Lecture 20 biotechnology

Chapter 20

Biotechnology

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Overview: The DNA Toolbox

• Sequencing of the human genome was completed by 2007

• DNA sequencing has depended on advances in technology, starting with making recombinant DNA

• In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule


• Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

• DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

• To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning


DNA Cloning and Its Applications: A Preview

• Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids

• Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome

• Cloned genes are useful for making copies of a particular gene and producing a protein product


• Gene cloning involves using bacteria to make multiple copies of a gene

• Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell

• Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA

• This results in the production of multiple copies of a single gene

Fig. 20-2a

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Plasmid

2

1

2

Fig. 20-2b

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

4

Recombinantbacterium

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

3


Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments

• DNA ligase is an enzyme that seals the bonds between restriction fragments


DNA technology allows us to study the sequence, expression, and function of a gene

• DNA cloning allows researchers to

– Compare genes and alleles between individuals

– Locate gene expression in a body

– Determine the role of a gene in an organism

• Several techniques are used to analyze the DNA of genes


Gel Electrophoresis and Southern Blotting

• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis

• This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size

• A current is applied that causes charged molecules to move through the gel

• Molecules are sorted into “bands” by their size

Fig. 20-9

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

RESULTS

1

2

+

+

–

–


• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

• The procedure is also used to prepare pure samples of individual fragments

Fig. 20-10

Normalallele

Sickle-cellallele

Largefragment

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene

Normal -globin allele

Sickle-cell mutant -globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp175 bp

DdeIDdeI

DdeI DdeI DdeI DdeI


Cloning Plants: Single-Cell Cultures

• Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

• One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism

• A totipotent cell is one that can generate a complete new organism


Cloning Animals: Nuclear Transplantation

• In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell

• Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg

• However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

Fig. 20-17

EXPERIMENT

Less differ-entiated cell

RESULTS

Frog embryo Frog egg cell

UV

Donornucleustrans-planted

Frog tadpole

Enucleated egg cell

Egg with donor nucleus activated to begin

development

Fully differ-entiated(intestinal) cell

Donor nucleus trans-planted

Most developinto tadpoles

Most stop developingbefore tadpole stage


Reproductive Cloning of Mammals

• In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell

• Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus

Fig. 20-18

TECHNIQUE

Mammarycell donor

RESULTS

Surrogatemother

Nucleus frommammary cell

Culturedmammary cells

Implantedin uterusof a thirdsheep

Early embryo

Nucleusremoved

Egg celldonor

Embryonicdevelopment Lamb (“Dolly”)

genetically identical tomammary cell donor

Egg cellfrom ovary

Cells fused

Grown inculture

1

33

4

5

6

2


• Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs

• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth

• CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”


Stem Cells of Animals

• A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types

• Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types

• The adult body also has stem cells, which replace nonreproducing specialized cells

• The aim of stem cell research is to supply cells for the repair of damaged or diseased organs

Fig. 20-20

Culturedstem cells

Early human embryoat blastocyst stage

(mammalian equiva-lent of blastula)

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Blood cellsNerve cellsLiver cells

Cells generatingall embryoniccell types

Adult stem cells

Cells generatingsome cell types

Embryonic stem cells

From bone marrowin this example


Medical Applications

• One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

• Genetic disorders can also be tested for using genetic markers that are linked to the disease-causing allele


Human Gene Therapy

• Gene therapy is the alteration of an afflicted individual’s genes

• Gene therapy holds great potential for treating disorders traceable to a single defective gene

• Vectors are used for delivery of genes into specific types of cells, for example bone marrow

• Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

Fig. 20-22

Bonemarrow

Clonedgene

Bonemarrowcell frompatient

Insert RNA version of normal alleleinto retrovirus.

Retroviruscapsid

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Inject engineeredcells into patient.

1

2

3

4


Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids

• Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains

• The Innocence Project

Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.

These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.

(a)

Semen on victim

Earl Washington

Source of sample

Kenneth Tinsley

STRmarker 1

STRmarker 2

STRmarker 3

(b)

17, 19

16, 18

17, 19

13, 16 12, 12

14, 15 11, 12

13, 16 12, 12


Environmental Cleanup

• Genetic engineering can be used to modify the metabolism of microorganisms

• Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

• Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels


Genetic Engineering in Plants

• Agricultural scientists have endowed a number of crop plants with genes for desirable traits

• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

• Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops

Fig. 20-25

Site whererestrictionenzyme cuts

T DNA

Plant with new trait

Tiplasmid

Agrobacterium tumefaciens

DNA withthe geneof interest

RecombinantTi plasmid

TECHNIQUE

RESULTS


• Most public concern about possible hazards centers on genetically modified (GM) organisms used as food

• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives


Genetic Engineering of MicroorganismsMany medicines & useful chemicals are now produced by genetically engineered bacteria. For instance, all insulin is

synthesized by transgenic E. coli that have the genes to produce human insulin. This has greatly reduced the cost and made insulinavailable to all diabetics who need it. Before the 1980s, all insulin was derived from the pancreases of animals (insulin from pigs differs from human insulin by only oneamino acid).


The leading artificial sweetener, aspartame, is made from two amino acids, aspartic acid and phenylalanine. Both amino acids are synthesized by bacteria, and the strain that produces the phenylalanine has been genetically engineered.

Genetic Engineering of Microorganisms


Genetic engineering of rice to contain vitamin A & increased levels of iron and zinc. “Golden rice”

How important is this?

Fig. 38.18

Vitamin A deficiency - 1 million childrenunder 5 die per year; many go blind.

Genetic engineering of corn toreduce the level of omega-6fatty acids.

http://www.nysaes.cornell.edu/comm/gmo/corn.jpg

Modifications to improve the nutritional value of food.

Iron deficiency - 2 billion affected; 100,000maternal deaths/yr in Africa & Asia.Severe mental impairment in women & children.


Some crop plants havebeen genetically engineered to extract limiting nutrients more efficiently from the soil.Other plants have beenengineered to increase thebioavailability of theiressential nutrients fordigestion and uptake.


Genetic engineering of crop plants to increase the quantity & quality of food that is harvested and reaches the consumer.

•Shift in emphasis from vegetative body to seed/fruit.

http://www.lsuagcenter.com/en/crops_livestock/crops/WheatOats/Diseases/Wheat+Diseases.htm

The Green Revolution was drivenin part by the introduction of genesfor dwarfism into cereal grainslike wheat. The same genesare now engineered intoother crops such as basmatirice.


- Salty soils (77 million hectares). Plants also remove the salt from the soil!

http://www.environment.gov.au/soe/2001/publications/theme-reports/water/water02-1c.html

•Decreased losses due to harsh

environmental conditions:


- Extreme heat or cold

http://cropwatch.unl.edu/photos/cwphoto/crop07-6wheat-3.jpg

http://web1.msue.msu.edu/vanburen/frstapp.htm

http://boxer.senate.gov/news/photos/events/2007/01/bigfreeze/16.html

•Decreased losses due to harsh environmental conditions:


Luciferaseengineeredin tobacco

There is interest in engineering plants to signal when they are water stressed or require more nutrients through the use of reporter genes (luciferase or one of the many fluorescent proteins that can be engineered toupregulate during a particular stress).

Luciferasein tobacco

Fig. 17.6


-Insects (Bt corn & Bt cotton).Environment cleaner, safer for farm workers,beneficial organisms spared,& decreased fossil fuel use.

http://www.agbioforum.org/v8n23/v8n23a12-f01.gif

•Decreased losses due to pests & pathogens:

http://www.whybiotech.com/html/images/cotton_ad.jpg

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

http://hgic.clemson.edu/factsheets/Graphics/cornins/borer.htm


Fungi (powdery mildews, potato blight, ergot, etc.)

http://www.potatomuseum.com/images/exblightfieldwithinsert.jpg

Fig. 31.25

- Pathogenic microorganisms

•Decreased losses due topests & pathogens:


•Increased shelf life for fruits and vegetables.

http://www.ces.ncsu.edu/depts/cs/biotech/pics/c2h.jpg

Normal

Flavor saver


Other genetic improvements to the foods and beveragesthat humans and animals consume.

To decrease the allergenicity of food. A transgenicsoybean was recently engineered that does not producea protein to which some individuals are allergic.Peanuts are currently a major target.

Decaffeinated coffee - “naturally”. A Japanese grouprecently engineered coffee that does not synthesize caffeine.

Several forage crops have been engineered to havereduced lignin content so that they are more easilydigested (by sheep, cows, etc.). Efforts are also underway to engineer grains to release more carbo-hydrates, or more essential

nutrients, for animal feed.

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummingshttp://arabidopsis.info/students/dom/mainpage.html

A number of species have been genetically engineered to more effectively clean uppollution or to detoxify specific toxins. One example is the transfer of genes from bacteria into plants, resulting in plants that can take up highly toxic methylmercury and convert it to elemental mercury.

Phytoremediation

Lecture 20 biotechnology

Technology

Transcript of Lecture 20 biotechnology