Chapter 19 Microbial Models: The Genetics of Viruses and Bacteria

1. Chapter 19 • Microbial Models: The Genetics of Viruses and Bacteria

2. Viral structure • Virus: “poison” (Latin); infectious particles consisting of a nucleic acid in a protein coat • Capsid; (viral envelopes); DNA or RNA • Bacteriophages (phages)

3. Viral reproduction: Lytic Cycle • Host range: infection of a limited range of host cells (receptor molecules on the surface of cells) • The lytic cycle: 1- attachment 2- injection 3- hydrolyzation 4- assembly 5- release • Results in death of host cell • Virulent virus (phage reproduction only by the lytic cycle)

4. Viral reproduction: Lysogenic Cycle • Genome replicated w/o destroying the host cell • Genetic material of virus becomes incorporated into the host cell DNA (prophage DNA) • Temperate virus (phages capable of using the lytic and lysogenic cycles) • May give rise to lytic cycle

5. RNA viruses • Retroviruses: transcribe DNA from an RNA template (RNA--->DNA) • Reverse transcriptase (catalyzing enzyme) • HIV--->AIDS

6. Viroids and Prions • Viroids: tiny, naked circular RNA that infect plants; do not code for proteins, but use cellular enzymes to reproduce; stunt plant growth • Prions:“infectious proteins”; “mad cow disease”; trigger chain reaction conversions; a transmissible protein

7. Bacterial genetics • Nucleoid: region in bacterium densely packed with DNA (no membrane) • Plasmids: small circles of DNA • Reproduction: binary fission (asexual)

8. Bacterial DNA-transfer processes • Transformation: genotype alteration by the uptake of naked, foreign DNA from the environment (Griffith expt.) • Transduction: phages that carry bacterial genes from 1 host cell to another •generalized - random transfer of host cell chromosome •specialized - incorporation of prophage DNA into host chromosome

9. Conjugation • direct transfer of genetic material; cytoplasmic bridges; pili; sexual

10. Bacterial Plasmids • Small, circular, self-replicating DNA separate from the bacterial chromosome • F (fertility) Plasmid: codes for the production of sex pili (F+ or F-) • R (resistance) Plasmid: codes for antibiotic drug resistance • Transposons: transposable genetic element; piece of DNA that can move from one location to another in a cell’s genome (chromosome to plasmid, plasmid to plasmid, etc.); “jumping genes”

11. Unit of genetic function consisting of coordinately related clusters of genes with related functions (transcription unit) Operons, I • Repressible (trp operon): • tryptophan (a.a.) synthesis • promoter: RNA polymerase binding site; begins transcription • operator: controls access of RNA polymerase to genes (tryptophan not present) • repressor: protein that binds to operator and prevents attachment of RNA polymerase - coded from a regulatory gene (tryptophan present - acts as a corepressor) • transcription is repressed • when tryptophan binds to a regulatory protein

12. Unit of genetic function consisting of coordinately related clusters of genes with related functions (transcription unit) Operons, II • Inducible (lac operon): - lactose metabolism • lactose not present: repressor active, operon off; no transcription for lactose enzymes • lactose present: repressor inactive, operon on; inducer molecule inactivates protein repressor (allolactose) • Transcription is stimulated when inducer binds to a regulatory protein

13. Chapter 19 • The Organization and Control of Eukaryotic Genomes

14. Chromatin • Def: complex of DNA and proteins • DNA Packing •histone protein (+ charged amino acids - phosphates of DNA are - charged) • Nucleosome •”beads on a string”; basic unit of DNA packing • Heterochromatin •highly condensed interphase DNA (can not be transcribed) • Euchromatin •less compacted interphase DNA (can be transcribed)

15. Molecular Biology of Cancer • Oncogene •cancer-causing genes • Proto-oncogene •normal cellular genes • How? 1-movement of DNA; chromosome fragments that have rejoined incorrectly 2-amplification; increases the number of copies of proto-oncogenes 3-proto-oncogene point mutation; protein product more active or more resistant to degradation • Tumor-suppressor genes •changes in genes that prevent uncontrolled cell growth (cancer growth stimulated by the absence of suppression)

16. Chapter 20 and 21 BioTechnology & Genomics

17. O.J. Simpson capital murder case,1/95-9/95 • Odds of blood in Ford Bronco not being R. Goldman’s: • 6.5 billion to 1 • Odds of blood on socks in bedroom not being N. Brown-Simpson’s: • 8.5 billion to 1 • Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson: • 21.5 billion to 1 • Number of people on planet earth: • 6.1 billion • Odds of being struck by lightning in the U.S.: • 2.8 million to 1 • Odds of winning the Illinois Big Game lottery: • 76 million to 1 • Odds of getting killed driving to the gas station to buy a lottery ticket • 4.5 million to 1 • Odds of seeing 3 albino deer at the same time: • 85 million to 1 • Odds of having quintuplets: • 85 million to 1 • Odds of being struck by a meteorite: • 10 trillion to 1

18. Recombinant DNA • Definition: DNA in which genes from 2 different sources are linked • Genetic engineering: direct manipulation of genes for practical purposes • Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

19. Bacterial plasmids in gene cloning

20. DNA Cloning • Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific • Restriction site: recognition sequence for a particular restriction enzyme • Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way • Sticky end: short extensions of restriction fragments • DNA ligase: enzyme that can join the sticky ends of DNA fragments • Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

21. Steps for eukaryotic gene cloning • Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest) • Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase • Introduction of cloning vector into cells (transformation by bacterial cells) • Cloning of cells (and foreign genes) • Identification of cell clones carrying the gene of interest

22. DNA Analysis & Genomics • PCR (polymerase chain reaction) • Gel electrophoresis • Restriction fragment analysis (RFLPs) • Southern blotting • DNA sequencing • Human genomeproject

23. Polymerase chain reaction (PCR) • Amplification of any piece of DNA without cells (in vitro) • Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers • Applications: fossils, forensics, prenatal diagnosis, etc.

24. DNA Analysis • Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

25. Restriction fragment analysis • Restriction fragment length polymorphisms (RFLPs) • Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence • DNA Fingerprinting

26. Southern Blotting • Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence • Southern blotting is a laboratory technique used to detect a specific DNA sequence in a blood or tissue sample. A restriction enzyme is used to cut a sample of DNA into fragments that are separated using gel electrophoresis. The DNA fragments are transferred out of the gel to the surface of a membrane. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the probe sequence is present in the sample.

27. Southern Blotting

28. DNA Sequencing • Determination of nucleotide sequences (Sanger method, sequencing machine) • Genomics: the study of genomes based on DNA sequences • Human Genome Project

29. Practical DNA Technology Uses • Diagnosis of disease • Human gene therapy • Pharmaceutical products (vaccines) • Forensics • Animal husbandry (transgenic organisms) • Genetic engineering in plants • Ethical concerns?

30. GENOMICS AP Biology Chap 21

31. Genomes – set of genes and their interactions Bioinformatics – computational methods of gene analysis - NCBI National Center Biotechnology Information – database of DNA sequences and proteins (proteomes) NCBI HomePage

32. The most ambitious mapping project to date has been the sequencing of the human genome Officially begun as the Human Genome Projectin 1990, the sequencing was largely completed by 2003 The project had three stages: Genetic (or linkage) mapping Physical mapping DNA sequencing

33. Fig. 21-2-4 Chromosome bands Cytogenetic map Genes located by FISH Fluorescence In Situ Hybridization Linkage mapping 1 Genetic markers Physical mapping 2 Overlapping fragments DNA sequencing 3

34. A linkage map (genetic map) maps the location of several thousand genetic markers on each chromosome • A genetic marker is a gene or other identifiable DNA sequence • Recombination frequencies are used to determine the order and relative distances between genetic markers

35. Fig. 21-3-3 1 Cut the DNA into overlapping fragments short enough for sequencing 2 Clone the fragments in plasmid or phage vectors. 3 Sequence each fragment. 4 Order the sequences into one overall sequence with computer software.

36. A complete haploid set of human chromosomes consists of 3.2 billion base pairs

37. By summer 2007, genomes had been sequenced for 500 bacteria, 45 archaea, and 65 eukaryotes including vertebrates, invertebrates, and plants

38. What do we know? • Humans have 20,488 genes • With alternate gene splicing, we can make 75,000 polypeptides • Genomes of most bacteria and archaea range from 1 to 6 million base pairs (Mb); genomes of eukaryotes are usually larger

39. Free-living bacteria and archaea have 1,500 to 7,500 genes Unicellular fungi have from about 5,000 genes and multicellular eukaryotes from 40,000 genes Number of genes is not correlated to genome size Humans and other mammals have the lowest gene density, or number of genes, in a given length of DNA

40. Table 21-1

41. About the human genome… • Only 1.5% codes for proteins, rRNA and tRNA • The rest is used for • regulatory sequences and introns 24% • pseudogenes (nonfunctioning genes) 15% • repetitive DNA 59%

42. Fig. 21-7 Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) L1 sequences (17%) Repetitive DNA unrelated to transposable elements (15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5–6%)

43. Repetitive DNA • 44% transposable elements (jumping genes) • - Transposons - cut and paste (ex Alu in primates) • - Most of these are retrotransposons – cut, copy to RNA, RT to DNA, and paste (ex Line1 or L1) • 15% – large segment and simple sequence DNA • - small ones STR - Short Tandem Repeats often used in centromeres and telomeres

44. Fig. 21-9 New copy of transposon Transposon DNA of genome Transposon is copied Insertion Mobile transposon (a) Transposon movement (“copy-and-paste” mechanism) New copy of retrotransposon Retrotransposon RNA Animation Quiz 5 - Transposons: Shifting Segments of the Genome Insertion Reverse transcriptase (b) Retrotransposon movement

45. “Jumping Genes” • The first evidence for wandering DNA segments came from geneticist Barbara McClintock’s breeding experiments with Indian corn

46. Fig. 21-8

47. Genes • Many eukaryotic genes are present in one copy per haploid set of chromosomes • More than ½ occur in multigene families – such as for RNA products and hemoglobin

48. Fig. 21-10 DNA RNA transcripts Heme -Globin Nontranscribed spacer Hemoglobin Transcription unit -Globin -Globin gene family -Globin gene family DNA Chromosome 16 Chromosome 11 5.8S 18S 28S G  2 A   1  2 1   rRNA  5.8S 28S Fetus and adult Embryo Fetus Embryo Adult 18S (a) Part of the ribosomal RNA gene family (b) The human -globin and -globin gene families

49. Genomic Evolution • Duplication of chromosome sets (polyploidy) • Chromosome alteration – duplications, inversions • Exon shuffling • Transposons

50. Humans have 23 pairs of chromosomes, while chimpanzees have 24 pairs Following the divergence of humans and chimpanzees from a common ancestor, two ancestral chromosomes fused in the human line Why we Are Smarter!