CH. 18: THE GENETICS OF VIRUSES & BACTERIA

1. CH. 18: THE GENETICS OF VIRUSES & BACTERIA Well-researched: Pneumococcus, Escherichia coli, bacteriophages, TMV

2. Bacteria

3. Bacterial cell shapes

4. Gramstaining Classification of bacteria according to what color stain they retain. A sample of bacteria (on a slide) is first stained with a violet dye. The slide is then rinsed with ethanol. If the violet stain washes off, a red dye is added (“counterstaining”). Depending on the structure of the cellwalls, some types of bacteria (such as staphylococcus and streptococcus) retain the violet stain and are called Gram-positive. Other types (such as pseudomonas and salmonella) retain the red, but not the violet, stain and are called Gram-negative. This technique is named after the Danish bacteriologist Hans C. J. Gram (1853-1938) who invented it in 1884. Gram + (top), Gram – (bottom)

5. The E.coli Chromosome: • Bacterial chromosome is GENERALLY a single, continuous (circular) thread of double- stranded DNA. • Approx. 1 mm long when fully extended (only 2mm in diameter); contains about 4.7 million base pairs. • More recently, it has been found that bacteria may actually have different kinds of chromosomal material

6. Prokaryotic Cells Replicate their DNA in a bidirectional fashion (replication) • Replication begins at a specific base sequence.diagram: “theta” replication

7. Transcription and Its Regulation • Transcription begins when RNA polymerase (enzyme) begins formation of an mRNA strand along DNA strand, beginning at promoter site. • A segment of DNA that codes for one specific protein is known as a structural gene • There may be several "start" and "stop" codons along the mRNA strand, marking the beginning and end of each structural gene. • "leader" sequence (of nucleotides) at 5’ end • "trailer" sequence (of nucleotides) at 3’ end

8. The Need for Regulation This is regulated by several means: • 1)  can be induced by presence of a material   (ex. Lactose presence induced E. coli to synthesize beta-galactosidase enzyme) • 2)  can be inhibited: presence of substance prevents formation of an enzyme "repressible" (ex: E. coli; tryptophan inhibits tryptophan forming enzymes)

9. The Lac Operon The Operon Model (1965 Nobel Prize) François Jacob, André Lwoff, and Jacques Monod "for their discoveries concerning genetic control of enzyme and virus synthesis" - arose from study of mutant cells studies done on E. coli cells making the enzyme: beta-galactosidase ; found it was "blocked" by a repressor binding to operator.

10. Operator – gene that activates transcription of a structural geneRegulator - This gene codes for a repressor proteinRepressor: protein that can bind to the operator gene, thus obstructing* the promotor (blocks the RNA polymerase from moving along ("reading") the molecule  no mRNA transcription can occur.*when the repressor is removed, mRNA transcription beginsanimations  of lac and tryp operons

11. CAP-cAMP Complex • The catabolite gene activator protein (CAP) is a sequence-specific DNA binding protein that starts transcription of some gene systems and represses others, when bound to cyclic adenosine monophosphate (cAMP) • this CAP-cAMP complex binds to the promotor and maximizes transcription. • It increases the binding of RNA polymerase, among other things

12. Bacterial plasmids are often used in genetic engineering, as a “gene of interest” can be inserted easily into them, then cloned into hundreds of copies when the bacteria multiply. Recombinant DNA: involves modifying/combining DNA from a variety of different sources and inserting these altered molecules into other cells, in which the "new "genes are expressed.

13. Bacterial Reproduction Binary fission • Bacteria cell goal: to grow and multiply • many can double number every 20 minutes!

14. Plasmids and Conjugation • Although bacterial chromosomes carry all the genes necessary for growth and reproduction of the cell, they also carry additional DNA molecules called Plasmids • Plasmids usually carry only between 2 to 30 genes; generally small) • 2 important types: • "sex factor" plasmids = F (fertility) • "drug resistance" plasmids = R (resistance)

15. The F Plasmid • We now know that the fertility factor or F factor is a very large (94,500 bp) circular dsDNA plasmid; it is generally independent of the host chromosome. http://www.mun.ca/biochem/courses/3107/Lectures/Topics/conjugation.html. • They make a cell capable of conjugation. • F+ (male) "donor" cells: make pili (protein "bridges" that form to connect 2 cells for transfer of genetic material) • F- cells lack the F plasmid and can’t form pili (female); these are "recipient" cells “rolling-circle replication”

16. Hfr Cell:    Sometimes the F factor gene can be incorporated within the main bacterial chromosome.  Because such strains transfer chromosomal genes very efficiently, they are called Hfr (high frequency of recombination).This can then transfer a portion of bacterial chromosome to a F- cell:

17. R  plasmids = resistance plasmids • Plasmids carrying genes responsible for antibiotic (or antibacterialdrug) resistance among bacteria may pass them to other species of bacteria as well! ex: E. coli  -------> Shigella (dysentery) • "Horizontal gene transfer“ also called Lateral gene transfer (LGT), is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism. • By contrast, vertical transferoccurs when an organism receives genetic material from its ancestor, e.g. its parent or a species from which it evolved. • It is most often thought of as a sexual process that requires a mechanism for the mobilization of chromosomal DNA among bacterial cells. • http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/genetic-exchange/exchange/exchange.html

18. Because they are unable to reproduce sexually, bacterial species have acquired several mechanisms by which to exchange genetic materials • Transformation - the uptake of naked DNA is a common mode of horizontal gene transfer that can mediate the exchange of any part of a chromosome; this process is most common in bacteria that are naturally transformable; typically only short DNA fragments are exchanged. • Conjugation - the transfer of DNA mediated by conjugal plasmids or conjugal transposons; requires cell to cell contact but can occur between distantly related bacteria or even bacteria and eukaryotic cells; can transfer long fragments of DNA. • Transduction - the transfer of DNA by phage requires that the donor and recipient share cell surface receptors for phage binding and thus is usually limited to closely related bacteria; the length of DNA transferred is limited by the size of the phage head.

19. VIRUSES diagram:  bacteriophages                                                        TMV • consists of a molecule of nucleic acid encased in a protein coat (capsid) contain no other "cell machinery", but can move from cell to cell and utilize the host cell’s "machinery" to replicate the viral genes "obligate intracellular parasites" • Viral nucleic acids vary: may be either DNA or RNA; double or single-stranded, circular or linear. Viral classifications • Viruses are generally classified by size (nm), shape/capsid, and type of nucleic acid they contain. CHARACTERISTICS: 1. Contain a varying, but small amount of DNA (or RNA) surrounded by protein         T7 bacteriophage has DNA and ~100 genes; The poliovirus has 7,600 RNA nucleotides; the vaccinia (cowpox) virus has 240,000 DNA nucleotide pairs 2. Small viruses that don’t have room for a lot of DNA use overlapping genes 3. Retroviruses (ex: HIV) are RNA viruses that use an enzyme called Reverse Transcriptase to make DNA to replicate itself during infection stage)

20. Viroids and Prions • Viroids:(mostly cause plant diseases) • are tiny strands of RNA, usually only a few hundred nucleotides long. • Viroids can interfere with a plant's metabolism.  • Generally speaking, where viroids come from and how they can disrupt the host cell are not known.

21. Prions • Prions are infectious agent, a misshapen protein, which have been defined as "small proteinaceous infectious particles which resist inactivation by procedures that modify nucleic acids". • The discovery that proteins alone can transmit an infectious disease has come as a considerable surprise to the scientific community. • Prion diseases are often called spongiform encephalopathies because of the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum. • Probably most mammalian species develop these diseases. Specific examples include: • Scrapie: sheep • TME (transmissible mink encephalopathy): mink • CWD (chronic wasting disease): mule deer, elk • BSE (bovine spongiform encephalopathy): cows • Humans are also susceptible to several prion diseases: • CJD: Creutzfeld-Jacob Disease • GSS: Gerstmann-Straussler-Scheinker syndrome • FFI: Fatal familial Insomnia • Kuru • Alpers Syndrome • http://www-micro.msb.le.ac.uk/3035/prions.html

22. Viral Cycles • Lytic cycle- causes destruction of the host cell quickly • Attachment- phage attaches to cell surface • Entry- phage injects DNA into host cell • Replication- phage DNA "tells" host to make more phage DNA and protein coats • Assembly- new viruses are assembled; host cell becomes a "virus factory" • Release- cell lyses (breaks open), releasing viruses

23. Lysogenic Cycle • some viruses incorporate their DNA into a cell’s chromosome. • The cell may then cause a sudden eruption of viral activity (can remain latent for many generations) • Temperate bacteriophages- viruses that can integrate their DNA into bacterial chromosome at specific sites • Prophage- an integrated bacteriophage

24. Viral Transduction • the transfer of cellular DNA from one host cell to another by means of viruses = recombinant DNA

25. Transduction 1. General Transduction Transduction is bacterial chromosome DNA transfer mediated by a virus. This is a one way nonreciprocal virus mediated transfer from one bacterial cell to another.  2.Restricted (specialized) Transduction The situation in which a particular phage will transduce only specific regions of the bacterial chromosome. * can you think of the implications to genetic engineering?  gene therapy?

26. Introducing Lambda • Lambda is the best studied of the temperate bacteriophages • Viral form: linear, double-stranded (2 free ends) • When inserted into a bacterial cell, it becomes circular • integrase:  enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell. Lambda plaques on an E.coli culture

27. Transposons  • discovered by Barbara McClintock • Are segments of DNA that can move around to different positions in the genome of a single cell. • These mobile segments of DNA are sometimes called "jumping genes". • In the process, they may cause mutations increase (or decrease) the amount of DNA in the genome. • Also contain a gene for enzyme "transposase"At each end, they contain a sequence of repeats • direct repeats -ATTCAG-ATTCAG- • often used to I.D. insertion points • indirect repeats -ATTCAG-GACTTA- • Can carry genes for mutations, protein synthesis, drug resistance, etc...from one place in the genome to another

28. Many transposons move by a "cut and paste" process: the transposon is cut out of its location (like command/control-X on your computer) and inserted into a new location (command/control-V). This process requires an enzyme — a transposase — that is encoded within some of these transposons.