.14B). The 1.5 kb band (Fig.14A) may be the same band observed with the
degenerate oligo probe (data not shown).
The repeated sequence was, however, interesting in its own right in that
it consisted of an unusual size (28 bp) repeated element. It was considered to
be to small for a minichromosome repeat and to large for a telomeric repeat.
The size was closest to a type of 50 bp repeat associated with some VSG
promoters and antigenic variation. BLAST analysis revealed that it had
significant homology to a single element found in a B. malayi suggesting that
the sequence may be conserved. This repeated element is an example of an
136
interesting sequence that was randomly extracted from a size selected
trypanosome genome, a strategy that has proven to be fruitful in the discovery
of novel trypanosome sequences (105,106).
A seemingly unrelated discovery of the 3' end of an ORF found at the 5'
end of Tb-24 was also found to have significant homology to B. malayi.
It's proximity to the 5' end of the calflagin locus suggested that it may be co-
transcribed with the calflagin genes perhaps as a house keeping gene or a
control/regulatory element. Strategies to PCR amplify the sequence between
the ORF and the calflagins from total mRNA were not with out concern due to
the conserved sequences 5' and 3' to the genes. In addition to understanding
the ORF's transcriptional status, was the desire to ultimately sequence the
entire calflagin locus as well as the 5' ORF sequence. A strategy was therefore
undertaken to isolate one or more fragments that would contain the entire
calflagin locus as well as the 5' ORF. Since the calflagin gene locus is
contained within Xba I-Xba I and Xho I-Xho I restriction fragments (Fig. 4) size
selected fragments would be taken from one of the digest. A consideration to
creating and screening such a library was that a significant number of
sequenceable clones would be generated. To sequence the clones from one
end (500 bp) would significantly contribute to trypanosome sequencing efforts.
The prospects of identifying novel genes that might be exploited for therapy was
also a motivating factor to sequence as many of the clones as possible.
Detection of novel T.brucei sequences using shotgun cloning of genomic DNA
has previously been reported using pCR-Script™ as a vector followed by
double stranded sequencing of the insert DNA (106). This approach worked
well due to the high density and close spacing of genes in T. brucei. The
137
decision to use the M13 bacteriophage as a shotgun vector was in part due to
the logistical situation at that moment, but was also based on several
advantages including: high copy numbers of the recombinant double stranded
replicative form (RF) per cell; no strict constraints on insert size due to the
filamentous nature of the phage; Isolation of single stranded templates resulting
in high quality sequencing reactions and clean sequence reads. It was further
considered that since the focus was extended to include a sequencing project
as well as an effort to isolate one or more calflagin containing fragments, a
further step could be taken to increase the number of different non-calflagin
containing fragments. By cloning a mixture of both Xba I-Xba I and Xho I-Xho I
fragments the variety of non-calflagin containing fragments would be increased.
While not completely random the use of two size selected restriction digests
increases the number of different non-calflagin fragments while keeping the
relative number of calflagin fragments the same. This could be accomplished
because Xba \-Xba I and Xho \-Xho I fragments containing the calflagin locus
are basically the same size (Fig.4). The DNA was size select between 10 and
14kilobases. For example:
Xho I fragments + calflagin fragments
Xba I fragments + calflagin fragments
2X DNA ss 2 fragment families + 2X calflagin fragments
Initially, all 455 sequences were analyzed by the TBLASTX algorithm
with the filters off. Each sequence was then submitted to the GSS database
and "Nearest Neighbor" analysis performed. Those sequences for which the
TBLASTX and Nearest Neighbor analysis gave different results were blasted
again using BLASTP and BLASTN searches with SEG (120,121) and DUST
138
(125) filters on, respectively. The differences between searches were largely
due to the parameter options of the blast source. All of the highest ranked
descriptors were considered while only one was reported for each sequence.
Those sequences chosen for inclusion in Table 1 would typically have low p
values (p»0.05), high complexity, and length of homology typically greater
than 30 amino acids. Some sequences with high ranking descriptors were
eliminated due to a highly repeated nature or low complexity of the homologous
region (i.e. proline rich, histidine rich, or glycine rich repeats). Some
sequences were included that only appeared in non-filtered BLAST formatting
but were of high enough homology over a long enough region to warrant
reporting. These sequences demonstrate that filtering can delete some useful
data. The initial BLAST analysis was done with filtering off to prevent loss of
these coding sequences and repeated elements in the genome. All of the
sequences were investigated for any mammalian contamination in the form of
repeated elements by the NCBI under the direction of Dr. David Litman. Only
one sequence was found to be remotely related to a mammalian repeated
element (mir), but the findings were not considered to be significant and the
sequence was left in the database.
In this report several protein coding regions previously unidentified in T.
brucei were of particular interest. Pyruvate: ferredoxin oxidoreductase (PFO),
Fe-superoxide dismutase, and a descriptor for the apoptosis inducer Nbk
warrant further discussion.
Pyruvate: ferridoxin oxidoreductase. In mammalian cells, oxidative
decarboxylation of pyruvate to form acetyl-CoA is accomplished by the enzyme
pyruvate dehydrogenase. However, a variety of anaerobic pathogens,
139
including Entamoeba histolytica, Trichomona vaginalis and Giardia lamblia
utilize PFO to conduct the same function (142,143,144). The biochemical
distinction between PFO and pyruvate dehydrogenase has been exploited in
the design of therapies against anaerobic pathogens since PFO can donate
electrons to nitroheterocyclic compounds (145). The resulting nitro-free radical
and subsequently formed superoxide anion are toxic to the cell (145).
Interestingly, nitroheterocylcic compounds such as Metronidazole are also
effective against T. brucei (146,147,148,149). In this case, it has been
assumed that trypanothione reductase is responsible for transfer of electrons to
the inactive drug (150). The trypanosome PFO might be used to generate free
radicals from nitroheterocyclic compounds, or might be targeted directly as a
critical component of metabolism. The absence of a functional mitochondrion in
bloodstream forms would suggest that the trypanosome PFO is likely to be
present during this stage and be developmental^ regulated.
Fe-superoxide dismutase. The efficacy of Metronidazole and other
nitroheterocyclic compounds depends in part on the rate of nitro free radical
production, and the rate of dismutation. Trypanosomes are lacking in some
enzymes which protect against oxidative stress, including catalase and
glutathione peroxidase (151). Defense against hydrogen peroxide relies
instead on the activity of a trypanothione dependent peroxidase (152,153,154).
A dismutase capable of destroying superoxide radicals has not been described
previously for T. brucei, although a Fe-superoxide dismutase has been
identified in Crithidia fasciculata and T. cruzi (129,130). The sensitivity of T.
brucei to free radicals is the basis for trypanotolerance in Cape buffalo (155)
and N'Dama cattle (156), where each of these organisms contain hydrogen
140
peroxide generating enzymes in their blood. An understanding of the oxidative
defense mechanisms of T. brucei is a first step towards exploiting these
pathways in therapy design.
A descriptor for apoptosis inducer Nbk. Xanthine oxidase is the enzyme
responsible for hydrogen peroxide production in Cape Buffalo blood (155). It
has been recently shown that exogenous xanthine oxidase/xanthine can kill
procyclic form trypanosomes in a manner which is blocked by catalase, and
involves fragmentation of the nuclear DNA and loss of mitochondrial function
(Dr. Larry Ruben, Southern Methodist University personal communication).
These data suggest that hydrogen peroxide can initiate an apoptosis-like event
in T. brucei. Apoptosis has already been described following treatment with the
lectin, concanavalin A (157,158). Vacuolization of the cytosol and
fragmentation of the nuclear DNA precedes cell death (157). Additionally, a
programmed pattern of RNA synthesis also precedes cell death (158).
Apoptosis-like events have also been described for T. cruzi (131) and
Leishmania (132).
The highly conserved nature of this DNA between humans and
trypanosomes suggests that it contains a critical regulatory motif or that it was
recently acquired by the trypanosome from the mammalian host. Activation of
apoptotic pathways may form the basis of interesting new therapies in which the
cell is essentially induced to commit suicide during the infection cycle.
Overall, this sequencing study reflects a continuation of work begun by
others (105,106). The goal has been to describe the calflagin gene locus and
sequence large quantities of the trypanosome genome in an effort to identify
new targets for diagnosis and treatment of trypanosomiasis. This is an ongoing
141
project that hopes to contribute to the elucidation of the entire trypanosome
genome. Sequencing of the entire genome might be accelerated by using
existing sequence data in conjunction with a cosmid (40 kb insert) or PAC
(140kb insert) library and constructing a minimal tiling path based on PCR
amplified sequence-tagged sites (STSs) and/or cosmid or PAC finger printing,
as described for Giardia lamblia (159). Select cosmids or PACs could then be
shotgun cloned into the M13 vector for sequencing and assembly. The 1X
deep cosmid library for T. brucei (Fig. 17) was created with this long term goal
in mind. Powerful new software is currently being developed to assemble
restriction fragments such as those shown in figure 17. Recombinant cosmid
DNA has been prepared to extend the depth of the library many fold if the
opportunity arises to apply this type of software or to create a working library for
sequencing. A set of filters for the cosmid library has also been stamped using
a Biomeck® robot for the purpose of hybridization experiments.
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