Chapter 18 and 20

1. Chapter 18 and 20 Viruses, Bacteria, DNA Technology, and Genomics

2. The Genetics of Virusesand Bacteria • Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli • E. coli and its viruses are called model systems because of their frequent use by researchers in studies that reveal broad biological principles • Beyond their value as model systems, viruses and bacteria have unique genetic mechanisms that are interesting in their own right

3. Bacteria are prokaryotes with cells much smaller and more simply organized than those of eukaryotes • Viruses are smaller and simpler than bacteria

4. Structure of Viruses • Viruses are not cells • Viruses are very small infectious particles consisting of nucleic acid enclosed in a protein coat and, in some cases, a membranous envelope

5. Viral Genomes • Viral genomes may consist of • Double- or single-stranded DNA • Double- or single-stranded RNA • Depending on its type of nucleic acid, a virus is called a DNA virus or an RNA virus

6. Capsids and Envelopes • A capsid is the protein shell that encloses the viral genome • A capsid can have various structures

7. Some viruses have structures have membranous envelopes that help them infect hosts • These viral envelopes surround the capsids of influenza viruses and many other viruses found in animals • Viral envelopes, which are derived from the host cell’s membrane, contain a combination of viral and host cell molecules

8. Concept 18.3: Rapid reproduction, mutation, and genetic recombination contribute to the genetic diversity of bacteria • Bacteria allow researchers to investigate molecular genetics in the simplest true organisms • The well-studied intestinal bacterium Escherichia coli(E. coli) is “the laboratory rat of molecular biology”

9. The Bacterial Genome and Its Replication • The bacterial chromosome is usually a circular DNA molecule with few associated proteins • Many bacteria also have plasmids, smaller circular DNA molecules that can replicate independently of the chromosome • Bacterial cells divide by binary fission, which is preceded by replication of the chromosome

10. Replication fork Origin of replication LE 18-14 Termination of replication

11. Mutation and Genetic Recombination as Sources of Genetic Variation • Since bacteria can reproduce rapidly, new mutations quickly increase genetic diversity • More genetic diversity arises by recombination of DNA from two different bacterial cells

12. Mechanisms of Gene Transfer and Genetic Recombination in Bacteria • Three processes bring bacterial DNA from different individuals together: • Transformation • Transduction • Conjugation

13. Transformation • Transformation is the alteration of a bacterial cell’s genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment • For example, harmless Streptococcus pneumoniae bacteria can be transformed to pneumonia-causing cells

14. Transduction • In the process known as transduction, phages carry bacterial genes from one host cell to another

15. Phage DNA A+ B+ A+ B+ LE 18-16 Donor cell A+ Crossing over A+ A– B– Recipient cell A+ B– Recombinant cell

16. Conjugation and Plasmids • Conjugation is the direct transfer of genetic material between bacterial cells that are temporarily joined • The transfer is one-way: One cell (“male”) donates DNA, and its “mate” (“female”) receives the genes

17. “Maleness,” the ability to form a sex pilus and donate DNA, results from an F (for fertility) factor as part of the chromosome or as a plasmid • Plasmids, including the F plasmid, are small, circular, self-replicating DNA molecules

18. The F Plasmid and Conjugation • Cells containing the F plasmid, designated F+ cells, function as DNA donors during conjugation • F+ cells transfer DNA to an F recipient cell • Chromosomal genes can be transferred during conjugation when the donor cell’s F factor is integrated into the chromosome

20. R plasmids and Antibiotic Resistance • R plasmids confer resistance to various antibiotics • When a bacterial population is exposed to an antibiotic, individuals with the R plasmid will survive and increase in the overall population

21. Restriction Enzymes • Bacteria’s defense against invasion – their immune system • Work by cutting up foreign DNA, a process called restriction

22. Chapter 20Overview: Understanding and Manipulating Genomes • 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

23. Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning

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

25. Cell containing gene of interest Bacterium Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell LE 20-2 Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

26. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at 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

27. Restriction site 5¢ 3¢ DNA 3¢ 5¢ Restriction enzyme cuts the sugar-phosphate backbones at each arrow. LE 20-3 Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

28. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

29. Producing Clones of Cells • Cloning a human gene in a bacterial plasmid can be divided into six steps: 1. Vector and gene-source DNA are isolated 2. Cut both DNA samples with the same restriction enzyme 3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place, ligase covalently bonds fragments 4. Recombinant plasmids are mixed with bacteria 5. The bacteria are plated and incubated 6. Cell clones with the right gene are identified

30. Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site LE 20-4_1 ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids

31. Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest LE 20-4_2 Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

32. lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. LE 20-4_3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

33. PCR – Polymerase Chain Reaction • PCR can make billions of copies of a target segment of DNA • Three cycles results in two copies of the target sequence which then will copy exponentially in each subsequent cycle • Each cycle is a series of three steps • Denaturation – heat separates the two strands of DNA • Annealing – Cooling allows primers to hydrogen bond to ends of target sequences • Extension – DNA polymerase adds nucleotides to the 3’ end of each primer (DNA always is built in the 5’ to 3’ direction) Things needed for PCR • Target sequence • Primers • Nucleotide triphosphates • Taq polymerase

34. Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites • Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules • Such analysis can rapidly provide comparative information about DNA sequences

35. 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 nuclei acids or proteins by size

36. Mixture of DNA molecules of differ- ent sizes Longer molecules Cathode LE 20-8 Shorter molecules Power source Gel Glass plates Anode

37. 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

38. Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele LE 20-9 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

39. Restriction Fragment Length Differences as Genetic Markers • Restriction fragment length polymorphisms (RFLPs, or Rif-lips) are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths • A RFLP can serve as a genetic marker for a particular location (locus) in the genome • RFLPs are detected by Southern blotting

40. Concept 20.3: Entire genomes can be mapped at the DNA level • The most ambitious mapping project to date has been the sequencing of the human genome • Officially begun as the Human Genome Project in 1990, the sequencing was largely completed by 2003 • Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

41. Future Directions in Genomics • Genomics is the study of entire genomes • Proteomics is the systematic study of all proteins encoded by a genome • Single nucleotide polymorphisms (SNPs) provide markers for studying human genetic variation

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

43. Diagnosis of Diseases • Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation • Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

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

45. Animal Husbandry and “Pharm” Animals • Transgenic organisms are made by introducing genes from one species into the genome of another organism • Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles) • Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use