Chapters 18 & 19

1. Chapters 18 & 19 Viruses, Bacteria & Eukaryotic Genomes

2. VIRUSES: First identified in tobacco plants (Tobacco Mosaic Virus) 1000’s can fit in one cell. Tiny: Two main parts: genome and protein capsid Obligate intracellular parasites: must be inside host cell to reproduce Host range varies: wide range = many host species, narrow = few (can be only one host species).

3. Evolutionary debate: May have evolved from plasmids (small circular DNA molecules in bacteria and yeasts) or transposons (DNA segments which move within a cell’s genome). Like viruses, these are both mobile genetic elements.

4. Viral Structure: Capsids are made from protein subunits called capsomeres. There is usually a large number of proteins, but the number of different kinds of proteins is usually small. Helical Viruses: capsid is formed from a repeating single type of protein with the overall shape of a rigid rod. Icosahedral Viruses: capsid is formed from 252 identical protein molecules arranged in a polyhedron with 20 triangular facets – an icosahedron. Example: Adenoviruses which cause respiratory infections in animals.

5. Influenza Viruses: capsid is surrounded by a membranous envelope derived from the membranes of the host (Such membranes prevent detection by the immune system). Bacteriophages: (Viruses which infect bacteria) Complex capsids. The first seven studied were nicknamed T1 – T7. They have an elongated icosahedral head and an elaborate protein tail. http://www.youtube.com/watch?v=41aqxcxsX2w

6. General Viral Replicative Cycles: Virus identifies host cell: lock and key fit with receptor molecules on host cell Viral Genome: Enters host via membrane fusion, endocytosis or injection (depending on type of virus). Viral Genome: Produces proteins which reprogram the cell to copy the viral genome and produce viral proteins using host: _nucleotides, enzymes, ribosomes, tRNAs, amino acids, ATP, etc. ___.

7. Replicative Cycles of Bacteriophages(the best understood of all viruses): Lytic: rapid destruction of host cell Phages that can only replicate this way are called virulent.

8. Lysogenic: Viral DNA integrates into host chromosomes (via crossing over), becomes a prophage and can reproduce (when cell divides) without destroying cell. Lysogenic viruses can: become lytic in response to environmental triggers Phages that can replicate in both ways are called ___ temperate ___.

9. Replicative Cycles of Animal Viruses: Animal Viruses are classified by their type of nucleic acid: ssDNA, dsDNA, ssRNA, dsRNA Most have RNA genome and an envelope. (See Table 18.1, p.340) Viral envelopes: Made of viral ___ glycoproteins ___ embedded in host membrane ___ (plasma or nuclear) ___. Help virus enter host cell. Enveloped viruses can exit host cells without killing them . Sometimes (as in herpes viruses) copies of the viral DNA can remain latent in host cells until a physical or emotional stress triggers active virus production.

10. RNA as Viral Genetic Material: Three types of single-stranded RNA animal viruses: Class IV viruses: Genome can directly serve as mRNA (translated into viral protein immediately after infection). Class V viruses: Genome serves as a template for mRNA synthesis (RNA to RNA transcription requires a special enzyme which is contained within the capsid).

11. Class VI Retroviruses (like HIV): can’t transcribe or translate their genome directly. Use reverse transcriptase (enzyme) to make DNA from RNA (backwards). They contain 2 identical ssRNAs and 2 molecules of reverse transcriptase within their enveloped capsids. Newly made viral DNA integrates into the host DNA -- called a ___ provirus ___ (Unlike prophages, however, it never leaves the host DNA.) Then, the proviral DNA uses host RNA polymerase to transcribe viral mRNA which can be translated into proteins or serve as new viral genomes. New viruses are assembled and released from the cell without lysis .

12. HIV Life Cycle

13. Three main ways viruses damage animal cells: Release of hydrolytic enzymes from cell’s lysosomes Viral activity causes cell to release: toxins Virus components are toxic: especiallyenvelope proteins

14. Emerging Viruses seem to appear suddenly: 1918 Flu, Ebola (1976), HIV (AIDS) in 1980’s, West Nile (1999), H1N1 in 2009, can cause epidemics, or even pandemics. “New” epidemics are caused by: mutation of existing viruses, dissemination from a small isolated group (globalization, drug use…), spread from one species to another (“swine flu”). West Nile Distribution

15. Plant Viruses: Cause agriculture damage. Most have RNA genomes and helical or icosahedral capsids. Two routes for spreading viral diseases: plant is infected from external source (often after epidermal damage) Horizontal = Vertical = plant inherits viral infection from parent Once a virus enters a plant cell and replicates, it can pass to other cells through plasmodesmata(some virally encoded proteins aid this movement).

16. The Simplest Infectious Agents: Pathogens that are even smaller and simpler than viruses include: Viroids: Naked circular RNA strands which infect plants, replicate, and cause errors in the regulatory systems which control plant growth.

17. Prions: proteins that cause degenerative brain diseases in animal hosts (such as mad cow disease): They are most likely transmitted in food. They act very slowly (incubation can lasts at least ten years) and are virtually indestructible. Current model on how they work: Prions are a misfolded form of a protein normally found in brain cells which triggers other proteins to convert to prions. The prion aggregation then interferes with normal cell functions. Creutzfeldt-Jakob disease (human variant of mad cow disease)

18. BACTERIA (Prokaryotes): 1 circular chromosome (dsDNA) plus accessory genes carried on small circular plasmids Associated proteins cause the DNA to form a dense “supercoil” region called the nucleoid Reproduce rapidly, short life span = new mutations can affect evolution quickly

19. Besides mutations, there are 3 are types of genetic recombination which increase genetic variation in bacteria: Transformation: Uptake of naked, foreign DNA Many bacteria have cell-surface proteins that recognize DNA from closely-related species and transport it inside. It is then incorporated into the genome by homologous DNA exchange.

21. Transduction: bacteriophages transfer bacterial genes between hosts accidentally.

22. Conjugation (primitive form of mating): direct transfer of genes via a pilus or “mating bridge” Transfer is one-way: donating cell requires a piece of DNA called the F factor F+ cell donates DNA to F- cell: If the F factor genes are on a plasmid, and the entire plasmid is transferred to an F- cell, it can be converted to an F+ cell. Cells with the F factor genes on their main chromosome are called Hfr cells due to their High Frequency of Recombination. R plasmids: carry resistance genes which code for enzymes that specifically destroy antibiotics. Many also carry genes which code for pili to allow conjugation.

23. Regulation of Gene Expression in Prokaryotes: Cells have 2 main ways of controlling metabolism. Regulation of enzyme activity (“feedback inhibition”): the end-product inhibits the enzyme ­­ at the beginning of the pathway. Good for: immediate, short-term response. Regulation of gene expression: end-product represses expression of genes for all the enzymes needed for the pathway (longer-term response).

24. SEE p. 353, Figure 18.20:

25. The Operon Model: Many Bacterial genes are turned on or off by changes in the metabolic status of the cell. In 1961, a basic mechanism for this control of gene expression was first described using E.coli bacteria. E.coli bacteria require the amino acid tryptophan. They can get it from their surroundings or they can produce it using a multistep pathway requiring 5 enzymes. The 5 genes that code for these enzymes are clustered together on the bacterial chromosome and make up one transcription unit. A single promoter serves all 5 genes.

26. Operator: segment of DNA, located within the promoter region, or between the promoter and genes, which switches the cluster on/off by controlling access of RNA polymerase to the genes. Operon = promoter, operator, and the cluster of related genes they control. Advantages of operon clustering: coordinated expression, single switch Separate regulatory genes (with their own promoters) encode proteins which turn the operator off or on: trpR gene encodes the repressor which, together with tryptophan, turns the trp operon off.

27. Remember Protein Synthesis? Transcription (modification) Translation (Where, why, how?)

28. Negative Gene Regulation: Certain operons are switched OFF by the active form of repressor proteins. There are two types: Repressible operons are usually “on” but can be inhibited when a small molecule binds allosterically to a regulatory protein: tryptophan (corepressor) binds the trp repressor protein which can only then bind the operator and block transcription. Inducible operons are usually “off” but can be stimulated when a small molecule interacts with a regulatory protein: lactose (lac) operon contains genes needed for the digestion of lactose. These genes are not transcribed unless lactose is present. It converts to its isomer allolactose which binds directly to the lac repressor, removing it from the operator. Allolactose is an _________________ inducer. http://www.youtube.com/watch?v=oBwtxdI1zvk lac operon 3:08

29. Positive Gene Regulation: Some operons are switched ON by the active form of activatorproteins. cyclic AMP (cAMP) accumulates when glucose is scarce in E.coli cells. This binds to the repressor: catabolite activator protein(CAP) which in turn attaches upstream of the lac operon promoter and directly stimulates transcription by increasing affinity for RNA polymerase. Purpose – limit use of lactose for food unless glucose is scarce. Dual Control: The lac operon has negative control by the lac repressor and positive control by CAP.

30. EUKARYOTES Eukaryotic Genomes: Genome Organization is more complex: Histone proteins help with coiling. They are positively charged so they bind tightly to negatively-charged DNA. Gene Regulation is more elaborate due to: larger genome, cell differentiation

31. Genome Organization: Due to the enormous amount of DNA, an elaborate system of DNA packing is required (4 levels). SEE p.361, Figure 19.2 1st Level: Nucleosomes or “Beads on a String” Made up of: DNA wound twice around 8 histones(2 of each of 4 main types) with “linker” DNA between the beads. 2nd Level: 30nm Chromatin Fiber Made up of: tightly wound coil of nucleosomes held in position by a fifth histone (H1)

32. 3rd Level: Looped Domains Made up of: loops held in place by scaffolding (nonhistone) proteins. 4th Level: Chromosomes as visible during mitosis Made up of: highly condensed, compact domains

33. Gene Regulation: Cells must continually turn genes on and off in response to internal & external signals. Only about 20% of a specific human cell’s genes are expressed at any given time. Expression of specific genes is most commonly regulated at transcription.

34. SEE p.362, Figure 19.3 for a diagram of the following regulatory processes: Chromatin Structure Regulation: lnterphase chromatin, called ___ heterochromatin ___ is so highly condensed that transcription enzymes cannot access it. Histone Modifications: When acetyl groups attach to histones, called ___ acetylation, ___ it loosens chromatin packing so transcription enzymes can more easily access it.

35. DNA Methylation: When certain enzymes methylate DNA bases (usually cytosine), those sections are not transcribed. This seems to cause long-term inactivation of genes such as during: cell differentiation in embryos and X-inactivation. Epigenetic Inheritance: Chromatin modifications that do NOT involve a change in the DNA nucleotide sequence may be passed to future generations of cells. These changes may affect gene expression and are sometimes reversible: (unlike mutations). Illustration of a DNA molecule that is methylated at the two center cytosines. DNA methylation plays an important role for epigenetic gene regulation in development and disease.

36. Transcription Initiation Regulation: Proteins can bind DNA and either inhibit or facilitate the binding of RNA polymerase. Activator proteins and DNA-bending proteins help “enhancers” on the DNA initiate transcription.   Coordinately Controlled Genes: Eukaryotic cells do not have operons. Instead a single signal (often from outside the cell), like a steroid hormone, can activate a group of genes that need to be co-expressed regardless of their location on diverse chromosomes. All co-expressed genes (such as genes coding for the enzymes of a metabolic pathway) have a common control element recognized by the same chemical signal.

37. Post-Transcriptional Regulation: Cells can regulate gene expression even after a gene is transcribed to rapidly respond to environmental changes by: RNA processing: alternate splicing mRNA Degradation: rate Translation Initiation: can be blocked by regulatoryproteins. Protein Processing & Degradation: regulation can occur during any of these final steps.

39. Transposons: • Discovered by Barbara McClintock (indian corn) • Non-coding DNA sequences which exert control over genes by jumping in or out of functional gene sequences: “Jumping Genes” Originally mocked for her conclusions about transposition in the 1950’s, she was awarded the Nobel Prize for Physiology or Medicine in 1983 (at the age of 81!); she is the only woman to receive an unshared Nobel Prize in that category. turning them on or off

40. Cancer Connections Proto-oncogenes: Normal cellular genes which code for proteins that stimulate normal growth and division. They can be converted to oncogenes. Oncogene: Gene responsible for a cell becoming cancerous. Oncogenes arise from 3 main causes: Movement of DNA within the genome. Amplificationof a proto-oncogene. Point mutations in a control element or the oncogene itself. Tumor-Suppressor Genes: Normal cellular genes which code for proteins that inhibit cell division. Mutations in these genes can also contribute to the onset of cancer.