Genetic Recombination Gene Expression: Transcription and ...jnkvv.org/PDF/31032020123011Bacterial...

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Chapter - III

BACTERIAL GENETICS

Genetic Recombination

Gene Expression: Transcription and Translation

From genes to proteins

Allele: An alternate form of a gene. Alleles occur at loci on chromosomes.

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Genetic Recombination

Genetic material, otherwise known as DNA (DeoxyriboNucleic Acid) is composed of millions ofgenetic instruction sets. Each "set" is actually a specific ordered sequence of only four differentamino acids (Adenine, Thymine, Guanine, and Cytosine). Each instruction set may be severalhundred acid molecules long.

A "set" of instruction "code" (as it is sometimes referred to) is known as an Allele.Connect a large number of alleles together into a long "strand" and you have half of aChromosome.

When you put two matching chromosome strands together, the "genetic code" or allelesmatch up on both sides, and together the two alleles are referred to as a Gene. In most bodycells, chromosomes occur in pairs. This is what leads to the term "gene pair". Each "gene",therefore, is actually comprised of a pair of alleles.

A particular area or location on a chromosome, is called a "locus" (plural = "loci"). Thecombination of both alleles at a specific locus determine a particular Trait or Characteristic ofthe animal. Hair color and eye color are examples of traits.

Most of the cells in the body contain all of chromosomes. The chromosomes act as"instruction sets" which both tell the body how it will grow, develop and operate the traits of theindividual.

Fig.1. Each "color band" pair represents a Locus. The two alleles at each location, together represent aGene. When both alleles are the same, they are referred to as homozygous. When each allele within a

pair is different, it is known as heterozygous.

Since the chromosomes occur in pairs, each "half" of the chromosome pair has thesame order/arrangement of loci as the other. The exception to this, is the chromosome pairknown as the Sex Chromosomes in the specialized sex cells used for reproduction.

These chromosomes are often referred to as the X and Y chromosomes. The Y chromosomeis shorter and contains less material than the X chromosome. The specific combination of the

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sex chromosomes determine the gender or sex of the individual. Two X chromosomes (XX) willresult in a female. The XY combination will result in a male.

The reproductive cells, known as gametes, (which take the form of ova in females orspermatozoa in males) carry only half of the animal's genes. In all the higher species (such asmammals) the offspring will receive only one half of a gene pair from each parent.

In the process of cell division that creates these gametes, the existing chromosomes ofthe parent are allowed to "cross-over". That is, some of the alleles found on one side of thechromosome pair will randomly trade places with the alleles found on the other side. After thisexchange, the cell divides, and each resulting gamete cell has only half of the genetic materialof the parent. As a result, each gamete contains a unique grouping of half of the genes of theparent animal. This special process of recombining the alleles to form gametes is known as

Meiosis.

Genetic recombination is the name given to a group of reactions during which cellularmachinery uses DNA to alter or "recombine" with a similar (homologous) sequence. Theprocess involves pairing between complementary strands of DNA, and results in a physicalexchange of chromosome material. Genetic information is recombined by the cell for severalreasons including the repair of damaged DNA, and the production of population variabilityduring sexual reproduction. In some cases, recombination is known to change genes, addingnew alleles to the population.

Creationists generally believe that this mechanism was designed to generate thetremendous variety that is evident within each kind, whereas evolutionists attribute suchvariability ultimately to random mutagenesis. However, many creationists contend thatrecombination processes add nothing new to the gene pool.

The position at which a gene is located on a chromosome is called a locus. In agiven individual, one might find two different versions of this gene at a particular locus.

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These alternate gene forms are called alleles. During Meiosis, when the chromosomesline up along the metaphase plate, the two strands of a chromosome pair mayphysically cross over one another, and during these events genetic recombination isperformed by the cell.

Recombination results in a new arrangement of maternal and paternal alleles onthe same chromosome. Although the same genes appear in the same order, the allelesare different. This process explains why offspring from the same parents can look sodifferent. In this way, it is theoretically possible to have any combination of parentalalleles in an offspring, and the fact that two alleles appear together in one offspring doesnot have any influence on the statistical probability that another offspring will have thesame combination. This theory of "independent assortment" of alleles is fundamental togenetic inheritance.

The frequency of recombination is actually not the same for all genecombinations. This is because recombination is greatly influenced by the proximity ofone gene to another. If two genes are located close together on a chromosome, thelikelihood that a recombination event will separate these two genes is less than if theywere farther apart. Linkage describes the tendency of genes to be inherited together asa result of their location on the same chromosome. Linkage disequilibrium describes asituation in which some combinations of genes or genetic markers occur more or lessfrequently in a population than would be expected from their distances apart. Scientistsapply this concept when searching for a gene that may cause a particular disease. Theydo this by comparing the occurrence of a specific DNA sequence with the appearanceof a disease. When they find a high correlation between the two, they know they aregetting closer to finding the appropriate gene sequence.

Evolutionary Assumptions

Chromosomes have genes arranged along their length. During meiosis, it is believed theintended function of recombination is to leave existing genes unchanged by performing reactingin the neutral regions between reading frames.

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Recombination within genes is able to create new alleles, however, it has beenassumed this is not the cell's intent, and any changes to gene sequence are believed tobe mutations resulting from mistakes during recombination or replication.

The theory of evolution has led to the assumption that recombination originallyoccurred by mistake, instead of being an intelligently designed process. During sexualreproduction, gametes (egg, sperm) are produced during a cell division process calledmeiosis. Prior to meiotic division, homologous chromosomes unite at the axis beforedividing to opposite poles. It is believed that this homologous pairing was originallyperformed simply to insure an equivalent division of genetic information. But, anexchange of DNA accidentally occurred during this process, which provided beneficialvariability and was naturally selected to became a regular part of gamete formation. Itremains generally assumed that recombination events are rather random, andtherefore, the phenotypes produced by these reaction are also random.

The DNA used for meiotic recombination possess homology or sequences thatare very similar, and also code for variations of the same characteristic. Before thechromosomal DNA is distributed into new daughter cells, the homologues pair and arespliced together at multiple locations. During these interactions, entire regions andmany genes are frequently exchanged. Offspring are always genetically unique due torecombination. However, it is now clear that recombination is a powerful source of newalleles.

The knowledge of recombination comes predominantly from the bacteria E. coli,and its effect during sexual reproduction (meiosis) has been studied mostly using lowereukaryotes such as baker's yeast, as well as fruit flies. Recent work with mice hasprovided additional information from mammals, and shown that substantial differencesexist between unicellular and multicellular organisms. The basic details and manygenes involved in homologous recombination (HR) appear conserved among themultitude of life forms on earth. It is now widely recognized that genetic editions throughHR are part of a highly coordinated process involving a cascade of specificmacromolecule interactions (genes), and controlled by highly organized regulatorysystems.

Non-Random Recombination

It was assumed that gene crossovers during meiosis occurred at random intervals alongchromosomes. It was believed that the frequency of gene crossovers was directlyrelated to the distance between genes, but a variety of discoveries have illustrated theexistence of differential recombination rates and patterns, and forced a revision of mapdistances. It is now a well-known fact that recombination frequency is not constant inany one particular cell. Reactions occur more frequently in some regions of the genomethan in others with variations of several orders of magnitude observed. These

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hyperactive regions have been termed as "hot spots" as opposed to inert "cold spots"where little to no exchange is found.

The frequencies of recombination events are also non-random. The rates arefound to be significantly higher when comparing germ-line with somatic cell types. Sex-specific differences in recombination frequency have also been elucidated. Standardlinkage analysis was used to confirm that females have a higher recombination ratethan males, and males recombine preferentially in the distal regions of thechromosome.

In addition to exchanges during cell division, HR is involved with many otherforms of genomic DNA editing. For example, recombination is induced or shut off as apreprogrammed cell function during differentiation and development. It is also used toperform error-free DNA repair, which in this case serves to prevent unintentionalvariability. In fact, HR maintains the integrity of the genome through the correction ofseveral different types of DNA damage. Homologous recombination is stimulated bydouble-stranded breaks during any stage of the cell cycle, and is also responsible forperforming deletions, duplications, and translocations between dispersed homologous,which are frequently a response to stress. The specific details or exact sequencehomology required for recombination remain largely unknown, but the plethora offunctions accomplished by these reactions has elevated them to the position of mastermechanic responsible for virtually all forms of sequence editing and maintenance.

New Alleles

There is an interesting new class of HR only recently recognized that sharescommon mechanisms with meiotic crossovers, and is likely responsible for theformation of new alleles. The process known as gene conversion uses template DNA toedit active sequences. During this process, pseudo genes previously referred to as junkDNA is frequently used to make these changes. Gene conversion can be easilydistinguished from crossovers in most cases because only one of the homologues isaltered. It has now been thoroughly documented that mitotic recombination via geneconversion is able to create genetically altered cells, and researchers have suggestedthat this process can generate a gene with novel functions by rearranging various partsof the parental reading frames. DNA is also repaired through conversion when an intactcopy from the sister chromatid or homologous chromosome is used to replace thedamaged region. Gene conversion is now understood to be responsible for performingmany alterations that were previously attributed to mutations or other repairmechanisms.

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Crossing-over is an exchange of sequences between two homologous regions, butduring gene conversion only one of the homologues is altered. Regions elsewhere onthe same chromosome are instead typically used to convert the gene, and therebyintroduce new alleles into the population. This mechanism is responsible for the creationof new alleles in immuno-globulins, the MHC loci, and others.

Variable Genes

Diversification within a population occurs because the genes involved with theproduction of characteristics exist as a variety of alleles, and therefore traits arepolymorphic or available in more than one form. Closely related species are commonlyfound with extremely high numbers of alleles. Evolutionists generally assume that newalleles are the result of random mutations that have accumulated gradually over millionsof years. However, it was discovered that many genes in every genome are highlydiverse (hypervariable) in comparison to others.

Not all genes are variable. The majority of genes in the genome is involved withhousekeeping functions, and is commonly found unchanged even when comparingvastly different organisms. In contrast, variable genes change significantly from onegeneration to the next and show nonrandom patterns within any given gene. Thisdiversity is systematically produced through gene conversion while under tight cellularcontrol. For example, variable genes have hot and cold spots of activity similar to thosefound among gene crossovers in meiosis. A preponderance of non-synonymoussubstitutions over synonymous has provided even further evidence againstrandomness. It is becoming increasingly questionable that variability is the result ofrandom mutations as commonly claimed by evolutionists.

Adaptation

Adaptation to a particular habitat or niche involves largely uncharacterized modificationsof the genome, and much of what is learnt about genetic heredity has come fromtheorists who do not believe the cell was designed to perform such changes with intent.The ability of the cell to produce new alleles has probably remained misunderstood forso long because the products of these reactions are being attributed to a source that is

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independent of cellular purpose (mutations). The mechanisms behind this type of geneconversion are not yet understood, but clearly illustrate the ability of the cell tospecifically edit genes, and thereby rapidly multiply the number of alleles in apopulation. Further characterization should prove to be valuable evidence that cellulardesign governs the production of genetic variability, and adaptive evolution that occursas a result.

The Central Dogma of genetic expression

Protein synthesis requires two steps: transcription and translation.

DNA contains codes: The simplistic diagram below illustrates the concept that threebases in DNA code for one amino acid. The DNA code is copied to produce mRNA. Theorder of amino acids in the polypeptide is determined by the sequence of 3-letter codesin mRNA.

Genetic Recombination: overview

The genetic information can be changed: either by mutation or by the transfer of genesfrom one organism to another. The successful transfer of genetic information includestwo elements: the introduction of genes from a donor cell into a recipient andrecombination of those introduced genes into the recipient's genome. Microbialgenetics are important because the genes are the basis for cell function andmicroorganisms are excellent tools for studying gene function.

What are Mutations?

A mutation is any physical change in the genetic material (such as a gene or achromosome). When a gene contains a mutation, the protein encoded by thatgene will be abnormal. Some protein changes are insignificant, others aredisabling.

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More than 4,000 diseases are thought to stem from mutated genes inherited fromthe parents.

A mutation may or may not affect the phenotype. A mutation is not necessarily bad. It may even be good.

Mutations are inheritable changes in the base sequence of nucleic acid -- thegenetic material. An organism with these changes is called a mutant. Geneticrecombination is the process where genes from two genomes are combined together. Amutant will be different from its parent, its genotype or genetic makeup has beenaltered. The phenotype or visible properties of the mutant may or may not be altered.The genotype of a strain is indicated by use of three small italics letters followed by acapital letter and indicates the gene involved in a process (hisC indicates the gene forHisC protein). The phenotype of the strain is indicated by three letter code that ends in a+/-. For example Thr+ indicates a strain can make its own threonine while Thr- indicatesthat it cannot. An auxotroph is formed when a required nutritional material (amino acidfor example) that the parent strain, prototroph, could make is no longer formed.

General Types of Mutations

1. Chromosomal Mutations

Changes in chromosome structureo Deletion, duplication, inversion, or translocation.

Changes in chromosome numbero Polyploidy, aneuploidy (autosomes or sex chromosomes).

2. Point Mutations

Changes made by substituting a single base with another or by adding ordeleting one or more nucleotides.

o Sickle cell disease results from a single base change (Remember in RNA,the nucleotide base uracil replaces thymine).

Genetic Mutations and their Effects on Proteins

Point Mutations: Changes in single DNA nucleotides.

A missense mutation substitutes a different amino acid for the original one.

TEMPLATE DNA code GAG (leucine - leu) -mutation->TEMPLATE DNA code GTG (histidine - his)

A nonsense mutation results in a stop codon being inserted someplace beforethe end of the gene.

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TEMPLATE DNA code ATG (tyrosine - tyr) -mutation->TEMPLATE DNA code ATT (STOP)

Silent mutations are point mutations that do not change the amino acidsequence of the protein. These are most likely to have no effect. Redundancy ofthe Genetic Code reduces the chance that point mutations do not alter thespecified amino acids.

The mRNA codons GAA and GAG code for the amino acid Glutamic Acid (Glu).The mRNA codons GCU, GCC, GCA, and GCG all code for the amino acidAlanine (Ala).The mRNA codons GGU, GGC, GGA, and GGG all code for the amino acidGlycine (Gly).

Frameshift Mutations: Additions or deletions of one or more nucleotides.o May result in "garbage" genes, as the entire amino acid sequence in the

code after the change is devastated.

Large deletions may remove a single amino acid, or an entire chunk of chromosome.The most common mutation that causes severe cystic fibrosis deletes only a singlecodon.

Mutations can occur spontaneously, because of mistakes during replication ordue to natural radiation at a frequency of about one in 1,000,000, or may beexperimentally induced using mutagens. Mutations can be chemically induced by baseanalogs, compounds that are structurally similar to the purines and pyrimidines in DNA.The cell incorporates them into DNA, but during subsequent replication, the analogshave a higher probability of base pairing incorrectly, thereby inserting the wrong baseinto the new DNA strand. Other chemical mutagens react directly with DNA to alter thebases. UV radiation is absorbed by the purines and pyrimidines in DNA, and one of its

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effects is to form pyrimidine dimers in one strand, which prevents these thymine basesfrom pairing correctly during replication.

Pyrimidine dimers

DNA Lesion-Thymine Dimer

Pyrimidine dimers are molecular lesions formed from thymine or cytosine bases in DNAvia photochemical reactions. Ultraviolet light induces the formation of covalent linkagesby reactions localized on the C=C double bonds. In dsRNA, uracil dimers may alsoaccumulate as a result of UV radiation. Two common UV products are cyclobutanepyrimidine dimers (CPDs, including thymine dimers) and 6,4 photoproducts. Thesepremutagenic lesions alter the structure of DNA and consequently inhibit polymerasesand arrest replication. Dimers may be repaired by photoreactivation or nucleotideexcision repair, but unrepaired dimers are mutagenic. Pyrimidine dimers are the primarycause of melanomas in human beings.

Types of dimers

Left: Spore photoproduct, Right: Cyclobutane pyrimidine dimer

A cyclobutane pyrimidine dimer (CPD) contains a four membered ring arising from the couplingof the C=C double bonds of pyrimidines. Such dimers interfere with base pairing during DNAreplication, leading to mutations.

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Pyrimidine dimers



Types of dimers



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Pyrimidine dimers



Types of dimers



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6,4-photoproducts, or 6,4 pyrimidine-pyrimidones, occur at one third the frequency ofCPDs but are more mutagenic. Spore photoproduct lyase provides another enzymaticpathway for repair of thymine photodimers.

Ionizing radiation generates free radicals in cells, and these can react with the DNAbackbone to cause breaks. Biological agents, such as transposons and thebacteriophage Mu, cause mutations by inserting DNA sequences into genes, andthereby disrupting the coding information.

Mu phageVirus classificationGroup: Group I (dsDNA)Order: CaudoviralesFamily: MyoviridaeGenus: Mu-like virusesSpecies: Mu Phage

Bacteriophage Mu or phage Mu is a temperate bacteriophage, a type of virus thatinfects bacteria. It has an icosahedral head, a contractile tail and 6 tail fibres. It usesDNA-based transposition to integrate its genome into the genome of the host cell that itis infecting. It can then use transposition to initiate its viral DNA replication. Once theviral DNA is inserted into the bacteria, the Mu transposase protein/enzyme in the cellrecognizes the recombination sites at the ends of the viral DNA (gix-L and gix-R sites)and binds to them, allowing the process of replicating the viral DNA or embedding it intothe host genome.

Fig. Schematic illustration of bacteriophage Mu based on electron microscopic observations. The letters,a, b, c, and d indicate the head, tail, baseplate, and tail fibers, respectively

Bacteriophase Mu

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A mutation may be a change in a single base pair (point mutation) or involve largedeletions or insertions of base pairs. The insertion of a single additional base into agene can have dramatic effects upon the amino acid sequence of the protein producedfrom that gene, due to the reading-frame shift this causes in the translation of themRNA produced from the gene. It is possible to move large sections of DNA to asecond location and the process is termed translocation. If the mutated gene is part ofan operon the mutation may exert polar effects upon other genes in the operon.

Operon - a segment of DNA containing adjacent genes including structural genes andan operator gene and a regulatory gene. An operon is made up of 3 basic components:

Promoter – a nucleotide sequence that enables a gene to be transcribed. Thepromoter is recognized by RNA polymerase, which then initiates transcription. In RNAsynthesis, promoters indicate which genes should be used for messenger RNAcreation – and, by extension, control which proteins the cell produces.

Operator – a segment of DNA that a regulator binds to. It is classically defined in thelac operon as a segment between the promoter and the genes of the operon. In thecase of a repressor, the repressor protein physically obstructs the RNA polymerasefrom transcribing the genes. A gene that activates the production of messenger RNAby adjacent structural genes.

Structural genes – the genes that are co-regulated by the operon.

Not always included within the operon, but important in its function is a regulatorygene, a constantly expressed gene which codes for repressor proteins. A gene thatproduces a repressor substance that inhibits an operator gene. The regulatory genedoes not need to be in, adjacent to, or even near the operon.

Fig. A typical Operon

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Fig. 1: RNA Polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7:lacY, 8: lacA. Top: The gene is essentially turned off. There is no lactose to inhibit therepressor, so the repressor binds to the operator, which obstructs the RNA polymerasefrom binding to the promoter and making lactase. Bottom: The gene is turned on.Lactose is inhibiting the repressor, allowing the RNA polymerase to bind with thepromoter, and express the genes, which synthesize lactase. Eventually, the lactase willdigest all of the lactose, until there is none to bind to the repressor. The repressor willthen bind to the operator, stopping the manufacture of lactase.

In genetics, an operon is a functioning unit of genomic DNA containing a cluster ofgenes under the control of a single regulatory signal or promoter. The genes aretranscribed together into an mRNA strand and either translated together in thecytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translatedseparately, i.e. several strands of mRNA that each encode a single gene product. Theresult of this is that the genes contained in the operon are either expressed together ornot at all. Several genes must be both co-transcribed and co-regulated to define anoperon.

The effects of a specific mutation may be reversed by a second (suppressor)mutation in either the same gene, or in another gene. Note that cells do have DNArepair systems to correct damage to DNA. The SOS system is one of these, but it iserror-prone and the repaired DNA may still contain mutations.

The SOS response is a state of high-activity DNA repair, and is activated bybacteria that have been exposed to heavy doses of DNA-damaging agents. Their DNAis basically chopped to shreds, and the bacteria attempts to repair its genome at anycost (including inclusion of mutations due to error-prone nature of repairmechanisms). The SOS system is a regulon; that is, it controls expression of severalgenes distributed throughout the genome simultaneously.

The primary control for the SOS regulon is the gene product of lexA, whichserves as a repressor for recA, lexA (which means it regulates its own expression), andabout 16 other proteins that make up the SOS response. During a normal cell’s life, theSOS system is turned off, because lexA represses expression of all the critical proteins.However, when DNA damage occurs, RecA binds to single-stranded DNA (single-

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stranded when a lesion creates a gap in daughter DNA). As DNA damageaccumulates, more RecA will be bound to the DNA to repair the damage.

What is interesting is that RecA, in addition to its abilities in recombination repair,stimulates the autoproteolysis of lexA’s gene product. That is, LexA will cleave itself inthe presence of bound RecA, which causes cellular levels of LexA to drop, which, inturn, causes coordinate derepression (induction) of the SOS regulon genes.

As damage is repaired, RecA releases DNA; in this unbound form, it no longer causesthe autoproteolysis of LexA, and so the cellular levels of LexA rise to normal again,shutting down expression of the SOS regulon genes.

One use of mutant bacterial strains has been to determine the potential mutagenicity ofchemicals -- either manufactured or natural. The Ames test utilizes back mutation in astrain of bacteria that are auxotrophic for a nutrient. When auxotrophic cells (His-) arespread on a medium that lacks histidine no growth will occur. If, however, the cells aretreated with a chemical that causes a reversion mutation it can then grow.

General or homologous recombination requires extensive homology and is mediated byan enzyme, RecA protein. The sequence of events are (1) nicking of a DNA molecule,(2) opening of the DNA double helix, (3) pairing between homologous single strands oftwo DNA molecules (requires presence of RecA), and (4) breakage and rejoining ofDNA strands so that portions of the DNA molecule are exchanged. An important point isthat this process leads to new genotypes only if the two molecules that are recombiningdiffer genetically in regions outside those where breakage and rejoining occurred. Inorder to detect recombination or exchange of DNA, the offspring must be phenotypicallydifferent from the parent.

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Mechanisms of genetic recombination in bacteria

In bacteria, the gene transfer that precedes recombination can occur by threemechanisms: transformation, transduction, or conjugation. Transformation involves the uptakeby a recipient of free or naked DNA released from a donor. However, cells may only bephysiologically competent to take up DNA. Competence is related to changes in the cell surfacethat allow strong binding of DNA. In some organisms, such as E. coli, the transformationprocess can be enhanced by special pre-treatment of cells. The cell can undergoelectroporation where small holes or pores are open in the cell. A single strand of the

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transforming DNA is integrated into the chromosome, using general recombinationmechanisms. A cell with a new genotype is generated when this strand is replicated and theresulting molecule forms the genome of a new cell, at cell division. Eukaryotic cells can also betreated to take up free DNA, although the specific treatments are different from those used inbacteria.

Transformation

• Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters acompetent recipient bacterium and it is exchanged for a piece of the recipient's DNA.

• Involves 4 steps

1. A donor bacterium dies and is degraded2. A fragment of DNA from the dead donor

bacterium binds to DNA binding proteins onthe cell wall of a competent, living recipientbacterium

3. The Rec A protein promotes genetic exchangebetween a fragment of the donor's DNA and therecipient's DNA

4. Exchange is complete

Transformation: 4 steps

In transduction, the transferred DNA is carried in the capsid of a bacteriophage.The donor's DNA replaces part or all of the viral genome in the phage head. Thus, theparticle is probably defective in viral replication because essential viral genes aremissing. In the case of temperate phages (mild or normal) such as lambda, bacterialDNA becomes associated with the virus genome when the prophage (harmless geneticmaterial) is excised incorrectly from the bacterial chromosome. When this occurs, thesame set of bacterial genes is always incorporated into lambda phage. Thisphenomenon is specialized transduction, because it is only effective in transducing afew special bacterial genes. In contrast, generalized transduction can transfer anybacterial gene to the recipient. This process may occur with phages that degrade theirhost DNA into pieces the size of viral genomes.

So, there are two types of transduction:

generalized transduction: A DNA fragment is transferred from one bacterium toanother by a lytic bacteriophage that is now carrying donor bacterial DNA due to anerror in maturation during the lytic life cycle.

specialized transduction: A DNA fragment is transferred from one bacterium toanother by a temperate bacteriophage that is now carrying donor bacterial DNA dueto an error in spontaneous induction during the lysogenic life cycle.

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Some temperate phages cause phenotypic changes in the bacteria they infect even withouttransducing bacterial genes. In the lysogenic state, viral genes are expressed which confernew properties on the cell. Examples of this phage conversion are toxin production bypathogenic bacteria such as Corynebacterium diphtheriae and surface polysaccharide structurein Salmonella anatum.

Generalised Transduction: 7 steps

1. A lytic bacteriophage adsorbs toa susceptible bacterium.

2. The bacteriophage genome entersthe bacterium. The genomedirects the bacterium's metabolicmachinery to manufacturebacteriophagecomponents andenzymes

3. Occasionally, a bacteriophagehead or capsid assemblesaround a fragment of donorbacterium'snucleoid or arounda plasmid instead of a phagegenome by mistake.

Generalised Transduction

4. The bacteriophagesare released.

5. The bacteriophagecarrying the donorbacterium's DNAadsorbs to a recipientbacterium

G enera lised Tran sd uction

6 . T he bacter iophage insertsthe donor b acterium'sDN A it is carrying int othe rec ipient b acterium

7 . T he donor b acterium'sDN A is ex ch anged forsome of t he rec ip ient'sDN A

Specialised Transduction: 6 steps

1. A temperatebacteriophage adsorbsto a susceptiblebacterium and injectsits genome .

2. The bacteriophageinserts its genome intothe bacterium'snucleoid to become aprophage.

Specialised Transduction

3. Occasionally duringspontaneous induction, asmall piece of the donorbacterium's DNA ispicked up as part of thephage's genome in placeof some of the phageDNA which remains inthe bacterium's nucleoid.

4. As the bacteriophagereplicates, the segmentof bacterial DNAreplicates as part of thephage's genome. Everyphage now carries thatsegment of bacterialDNA.

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Conjugation, the third means of gene transfer is mediated by special genetic elementscalled plasmids. Plasmids are defined as small, circular DNA molecules that reproduceautonomously. While plasmids are DNA, they control their own replication separatelyfrom that of the chromosome.

The presence of plasmids in cells can be detected by techniques that separatethem from chromosomal DNA. This involves buoyant density differences due to the tightsupercoiling of these rather small DNA circles; the density difference can be enhancedby adding compounds that intercalate between DNA base pairs, such as ethidiumbromide. The tightly wound plasmid DNA cannot bind as much ethidium bromide as thechromosomal fragments. Adding ethidium bromide, or other treatments that affect DNA,to whole cells at the appropriate concentration may cure cells of their plasmids. Ifplasmid replication is more sensitive to these agents than chromosome replication,plasmids may not segregate to all progeny cells during cell division.

Some (but not all) have genes that can direct their transmission from one cell toanother by conjugation. Finally, plasmids may have genes that confer novel phenotypeson cells, such as resistance to antibiotics, production of toxins, or the capacity tometabolize unusual substrates such as pesticides or industrial solvents. Antibioticresistance is conferred by R plasmids. These plasmids have diminished theeffectiveness of antibiotics in combating infectious diseases because (i) they may conferresistance to as many as five different antibiotics at once upon the cell, and (ii) byconjugation, they can be rapidly disseminated through the bacterial population. Multipleantibiotic resistance is a consequence of their construction -- they contain severaltransposons, each of which confers resistance to a unique antibiotic. The genes in thetransposons generally specify an enzyme that inactivates the drug before it enters thecell and reaches its target. This differs from chromosomal mutations that result inantibiotic resistance. These generally are modifications of the antibiotic's target ofaction.

Specialised Transduction

5. The bacteriophageadsorbs to a recipientbacterium and injectsits genome.

6. The bacteriophagegenome carrying thedonor bacterial DNAinserts into therecipient bacterium'snucleoid.

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Transposon: A transposable element (TE) is a DNA sequence that can change itsposition within the genome, sometimes creating mutations and altering the cell'sgenome size. Transposition often results in duplication of the TE. They are generallyconsidered non-coding DNA. In Oxytricha, which has a unique genetic system, theyplay a critical role in development. They are also very useful to researchers as a meansto alter DNA inside a living organism.Transposable elements (TEs) represent one of several types of mobile geneticelements. TEs are assigned to one of two classes according to their mechanism oftransposition, which can be described as either copy and paste (class I TEs:retrotransposons) or cut and paste (class II TEs: DNA transposons).

Plasmids are autonomously replicating molecules. What elements are necessary tocontrol DNA replication? There must be an origin of replication, where the frequency ofreplication can be regulated. The number of plasmid copies is tightly regulated at a few copieswith some plasmids, whereas in others, initiation of replication is relatively uncontrolled, andtwenty to thirty plasmid copies may be present in a cell. In general, the enzymes used for DNAreplication are those coded by the chromosome -- it is the regulatory genes that are plasmidencoded.

Conjugative plasmids initiate gene transfer by altering the cell surface to allow contactbetween the plasmid-containing donor cell and a plasmid-less recipient. A plasmid gene codesfor the production of a sex pilus that initiates pair formation. Subsequently, a conjugationbridge is formed through which DNA is transferred. The transfer of plasmid DNA isaccompanied by its replication. That is, the donor cell does not lose its plasmid but transfers acopy to the recipient. In actual fact, replication is shared between donor and recipient. A singleDNA strand is transferred as a consequence of rolling circle replication in the donor; thisstrand is used as a template by the recipient to generate a double stranded DNA molecule.Therefore, the consequence of conjugation is that both the donor and the recipient cells containthe plasmid. The recipient is now competent to serve as a plasmid donor in other conjugations.

So, bacterial conjugation• Bacterial Conjugation is genetic recombination in which there is a transfer of DNA

from a living donor bacterium to a recipient bacterium. Often involves a sex pilus.• The 3 conjugative processes

I. F+ conjugationII. Hfr conjugationIII. Resistance plasmid conjugation

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I. F+ Conjugation ProcessF+ Conjugation- Genetic recombination in which there is a transfer of an F+ plasmid(coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium toa female recipient bacterium. Involves a sex (conjugation) pilus. Other plasmids presentin the cytoplasm of the bacterium, such as those coding for antibiotic resistance, mayalso be transferred during this process.

Some conjugative plasmids, such as the F factor in E. coli, can also direct transfer ofchromosomal genes by conjugation. E. coli strains which have this property are Hfr strains.The F factor can integrate into the chromosome to form one DNA molecule. This occurs atregions of homology between F and the chromosome. These regions are insertion sequenceslocated on both molecules. F factor can now transfer chromosomal genes during a conjugation,because in effect, the chromosome has become part of the F factor. It is the F factor that hasthe genetic information to drive gene transfer. Specifically, there is a nucleotide sequence on Fthat specifies the origin of transfer. The host chromosome was inserted just downstream fromthis region. DNA is transferred just as described above for plasmid transfer. It is important tonote that chromosomal genes are transferred before any of the plasmid genes. Thus, if thecytoplasmic bridge is broken before the entire chromosome is transferred, the recipient remains.

A high-frequency recombination cell (Hfr cell) (also called an Hfr strain) is abacterium with a conjugative plasmid (often the F-factor) integrated into its genomicDNA. The Hfr strain was first characterized by Luca Cavalli-Sforza. Unlike a normal F+

cell, hfr strains will, upon conjugation with a F− cell, attempt to transfer their entire DNAthrough the mating bridge, not to be confused with the pilus. This occurs because the Ffactor has integrated itself via an insertion point in the bacterial chromosome. Due to theF factor's inherent nature to transfer itself during conjugation, the rest of the bacterialgenome is dragged along with it, thus making such cells very useful and interesting interms of studying gene linkage and recombination. Because the genome's rate oftransfer through the mating bridge is constant, molecular biologists and geneticists canuse Hfr strain of bacteria (often E. coli) to study genetic linkage and map thechromosome. The procedure commonly used for this is called interrupted mating.

F+ Conjugation: 4 steps

1. The F+ male has an F+ plasmid codingfor a sex pilus and can serve as agenetic donor

2. The sex pilus adheres to an F- female(recipient). One strand of the F+plasmid breaks

3. The sex pilus retracts and a bridgeis created between the twobacteria. One strand of the F+plasmid enters the recipientbacterium

4. Both bacteria make a complementarystrand of the F+ plasmid and both arenow F+ males capable of producing asex pilus. There was no transfer ofdonor chromosomal DNA although otherplasmids the donor bacterium carriesmay also be transferred during F+conjugation.

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II. Hfr Conjugation

Genetic recombination in which fragments of chromosomal DNA from a male donorbacterium are transferred to a female recipient bacterium following insertion of an F+plasmid into the nucleoid of the donor bacterium. Involves a sex (conjugation) pilus.

The F plasmid is considered to be an episome which may become integrated into themain chromosome. When the F genes become integrated into the chromosome, the cellis said to be Hfr (high frequency of recombination). An Hfr cell may transfer F genes toan F− cell. During this transfer of genetic material, the F episome may takechromosomal DNA with it. The donor cell does not lose any genetic material as anythingtransferred is replicated concurrently. It is extremely rare that an Hfr cell's chromosomeis transferred in its entirety. Homologous recombination occurs when the newly acquiredDNA crosses over with the homologous region of its own chromosome.

Hfr Conjugation: 5 steps

1. An F+ plasmid inserts into thedonor bacterium's nucleoid toform an Hfr male.

2. The sex pilus adheres to an F-female (recipient). One donor DNAstrand breaks in the middle of theinserted F+ plasmid.

3. The sex pilus retracts and abridge forms between the twobacter ia. One donor DNA strandbegins to enter the recipientbacter ium. The two cells breakapa rt easily so the only a portionof the donor's DNA strand isusually transferred to therecipient bacterium.

4. The donor bacterium makes acom plementary copy of theremaining DNA strand and remainsan Hfr male. The recipientbacte rium makes a complementarystrand of the transferred donorDNA.

5. The donor DNA fragmentundergoes genetic exchange withthe recipient bacterium's DNA.Since there was transfer of somedonor chromosomal DNA butusually not a complete F+plasmid, the recipient bacteriumusually remains F-

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Fig. There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the topinstance, whereas episomes, the lower example, integrate into the host chromosome.

Transposons and insertion sequences are genetic elements capable of movingwithin cells. Transposons differ from insertion sequences in that they contain additionalgenes, such as ones for antibiotic resistance. The frequency with which these elementsmove is rather low, but 10 to 100 fold greater than the frequency of spontaneousmutation. The ends of these elements contain repeated sequences. In addition, theycode for a transposase enzyme that can insert the elements at any point into a DNAmolecule.When these transposable elements insert into a DNA target sequence, that targetsequence is duplicated. In addition, elements that undergo replicative transposition alsoare duplicated. That is, a copy remains at the original site, and the other copy is insertedat a new site. The transposase makes single strand cuts in the inverted repeatsequences at the ends of the transposable element, and at the target site. The elementis joined to the target via the single strand ends, and the gaps are filled in by DNAreplication. Finally, the cointegrate formed by recombination is resolved to generate acopy of the transposable element at the new site. In other transposons (such as Tn5),transposition is conservative, and the transposon is excised from its original location,and is reinserted at a new site. If the site of transposon insertion is within an existingbacterial gene, it is likely to be inactivated, and a mutation has occurred.

III. Resistant Plasmid ConjugationGenetic recombination in which there is a transfer of an R plasmid (a plasmid coding formultiple antibiotic resistance and often a sex pilus) from a male donor bacterium to afemale recipient bacterium. Involves a sex (conjugation) pilus

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Genetic Engineering

Genetic engineering, also called genetic modification, is the direct manipulationof an organism's genome using biotechnology. New DNA may be inserted in the hostgenome by first isolating and copying the genetic material of interest using molecularcloning methods to generate a DNA sequence, or by synthesizing the DNA, and theninserting this construct into the host organism. Genes may be removed, or "knockedout", using a nuclease. Gene targeting is a different technique that uses homologousrecombination to change an endogenous gene, and can be used to delete a gene,remove exons, add a gene, or introduce point mutations.

An organism that is generated through genetic engineering is considered to be agenetically modified organism (GMO). The first GMOs were bacteria in 1973; GM micewere generated in 1974. Insulin-producing bacteria were commercialized in 1982 andgenetically modified food has been sold since 1994.

Fig. Comparison of conventional plant breeding with transgenic and cisgenic genetic modification

Resistant Plasmid Conjugation: 4 steps

1. The bacterium with an R-plasmid is multiple antibioticresistant and can produce a sexpilus (serve as a genetic donor).

2. The sex pilus adheres to an F-female (recipient). One strand ofthe R-plasmid breaks.

3. The sex pilus retracts and abridge is created between thetwo bacteria. One strand ofthe R-plasmid enters therecipient bacterium.

4. Both bacteria make acomplementary strand of the R-plasmid and both are now multipleantibiotic resistant and capable ofproducing a sex pilus.

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If genetic material from another species is added to the host, the resulting organism iscalled transgenic. If genetic material from the same species or a species that cannaturally breed with the host is used the resulting organism is called cisgenic. Geneticengineering can also be used to remove genetic material from the target organism,creating a gene knockout organism. In Europe genetic modification is synonymous withgenetic engineering while within the United States of America it can also refer toconventional breeding methods. The Canadian regulatory system is based on whether aproduct has novel features regardless of method of origin. In other words, a product isregulated as genetically modified if it carries some trait not previously found in thespecies whether it was generated using traditional breeding methods (e.g., selectivebreeding, cell fusion, mutation breeding) or genetic engineering. Within the scientificcommunity, the term genetic engineering is not commonly used; more specific termssuch as transgenic are preferred.

Technique

Genetic engineering, also known as recombinant DNA technology, means altering thegenes in a living organism to produce a Genetically Modified Organism (GMO) with anew genotype. Various kinds of genetic modification are possible: inserting a foreigngene from one species into another, forming a transgenic organism; altering an existinggene so that its product is changed; or changing gene expression so that it is translatedmore often or not at all.

Techniques of Genetic EngineeringGenetic engineering is a very young discipline, and is only possible due to thedevelopment of techniques from the 1960s onwards. Watson and Crick have madethese techniques possible from our greater understanding of DNA and how it functionsfollowing the discovery of its structure in 1953. Although the final goal of geneticengineering is usually the expression of a gene in a host, in fact most of the techniquesand time in genetic engineering are spent isolating a gene and then cloning it. This tablelists the techniques that we shall look at in detail.Technique Purpose1 cDNA To make a DNA copy of mRNA2 Restriction Enzymes To cut DNA at specific points, making small fragments3 DNA Ligase To join DNA fragments together4 Vectors To carry DNA into cells and ensure replication5 Plasmids Common kind of vector6 Gene Transfer To deliver a gene to a living cells7 Genetic Markers To identify cells that have been transformed8 Replica Plating * To make exact copies of bacterial colonies on an agar plate9 PCR To amplify very small samples of DNA10 DNA probes To identify and label a piece of DNA containing a certain sequence11 Shotgun * To find a particular gene in a whole genome12 Antisense genes * To stop the expression of a gene in a cell13 Gene Synthesis To make a gene from scratch14 Electrophoresis To separate fragments of DNA* Additional information that is not directly included in AS Biology. However it can help to consolidate other techniques.

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1. Complementary DNAComplementary DNA (cDNA) is DNA made from mRNA. This makes use of the enzymereverse transcriptase, which does the reverse of transcription: it synthesises DNA froman RNA template. It is produced naturally by a group of viruses called the retroviruses(which include HIV), and it helps them to invade cells. In genetic engineering reversetranscriptase is used to make an artificial gene of cDNA as shown in this diagram.

Complementary DNA has helped to solve different problems in genetic engineering:It makes genes much easier to find. There are some 70,000 genes in the humangenome, and finding one gene out of this many is a very difficult (though not impossible)task. However a given cell only expresses a few genes, so only makes a few differentkinds of mRNA molecule. For example the b cells of the pancreas make insulin, somake lots of mRNA molecules coding for insulin. This mRNA can be isolated from thesecells and used to make cDNA of the insulin gene.

2. Restriction Enzymes

These are enzymes that cut DNA at specific sites. They are properly called restrictionendonucleases because they cut the bonds in the middle of the polynucleotide chain.Some restriction enzymes cut straight across both chains, forming blunt ends, but mostenzymes make a staggered cut in the two strands, forming sticky ends.

The cut ends are “sticky” because they have short stretches of single-stranded DNAwith complementary sequences. These sticky ends will stick (or anneal) to anotherpiece of DNA by complementary base pairing, but only if they have both been cut withthe same restriction enzyme. Restriction enzymes are highly specific, and will only cutDNA at specific base sequences, 4-8 base pairs long, called recognition sequences.

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Restriction enzymes are produced naturally by bacteria as a defense against viruses(they “restrict” viral growth), but they are enormously useful in genetic engineering forcutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out byrestriction enzymes are called restriction fragments. There are thousands of differentrestriction enzymes known, with over a hundred different recognition sequences.Restriction enzymes are named after the bacteria species they came from, so EcoR1 isfrom E. coli strain R, and HindIII is from Haemophilis influenzae.

3. DNA Ligase

This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It iscommonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. tojoin together complementary restriction fragments.The sticky ends allow two complementary restriction fragments to anneal, but only byweak hydrogen bonds, which can quite easily be broken, say by gentle heating. Thebackbone is still incomplete.DNA ligase completes the DNA backbone by forming covalent bonds. Restrictionenzymes and DNA ligase can therefore be used together to join lengths of DNA fromdifferent sources.

4. Vectors

In biology a vector is something that carries things between species. For example themosquito is a disease vector because it carries the malaria parasite into humans. Ingenetic engineering a vector is a length of DNA that carries the gene we want into ahost cell. A vector is needed because a length of DNA containing a gene on its ownwon’t actually do anything inside a host cell. Since it is not part of the cell’s normalgenome it won’t be replicated when the cell divides, it won’t be expressed, and in fact itwill probably be broken down pretty quickly. A vector gets round these problems byhaving these properties:

It is big enough to hold the gene we want (plus a few others), but not too big. It is circular (or more accurately a closed loop), so that it is less likely to be

broken down (particularly in prokaryotic cells where DNA is always circular).

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It contains control sequences, such as a replication origin and a transcriptionpromoter, so that the gene will be replicated, expressed, or incorporated into thecell’s normal genome.

It contain marker genes, so that cells containing the vector can be identified.Many different vectors have been made for different purposes in genetic engineering bymodifying naturally-occurring DNA molecules, and these are now available off the shelf.For example a cloning vector contains sequences that cause the gene to be copied(perhaps many times) inside a cell, but not expressed. An expression vector containssequences causing the gene to be expressed inside a cell, preferably in response to anexternal stimulus, such as a particular chemical in the medium. Different kinds of vectorare also available for different lengths of DNA insert:

Type of vector Max length of DNA insertPlasmid 10 kbpVirus or phage 30 kbpBacterial Artificial Chromosome(BAC)

500 kbp

5. Plasmids

Plasmids are by far the most common kind of vector, so we shall look at how they areused in some detail. Plasmids are short circular bits of DNA found naturally in bacterialcells. A typical plasmid contains 3-5 genes and there are usually around 10 copies of aplasmid in a bacterial cell. Plasmids are copied separately from the main bacterial DNAwhen the cell divides, so the plasmid genes are passed on to all daughter cells. Theyare also used naturally for exchange of genes between bacterial cells (the nearest theyget to sex), so bacterial cells will readily take up a plasmid. Because they are so small,they are easy to handle in a test tube, and foreign genes can quite easily beincorporated into them using restriction enzymes and DNA ligase.

One of the most common plasmids used is the R-plasmid (or pBR322). This plasmidcontains a replication origin, several recognition sequences for different restrictionenzymes (with names like PstI and EcoRI), and two marker genes, which conferresistance to different antibiotics (ampicillin and tetracycline).

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The diagram below shows how DNA fragments can be incorporated into a plasmidusing restriction and ligase enzymes. The restriction enzyme used here (PstI) cuts theplasmid in the middle of one of the marker genes (we’ll see why this is useful later). Theforeign DNA anneals with the plasmid and is joined covalently by DNA ligase to form ahybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA). Severalother products are also formed: some plasmids will simply re-anneal with themselves tore-form the original plasmid, and some DNA fragments will join together to form chainsor circles. These different products cannot easily be separated, but it doesn’t matter, asthe marker genes can be used later to identify the correct hybrid vector.

6. Gene Transfer

Vectors containing the genes we want must be incorporated into living cells so that theycan be replicated or expressed. The cells receiving the vector are called host cells, andonce they have successfully incorporated the vector they are said to be transformed.Vectors are large molecules which do not readily cross cell membranes, so themembranes must be made permeable in some way. There are different ways of doingthis depending on the type of host cell. Heat Shock. Cells are incubated with the vector in a solution containing calcium ions

at 0°C. The temperature is then suddenly raised to about 40°C. This heat shockcauses some of the cells to take up the vector, though no one knows why. This workswell for bacterial and animal cells.

Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disruptsthe membrane and allows the vector to enter the cell. This is the most efficient methodof delivering genes to bacterial cells.

Viruses. The vector is first incorporated into a virus, which is then used to infect cells,carrying the foreign gene along with its own genetic material. Since viruses rely ongetting their DNA into host cells for their survival they have evolved many successfulmethods, and so are an obvious choice for gene delivery. The virus must first begenetically engineered to make it safe, so that it can’t reproduce itself or make toxins.

Three viruses are commonly used:

1. Bacteriophages (or phages) are viruses that infect bacteria. They are a veryeffective way of delivering large genes into bacteria cells in culture.

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2. Adenoviruses are human viruses that causes respiratory diseases including thecommon cold. Their genetic material is double-stranded DNA, and they are idealfor delivering genes to living patients in gene therapy. Their DNA is notincorporated into the host’s chromosomes, so it is not replicated, but their genesare expressed.

The adenovirus is genetically altered so that its coat proteins are not synthesised, sonew virus particles cannot be assembled and the host cell is not killed.

3. Retroviruses are a group of human viruses that include HIV. They are enclosed ina lipid membrane and their genetic material is double-stranded RNA. On infectionthis RNA is copied to DNA and the DNA is incorporated into the host’schromosome. This means that the foreign genes are replicated into every daughtercell.

After a certain time, the dormant DNA is switched on, and the genes are expressedin all the host cells. Plant Tumours. This method has been used successfully to transform plant cells,

which are perhaps the hardest to do. The gene is first inserted into the Ti plasmidof the soil bacterium Agrobacterium tumefaciens, and then plants are infectedwith the bacterium. The bacterium inserts the Ti plasmid into the plant cells'chromosomal DNA and causes a "crown gall" tumour. These tumour cells can becultured in the laboratory and whole new plants grown from them bymicropropagation. Every cell of these plants contains the foreign gene.

Gene Gun. This extraordinary technique fires microscopic gold particles coatedwith the foreign DNA at the cells using a compressed air gun. It is designed toovercome the problem of the strong cell wall in plant tissue, since the particlescan penetrate the cell wall and the cell and nuclear membranes, and deliver theDNA to the nucleus, where it is sometimes expressed.

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Micro-Injection. A cell is held on a pipette under a microscope and the foreignDNA is injected directly into the nucleus using an incredibly fine micro-pipette.This method is used where there are only a very few cells available, such asfertilised animal egg cells. In the rare successful cases the fertilised egg isimplanted into the uterus of a surrogate mother and it will develop into a normalanimal, with the DNA incorporated into the chromosomes of every cell.

Liposomes. Vectors can be encased in liposomes, which are small membranevesicles (see module 1). The liposomes fuse with the cell membrane (andsometimes the nuclear membrane too), delivering the DNA into the cell. Thisworks for many types of cell, but is particularly useful for delivering genes to cellin vivo (such as in gene therapy).

7. Genetic Markers

These are needed to identify cells that have successfully taken up a vector and sobecome transformed. With most of the techniques above less than 1% of the cellsactually take up the vector, so a marker is needed to distinguish these cells from all theothers. We’ll look at how to do this with bacterial host cells, as that’s the most commontechnique.

A common marker, used in the R-plasmid, is a gene for resistance to anantibiotic such as tetracycline. Bacterial cells taking up this plasmid can make this geneproduct and so are resistant to this antibiotic. So if the cells are grown on a mediumcontaining tetracycline all the normal untransformed cells, together with cells that havetaken up DNA that’s not in a plasmid (99%) will die. Only the 1% transformed cells willsurvive, and these can then be grown and cloned on another plate.

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8. Replica Plating

Replica plating is a simple technique for making an exact copy of an agar plate. A padof sterile cloth the same size as the plate is pressed on the surface of an agar plate withbacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth isthen pressed onto a new agar plate, some cells will be deposited and colonies will growin exactly the same positions on the new plate. This technique has a number of uses,but the most common use in genetic engineering is to help solve another problem inidentifying transformed cells.

This problem is to distinguish those cells that have taken up a hybrid plasmidvector (with a foreign gene in it) from those cells that have taken up the normal plasmid.This is where the second marker gene (for resistance to ampicillin) is used. If the foreigngene is inserted into the middle of this marker gene, the marker gene is disrupted andwon't make its proper gene product. So cells with the hybrid plasmid will be killed byampicillin, while cells with the normal plasmid will be immune to ampicillin. Since thismethod of identification involves killing the cells we want, we must first make a masteragar plate and then make a replica plate of this to test for ampicillin resistance.

Once the colonies of cells containing the correct hybrid plasmid vector have beenidentified, the appropriate colonies on the master plate can be selected and grown onanother plate.

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The R-plasmid with its antibiotic-resistance genes dates from the early days of geneticengineering in the 1970s. In recent years better plasmids with different marker geneshave been developed that do not kill the desired cells, and so do not need a replicaplate. These new marker genes make an enzyme (actually lactase) that converts acolourless substrate in the agar medium into a blue-coloured product that can easily beseen. So cells with a normal plasmid turn blue on the correct medium, while those withthe hybrid plasmid can't make the enzyme and stay white. These white colonies caneasily be identified and transferred to another plate. Another marker gene, transferredfrom jellyfish, makes a green fluorescent protein (GFP).

9. Polymerase Chain Reaction (PCR)Genes can be cloned by cloning the bacterial cells that contain them, but this requiresquite a lot of DNA in the first place. PCR can clone (or amplify) DNA samples as smallas a single molecule. It is a newer technique, having been developed in 1983 by KaryMullis, for which discovery he won the Nobel prize in 1993. The polymerase chainreaction is simply DNA replication in a test tube. If a length of DNA is mixed with thefour nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, thenthe DNA will be replicated many times. The details are shown in this diagram:

1. Start with a sample of the DNA to be amplified, and add the four nucleotides and theenzyme DNA polymerase.

2. Normally (in vivo) the DNA double helix would be separated by the enzyme helicase,but in PCR (in vitro) the strands are separated by heating to 95°C for two minutes.This breaks the hydrogen bonds.

3. DNA polymerisation always requires short lengths of DNA (about 20 bp long) calledprimers, to get it started. In vivo the primers are made during replication by DNApolymerase, but in vitro they must be synthesised separately and added at this stage.

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This means that a short length of the sequence of the DNA must already be known,but it does have the advantage that only the part between the primer sequences isreplicated. The DNA must be cooled to 40°C to allow the primers to anneal to theircomplementary sequences on the separated DNA strands.

4. The DNA polymerase enzyme can now extend the primers and complete thereplication of the rest of the DNA. The enzyme used in PCR is derived from thethermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at atemperature of 90°C, so it is not denatured by the high temperatures in step 2. Itsoptimum temperature is about 72°C, so the mixture is heated to this temperature for afew minutes to allow replication to take place as quickly as possible.

5. Each original DNA molecule has now been replicated to form two molecules. Thecycle is repeated from step 2 and each time the number of DNA molecules doubles.This is why it is called a chain reaction, since the number of molecules increasesexponentially, like an explosive chain reaction. Typically PCR is run for 20-30 cycles.

PCR can be completely automated, so in a few hours a tiny sample of DNA can beamplified millions of times with little effort. The product can be used for further studies,such as cloning, electrophoresis, or gene probes. Because PCR can use such smallsamples it can be used in forensic medicine (with DNA taken from samples of blood,hair or semen), and can even be used to copy DNA from mummified human bodies,extinct woolly mammoths, or from an insect that's been encased in amber since theJurassic period. One problem of PCR is having a pure enough sample of DNA to startwith. Any contaminant DNA will also be amplified, and this can cause problems, forexample in court cases.

10. DNA Probes

These are used to identify and label DNA fragments that contain a specific sequence. Aprobe is simply a short length of DNA (20-100 nucleotides long) with a label attached.There are two common types of label used: A radioactively-labelled probe (synthesised using the isotope 32P) can be visualised

using photographic film (an autoradiograph). A fluorescently-labelled probe will emit visible light when illuminated with invisible

ultraviolet light. Probes can be made to fluoresce with different colours.Probes are always single-stranded, and can be made of DNA or RNA. If a probe isadded to a mixture of different pieces of DNA (e.g. restriction fragments) it will anneal(base pair) with any lengths of DNA containing the complementary sequence. These

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fragments will now be labelled and will stand out from the rest of the DNA. DNA probeshave many uses in genetic engineering: To identify restriction fragments containing a particular gene out of the thousands of

restriction fragments formed from a genomic library. This use is described inshotguning below.

To identify the short DNA sequences used in DNA fingerprinting. To identify genes from one species that are similar to those of another species. Most

genes are remarkably similar in sequence from one species to another, so forexample a gene probe for a mouse gene will probably anneal with the same genefrom a human. This has aided the identification of human genes.

To identify genetic defects. DNA probes have been prepared that match thesequences of many human genetic disease genes such as muscular dystrophy, andcystic fibrosis. Hundreds of these probes can be stuck to a glass slide in a gridpattern, forming a DNA microarray (or DNA chip). A sample of human DNA is addedto the array and any sequences that match any of the various probes will stick to thearray and be labelled. This allows rapid testing for a large number of genetic defects ata time.

11. Shotguning

This is used to find one particular gene in a whole genome, a bit like finding theproverbial needle in a haystack. It is called the shotgun technique because it starts byindiscriminately breaking up the genome (like firing a shotgun at a soft target) and thensorting through the debris for the particular gene we want. For this to work a gene probefor the gene is needed, which means at least a short part of the gene’s sequence mustbe known.

12. Antisense Genes

These are used to turn off the expression of a gene in a cell. The principle is verysimple: a copy of the gene to be switch off is inserted into the host genome the “wrong”way round, so that the complementary (or antisense) strand is transcribed. Theantisense mRNA produced will anneal to the normal sense mRNA forming double-stranded RNA. Ribosomes can’t bind to this, so the mRNA is not translated, and thegene is effectively “switched off”.

13. Gene Synthesis

It is possible to chemically synthesise a gene in the lab by laboriously joiningnucleotides together in the correct order. Automated machines can now make thismuch easier, but only up to a limit of about 30bp, so very few real genes could be madethis way (anyway it’s usually much easier to make cDNA). The genes for the two insulinchains (xx bp) and for the hormone somatostatin (42 bp) have been synthesisied thisway. It is very useful for making gene probes.

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14.Electrophoresis

This is a form of chromatography used to separate different pieces of DNA on the basisof their length. It might typically be used to separate restriction fragments. The DNAsamples are placed into wells at one end of a thin slab of gel made of agarose orpolyacrylamide, and covered in a buffer solution. An electric current is passed throughthe gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphategroup, so DNA is attracted to the anode (the positive electrode). The molecules have todiffuse through the gel, and smaller lengths of DNA move faster than larger lengths,which are retarded by the gel. So the smaller the length of the DNA molecule, thefurther down the gel it will move in a given time. At the end of the run the current isturned off.

Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There arethree common methods for doing this: The gel can be stained with a chemical that specifically stains DNA, such as ethidium

bromide or azure A. The DNA shows up as blue bands. This method is simple but notvery sensitive.

The DNA samples at the beginning can be radiolabelled with a radioactive isotopesuch as 32P. Photographic film is placed on top of the finished gel in the dark, and theDNA shows up as dark bands on the film. This method is extremely sensitive.

The DNA fragments at the beginning can be labelled with a fluorescent molecule. TheDNA fragments show up as coloured lights when the finished gel is illuminated withinvisible ultraviolet light.

Gene Therapy

This is perhaps the most significant, and most controversial kind of genetic engineering.It is also the least well-developed. The idea of gene therapy is to genetically alterhumans in order to treat a disease viz., Cystic Fibrosis (CF), Severe CombinedImmunodefficiency Disease (SCID), etc. This could represent the first opportunity tocure incurable diseases. Note that this is quite different from using genetically-engineered microbes to produce a drug, vaccine or hormone to treat a disease byconventional means. Gene therapy means altering the genotype of a tissue or even awhole human.

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