UNIT VII – MOLECULAR GENETICS

1. UNIT VII – MOLECULAR GENETICS Big Campbell – Ch 18, 19, 20 Baby Campbell – Ch 10, 11, 12

2. UNIT OVERVIEW • Microbial Genetics • Viruses • Bacteria • Gene Expression in Prokaryotes • Regulation of Gene Expression • DNA Technology • DNA Testing Techniques • PCR • Recombinant DNA • Extensions

3. I. MICROBIAL GENETICS – VIRUSES • Discovery of Viruses • First isolated by Ivanowsky in 1890s from infected tobacco leaves • Crystallized by Stanley in 1935 – proved viruses were not cells • Not organisms; correctly referred to as particles • Not capable of carrying out life processes without a host cell • Parasites

4. I. VIRUSES, cont

5. I. VIRUSES, cont • Characteristics • Viral genome may be either single-stranded or double-stranded DNA or RNA. • Protein coat surrounding virus is known as a capsid made up of protein subunits called capsomeres. • Some viruses are also surrounded by a viral envelope • Typically derived from host cell membrane • Exception is Herpes virus, synthesized from nuclear envelope of host cell • Aid in attachment. Envelope glycoproteins bind to receptor molecules on host cell • Most viruses that infect animals have envelope

6. I. VIRUSES, cont • Bacteriophages • Defense Mechanisms

7. I. VIRUSES, cont - Reproduction

8. I. VIRUSES, cont - Reproduction Viral Entrance into Host Cell

9. I. VIRUSES, cont - Reproduction LYTIC CYCLE 1. Lytic Cycle – Results in death of host cell.

10. I. VIRUSES, cont - Reproduction LYSOGENIC CYCLE

11. I. VIRUSES, cont - Retroviruses

12. I. VIRUSES, cont – Animal Viruses • Coronavirus • Filovirus • Herpesvirus • Herpes simplex I and II • Epstein-Barr virus • Varicella zoster

13. I. VIRUSES, cont – Animal Viruses • Influenza Virus • Papillomavirus • Rhinovirus

14. II. OTHER INFECTIOUS AGENTS • Viroids • Prions

15. III. MICROBIAL GENETICS - BACTERIA • Genetic Make-up of Bacteria • Prokaryotic • Single chromosome wrapped in much less protein than found in euks • DNA concentrated in region known as nucleoid • Reproduction • Reproduce asexually • Advantages of asexual reproduction • Disadvantages of asexual reproduction • Have developed mechanisms for genetic variability . . .

16. III. BACTERIA, cont • Transformation • Ability to take up DNA from surrounding environment • Streptococcus pneumoniae • Used to introduce human genes into bacterial cells to produce human protein • Transduction • Use of viruses to carry bacterial genes from one host cell to another • Conjugation • “Bacterial sex”  • One-way transfer of a self-replication piece of DNA known as a plasmid • Donor (male) extends pilus • Pulls cells together • Cytoplasmic bridge forms • Plasmid is transferred

17. III. BACTERIA, cont • A Closer Look at Plasmids • Bacterium’s ability to produce plasmids and form pili due to specific piece of DNA known as the F factor • F factor may be integrated into chromosome, or separate as a piece of plasmid • Contains an origin of replication • Copy of F factor may be transferred to recipient cell; allow recipient cell to become “male” • R Plasmids – Carry genes for enzymes that destroy antibiotics

18. III. BACTERIA, contA Quick Look Back at Protein Synthesis in Euks

19. III. BACTERIA, cont • Protein Synthesis • Similar to euks • Different in that transcription & translation occur virtually simultaneously

20. IV. REGULATION OF GENE EXPRESSION IN PROKARYOTES • Important adaptation for bacteria • Two basic mechanisms for metabolic control • Regulation of Enzyme Activity • Feedback Inhibition • Regulation of Gene Expression • Operons

21. IV. PROKARYOTE GENE EXPRESSION, cont • Operon Model • Operon = Promoter + Operator + all genes required for a given metabolic pathway • Operon acts as a single transcription unit • Promoter → Binding site for RNA polymerase • Operator → “On-off” switch located either close to or within the promoter • Operator controls whether or not RNA polymerase can bind to the promoter region • Therefore operator determines whether operon genes are transcribed & translated

22. IV. PROKARYOTE GENE EXPRESSION, cont • Operon Control • Operon can be turned off by a protein known as a repressor • Repressor binds to operator and prevents attachment of RNA polymerase to promoter • Repressor is a protein controlled by a gene known as a regulatory gene in a different location on chromosome; not part of operon • Expressed continuously • Always a small supply of repressor protein present

23. IV. PROKARYOTE GENE EXPRESSION, cont • Types of Operons • Inducible Operons • Operons that are usually off; that is, not usually transcribed • Can be stimulated when a specific molecule interacts with regulatory protein • Example is the lac Operon • Repressible Operons • Transcription normally occurs • Can be inhibited when a specific molecule binds allosterically to regulatory protein • Example is the trp Operon

24. IV. PROKARYOTE GENE EXPRESSION, cont Inducible Operons • lac Operon • Regulates transcription of genes required for breakdown of lactose • Inducible – Typically off • Bacterium is metabolizing glucose, other carbs; lactose is not present

25. IV. PROKARYOTE GENE EXPRESSION, cont Inducible Operons • lac Operon, cont • When lactose is available, lactose itself binds with repressor; inactivates it by changing its shape • Repressor cannot bind to regulator • Therefore, RNA polymerase is able to bind to promoter; operon is “on” • 3 enzymes required to metabolize lactose are synthesized

26. IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons • Tryptophan (trp) Operon • Operon controls production of 5 enzymes required to synthesize amino acid, tryptophan when it is not available to bacterium in surrounding • Operon normally on; repressor inactive

27. IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons • When tryptophan is present, it binds to the repressor of the trp operon, activating the repressor, and turning off enzyme production. • Tryptophan acts as a co-repressor, a molecule that works with a repressor protein to switch an operon off.

28. IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons

29. IV. PROKARYOTE GENE EXPRESSION, cont • Positive Gene Regulation • In addition to repressors, some operons are also under the control of proteins known as activators • Essentially the opposite of repressors • They “turn up” an operon by making it easier for RNA polymerase to bind to DNA, therefore facilitating transcription of operon genes • In the lac operon . . . • If both glucose and lactose are available, bacterium utilizes glucose until its supplies are depleted • As glucose ↓, concentration of cyclic AMP (cAMP) ↑ • Increase in cAMP triggers release of activator protein known as CAP; CAP binds to promoter, facilitates binding of RNA polymerase to promoter of operon to enhance synthesis of enzymes of lac operon • When glucose concentration is high, decrease in cAMP results in decrease in CAP → RNA polymerase has very low affinity for lac operon promoter so lactose metabolism does not occur

30. IV. PROKARYOTE GENE EXPRESSION, cont

31. DNA TECHNOLOGY & GENOMICS

32. V. TECHNIQUES IN DNA TECHNOLOGY • Restriction Enzymes • Used by bacteria to “chop up” viral DNA • Bacterial DNA protected by _________ • Very specific • Each enzyme recognizes a particular nucleotide sequence • Called a restriction sequence or restriction site • Palindromic • Cuts made at specific points • May create “sticky ends” • Used in gel electrophoresis • Also used to form recombinant DNA • Fragments may be pasted together with DNA ligase to form recombinant DNA

33. V. TECHNIQUES, cont • Polymerase Chain Reaction (PCR) • In vitro method of amplifying small amounts of DNA • DNA is heated to separate the double helix. • Mixture is allowed to cool, DNA primers attach to target • Heat-stable polymerase is used to extend the primers in the 5’–3’ direction.

34. V. TECHNIQUES, cont • Gel Electrophoresis • Separates DNA fragments based on size • Restriction fragment analysis • DNA treated with restriction enzymes • Resulting fragments migrate based on size • Produce a pattern characteristic of original DNA and restriction enzyme used

35. Southern Blotting Designed by Dr. Southern Detects particular DNA sequences Northern Blotting Detects particular mRNA sequences Western Blotting Used to detect proteins V. TECHNIQUES, cont

36. VI. EXTENSIONS IN DNA TECHNOLOGY • Recombinant DNA • DNA containing nucleotides from other sources • Process utilizes restriction enzymes that make jagged cuts in DNA; creates sticky ends • When DNA from different sources treated with same restriction enzyme, sticky ends “mix & match” • Often use reporter genes to determine success; for example, ampicillin resistance

37. VI. EXTENSIONS, cont • cDNA - complementary DNA • Procedure for “cloning DNA” that uses mRNA, reverse transcriptase • STRs – short tandem repeats • Short segments of DNA that are highly repetitive, polymorphic • Repeat patterns are inherited • Useful for identifying individuals • SNPs – single nucleotide polymorphisms • Single base-pair that shows variation in a significant % of population • SNPs that alter the fragment length following exposure to restriction enzymes called RFLPs (restriction fragment length polymorphisms) • Genetic markers

38. VI. EXTENSIONS, cont • DNA Microarray Assays • AKA DNA Chips • Test used to determine gene function, gene interactions • May be used to determine agressiveness of cancers, method of treatment, etc

39. VI. EXTENSIONS, cont • Gene Cloning • Process of preparing multiple copies of a particular segment of DNA • Requires host and vector • Hosts • Initially done using bacterial cells • Now eukaryotic hosts are used • Yeast • Plants • Vector • Should have 4 characteristics • Ability to replicate independently of host cell DNA • Recognition sequence • Reporter gene • Small size • Possible vectors include • Plasmids • Viruses • YAC = Yeast Artificial Chromosome

40. VI. EXTENSIONS, cont • Gene Cloning • Use of plasmid as vector • Plasmid isolated from bacterial cell • Foreign DNA inserted into plasmid • Plasmid returned to bacterial cell; described as recombinant bacterium • Foreign gene is cloned as bacteria reproduce • Common bacterium used for plants is Agrobacterium tumefactiens

41. VI. EXTENSIONS, cont A CLOSER LOOK AT GENE CLONING

42. VI. EXTENSIONS, cont • Reproductive Cloning • Nuclear Transplantation • Process of using unfertilized egg cell & replacing nucleus with DNA • In 1997, scientists were able to produce first reproductive clone, “Dolly”, by culturing somatic cells in a nutrient-poor medium to de-differentiate them and force them back to totipotency. • Reproductive cloning in animals has enjoyed limited success.

43. VI. EXTENSIONS, cont • Gene Silencing • Knockout Genes • Use of genetic recombination to create an inactive , “knocked out” gene • Based on principal of homologous recombination • Mutated allele introduced into embryonic stem cells • Forms chimeras • Often used in mice to study gene expression • RNAi • Based on principal of microRNA • Small-interfering RNA (siRNA) synthesized complementary to mRNA • Base-pairing occurs • Translation is blocked • Has been used to block production of growth factors in certain cancers

44. VII. GENOMICS • Human Genome Project • International government effort begun in 1990 • Goals • identify all the approximately 20,000-25,000 genes in human DNA, • determine the sequences of the 3 billion chemical base pairs that make up human DNA, • store this information in databases, • improve tools for data analysis, • transfer related technologies to the private sector, and • address the ethical, legal, and social issues (ELSI) that may arise from the project. • Celera Genomics • Shotgun sequencing • Completed early and under-budget in 2003 • Genomics has given rise to proteonomics