Frontiers of Frontiers of GeneticsGenetics

Chapter 13Chapter 13

• Bacteria is a very important organism used in DNA technology

• Specifically Escherichia coli

• Bacteria can easily exchange genes

How can bacteria exchange genes?

Through tunnel like connectionsViruses carry bacterial genesBacteria take up DNA from surrounding

environment

I. Biologists have learned to manipulate I. Biologists have learned to manipulate DNADNA

A. The Beginnings of DNA A. The Beginnings of DNA TechnologyTechnology

1. Biotechnology- the use of 1. Biotechnology- the use of organisms to perform practical organisms to perform practical tasks for humanstasks for humans

2. Recombinant DNA 2. Recombinant DNA technology- combines genes technology- combines genes from different sources into a from different sources into a single DNA moleculesingle DNA molecule

II. Biologists can engineer bacteria to make II. Biologists can engineer bacteria to make useful productsuseful products

A. Engineering Bacteria: An IntroductionA. Engineering Bacteria: An Introduction

1. Plasmid- a small, circular DNA 1. Plasmid- a small, circular DNA molecule separate from the much molecule separate from the much larger bacterial chromosomelarger bacterial chromosome

a) May carry a number of a) May carry a number of genes and can make copies of genes and can make copies of itself.itself.

b)Some Bacteria are used to mass produce specific desirable genes and proteins

c) When a plasmid replicates, one c) When a plasmid replicates, one copy can pass from one bacterial copy can pass from one bacterial cell to another, resulting in gene cell to another, resulting in gene “sharing” among bacteria.“sharing” among bacteria.

2. Biologists use plasmids to move 2. Biologists use plasmids to move pieces of DNA into bacteria.pieces of DNA into bacteria.

a) First, a plasmid is removed a) First, a plasmid is removed from a bacterial cell and the desired from a bacterial cell and the desired gene is inserted into the plasmid.gene is inserted into the plasmid.

b) The plasmid is now a combination of its b) The plasmid is now a combination of its original DNA and the new DNA - it is called original DNA and the new DNA - it is called recombinant DNA.recombinant DNA.

c) Then, the recombinant DNA is put back into c) Then, the recombinant DNA is put back into a bacterial cell, where it can replicate many a bacterial cell, where it can replicate many times as the cell reproduces, making many times as the cell reproduces, making many copies of the desired gene. This is called copies of the desired gene. This is called gene cloning.gene cloning.

Figure 13-4Figure 13-4Plasmids can serve as carriers of genetic information. This Plasmids can serve as carriers of genetic information. This diagram shows the basic technique for creating a genetically diagram shows the basic technique for creating a genetically engineered bacterial cell.engineered bacterial cell.

B. “Cutting and Pasting” DNAB. “Cutting and Pasting” DNA

1. First, a piece of DNA containing the 1. First, a piece of DNA containing the desired gene must be “cut” desired gene must be “cut” out of a out of a

much longer DNA molecule.much longer DNA molecule.

a) restriction enzyme- an enzyme that a) restriction enzyme- an enzyme that chops up foreign DNA into small pieces chops up foreign DNA into small pieces at specific spots in the DNA sequenceat specific spots in the DNA sequence

2. Most restriction enzymes make staggered 2. Most restriction enzymes make staggered cuts. These staggered cuts leave single-cuts. These staggered cuts leave single-stranded DNA hanging off the ends of the stranded DNA hanging off the ends of the fragments. This is called a “sticky end” fragments. This is called a “sticky end” because it is available to stick to any because it is available to stick to any sequence that is complementary to it.sequence that is complementary to it.

a) DNA ligase (another enzyme) is used a) DNA ligase (another enzyme) is used to join the sticky ends together.to join the sticky ends together.

Restriction Restriction enzymes cut DNA enzymes cut DNA molecules at molecules at specific locations. specific locations. Splicing together Splicing together fragments of DNA fragments of DNA from two different from two different sources produces sources produces a recombinant a recombinant DNA molecule.DNA molecule.

C. Cloning Recombinant DNAC. Cloning Recombinant DNA

1. Libraries of Cloned Genes1. Libraries of Cloned Genes

a) Genomic Library- the a) Genomic Library- the complete collection of cloned complete collection of cloned DNA fragments from an DNA fragments from an organismorganism

2. Identifying Specific Genes with Probes2. Identifying Specific Genes with Probesa) One method requires knowing at least a) One method requires knowing at least part of the gene’s nucleotide sequence.part of the gene’s nucleotide sequence.

1) Knowing this, a biologist can use 1) Knowing this, a biologist can use nucleotides labeled with a nucleotides labeled with a radioactive isotope to build a radioactive isotope to build a complementary single strand of complementary single strand of DNA.DNA.

2) Nucleic acid probe- used to locate 2) Nucleic acid probe- used to locate specific genesspecific genes

b) Next, the biologist treats the DNA being b) Next, the biologist treats the DNA being searched with chemicals or heat to separate searched with chemicals or heat to separate the 2 DNA strands. The nucleic acid probe is the 2 DNA strands. The nucleic acid probe is mixed in with these single strands.mixed in with these single strands.

c) The probe tags the correct DNA portion by c) The probe tags the correct DNA portion by pairing with the complementary sequence in pairing with the complementary sequence in the protein-V gene.the protein-V gene.

d) Once the biologist uses this d) Once the biologist uses this radioactive marker to identify the radioactive marker to identify the bacterial cells with the desired gene, bacterial cells with the desired gene, those cells are allowed to multiply those cells are allowed to multiply further, producing the desired gene further, producing the desired gene in large amounts.in large amounts.

D. Useful Products from Genetically D. Useful Products from Genetically Engineered MicroorganismsEngineered Microorganisms

1. Genetically engineered bacteria 1. Genetically engineered bacteria used to make medicine (ex: insulin)used to make medicine (ex: insulin)

2. Recombinant DNA technology is 2. Recombinant DNA technology is also helping to develop effective also helping to develop effective

vaccines (ex: hepatitis B)vaccines (ex: hepatitis B)

III. Biologists can genetically engineer plants III. Biologists can genetically engineer plants and animalsand animals

A. Producing Genetically Modified PlantsA. Producing Genetically Modified Plants

1. Genetically modified organism 1. Genetically modified organism (GMO)- any organism that has (GMO)- any organism that has acquired one or more genes by acquired one or more genes by artificial meansartificial means

a) Transgenic- a GMO whose a) Transgenic- a GMO whose source of new genetic materials source of new genetic materials is from a different speciesis from a different species

Figure 13-11Figure 13-11To genetically modify a plant, researchers insert a plasmid To genetically modify a plant, researchers insert a plasmid containing the desired gene into a plant cell. There, the gene containing the desired gene into a plant cell. There, the gene is incorporated into the plant cell's DNA. The engineered is incorporated into the plant cell's DNA. The engineered plant cell then grows into a genetically modified plant.plant cell then grows into a genetically modified plant.

B. Producing Genetically Modified AnimalsB. Producing Genetically Modified Animals1. First step is to extract an egg cell from 1. First step is to extract an egg cell from a female.a female.

2. Sperm from the same species is used 2. Sperm from the same species is used to fertilize the egg in a “test-tube” to fertilize the egg in a “test-tube”

environment.environment.

3. Then the desired gene is injected into 3. Then the desired gene is injected into the fertilized egg and the egg is returned the fertilized egg and the egg is returned to a uterus where it can develop into an to a uterus where it can develop into an embryo.embryo.

C. Animal CloningC. Animal Cloning

1. The first successful clone was the 1. The first successful clone was the sheep named Dolly.sheep named Dolly.

2. The procedure for cloning is the same 2. The procedure for cloning is the same as producing a GM animal, except as producing a GM animal, except

that instead of inserting a gene into an that instead of inserting a gene into an egg, an entire foreign nucleus replaces egg, an entire foreign nucleus replaces the the egg’s own nucleus.egg’s own nucleus.

D. The GMO ControversyD. The GMO Controversy

1. Can GM crops pass their new 1. Can GM crops pass their new genes to closely related plants in the genes to closely related plants in the nearby wild areas?nearby wild areas?

2. Another concern is the GM plants 2. Another concern is the GM plants and animals could have unknown risks and animals could have unknown risks to human consumers.to human consumers.

IV. DNA technologies have many applicationsIV. DNA technologies have many applications

A. Mass-Producing DNA in a Test TubeA. Mass-Producing DNA in a Test Tube

1. Polymerase chain reaction (PCR)- 1. Polymerase chain reaction (PCR)- a technique that makes many copies a technique that makes many copies of a certain segment of DNA without of a certain segment of DNA without using living cellsusing living cells

Figure 13-15Figure 13-15PCR produces multiple copies of a segment of DNA. PCR produces multiple copies of a segment of DNA.

B. Comparing DNAB. Comparing DNA

1. Gel electrophoresis- a technique for 1. Gel electrophoresis- a technique for sorting molecules or fragments of sorting molecules or fragments of

molecules by lengthmolecules by length

a) First, each DNA sample is cut up a) First, each DNA sample is cut up into fragments by a group of into fragments by a group of restriction enzymes.restriction enzymes.

b) Next, a few drops of each sample are b) Next, a few drops of each sample are placed in a small pocket or well at one placed in a small pocket or well at one end of a gel. The other end of the gel end of a gel. The other end of the gel has a positive charge. All DNA has a positive charge. All DNA molecules are negatively charged, so molecules are negatively charged, so they move through pores in the gel they move through pores in the gel toward the positive pole.toward the positive pole.

c) The shorter DNA fragments slip more c) The shorter DNA fragments slip more easily through the pores of the gel. easily through the pores of the gel. Therefore, the shorter DNA fragments Therefore, the shorter DNA fragments will travel faster through the gel and be will travel faster through the gel and be closer to the positive end of the gel than closer to the positive end of the gel than the longer fragments.the longer fragments.

d) Lastly, the gel is treated with a stain d) Lastly, the gel is treated with a stain that makes the DNA visible under that makes the DNA visible under ultraviolet light. The fragments show up ultraviolet light. The fragments show up as a series of bands in each “lane” of the as a series of bands in each “lane” of the gel.gel.

Figure 13-16Figure 13-16The gel electrophoresis technique shown above can The gel electrophoresis technique shown above can be used to compare DNA of individuals or species.be used to compare DNA of individuals or species.

2. Genetic markers- particular stretches 2. Genetic markers- particular stretches of DNA that are variable among of DNA that are variable among individuals (easy way to tell if an individuals (easy way to tell if an individual is a carrier of a disease)individual is a carrier of a disease)

3. DNA fingerprints- an individual’s 3. DNA fingerprints- an individual’s unique banding pattern unique banding pattern

V. Control mechanisms switch genes on and V. Control mechanisms switch genes on and offoff

A. Regulation of Genes in ProkaryotesA. Regulation of Genes in Prokaryotes

1. Operon- cluster of genes and 1. Operon- cluster of genes and their controlled sequencestheir controlled sequences

2. Promoter- control sequence on an 2. Promoter- control sequence on an operon where RNA polymerase operon where RNA polymerase attaches to the DNAattaches to the DNA

Figure 13-18Figure 13-18E. coliE. coli bacteria, natural inhabitants of your intestine, break bacteria, natural inhabitants of your intestine, break down the sugar lactose. The genes that code for lactose-down the sugar lactose. The genes that code for lactose-processing enzymes are located next to control sequences. processing enzymes are located next to control sequences. Altogether, this stretch of DNA is called the Altogether, this stretch of DNA is called the laclac operon. operon.

3. Operator- a control sequence that acts like 3. Operator- a control sequence that acts like a switch, determining whether or not RNA a switch, determining whether or not RNA polymerase can attach to the promoterpolymerase can attach to the promoter

4. Repressor- a protein that functions by 4. Repressor- a protein that functions by binding to the operator and blocking the binding to the operator and blocking the attachment of RNA polymerase to the attachment of RNA polymerase to the promoter; turns off transcriptionpromoter; turns off transcription

Figure 13-19Figure 13-19The The laclac operon is inactive in the absence of lactose (top) because a operon is inactive in the absence of lactose (top) because a repressor blocks attachment of RNA polymerase to the promoter. With repressor blocks attachment of RNA polymerase to the promoter. With lactose present (bottom), the repressor is inactivated, and transcription of lactose present (bottom), the repressor is inactivated, and transcription of lactose-processing genes proceeds.lactose-processing genes proceeds.

B. Regulation of Genes in EukaryotesB. Regulation of Genes in Eukaryotes

1. Transcription factors- proteins that 1. Transcription factors- proteins that regulate transcription by binding to those regulate transcription by binding to those promoters or to RNA polymerases; are promoters or to RNA polymerases; are activated and deactivated by chemical activated and deactivated by chemical signals in the cellsignals in the cell

2. Gene expression- the transcription and 2. Gene expression- the transcription and translation of genes into proteinstranslation of genes into proteins

C. From Egg to OrganismC. From Egg to Organism

1. Cellular differentiation- when 1. Cellular differentiation- when cells become increasingly cells become increasingly specialized specialized in structure and functionin structure and function

Figure 13-21Figure 13-21Though all the genes of the genome are present in every type of Though all the genes of the genome are present in every type of cell, only a small, specific fraction of these genes are actually cell, only a small, specific fraction of these genes are actually expressed in each type of cell. The yellow color indicates a gene expressed in each type of cell. The yellow color indicates a gene that is "turned on" (expressed).that is "turned on" (expressed).

D. Stem CellsD. Stem Cells

1. Cells that remain undifferentiated; 1. Cells that remain undifferentiated; they have the potential to they have the potential to

differentiate into various types of differentiate into various types of cells; may be able to help people cells; may be able to help people with with disabling diseasesdisabling diseases

Figure 13-22Figure 13-22Present at a very early stage of human development, stem Present at a very early stage of human development, stem cells have the potential to develop into any type of human cells have the potential to develop into any type of human cell.cell.

E. Homeotic GenesE. Homeotic Genes

1. Master control genes that direct 1. Master control genes that direct development of body parts in development of body parts in

specific locations in many specific locations in many organismsorganisms

Figure 13-24Figure 13-24The highlighted The highlighted portions of the portions of the fruit fly and fruit fly and mouse mouse chromosomes chromosomes carry very carry very similar homeotic similar homeotic genes. The color genes. The color coding identifies coding identifies the parts of the the parts of the embryo and embryo and adult animals adult animals that are affected that are affected by these genes.by these genes.