Microbial Genomics II

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Microbial Genomics II
2011-11-09 04:39:08
PMB 112 midterm2

general microbiology midterm 2
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  1. bacterial genome size
    • > 20-fold range
    • in general, species-specific trait
    • number of genes and total number of ORFs in genome is proportional to genome size
    • - not true for eukaryotes because have splicing
    • coding density for prokaryotic gene is consistent ~1kb/gene
  2. chromosome map
    • initiation codon for the dnaA gene - hallmark for the origin of replication = 0
    • -oriC harder to locate size marked on outer ring
    • genes tend to be oriented in the direction of replication (leading strand - clockwise)
  3. gene orientation
    genes tend to be oriented in direction of replication, transcribing away from the origin of replication (leading strand bias)

    • highly expressed genes tend to be on leading strand essential genes tend to be on leading strand
    • presumably
    • because leading strand arrangement allows RNA pol to avoid collision with the replication fork, with would be moving in the same direction
  4. dominant negative
    • gene product that adversely affects the normal wild type gene product
    • this usually occurs if the product can still interact with the wild type gene products or interactors of wild type gene product thus blocking some aspect of wild type gene product function
  5. How could a truncated essential gene act as a dominant negative?
    essential metabolic protein that operates as a dimer, containing dimerization domain and catalytic domain. catalytic domain of each monomer is required for function.

    • if get truncated protein with only dimerization, no catalytic activity.
    • And, the truncated protein will bind wild type copies of the protein making them inactive.
  6. genes may be clustered yielding polycistronic mRNA
    • biosynthetic genes may be clustered - eg an operon
    • however, most transcriptional units are small
  7. synteny
    • conservation of gene order
    • useful indicator in assessing genome evolution
    • can be useful in predicting gene function

    • dot matrix:
    • axes represent encoded proteins in order they appear on chromosome
    • for each chromosome DnaA is centrally located
    • dots represent a reciprocal best match by FASTA comparison between protein sets
  8. evidence of horizontal gene transfer
    • genes with different %GC content
    • genes with different codon usage bias
    • these regions can be quite large
  9. horizontal transfer
    most frequent between closely related species or between distantly related species that share a common habitat

    transferred genes tend to be "operational"
  10. How does a genome give us insight into the biology of the organism?
    • phylogeny
    • horizontal gene transfer
    • metabolic and regulatory pathways
    • environment
  11. gene content of bacterial genome
    • need more crystal structure - help get at function of unknown proteins, tertiary structure conserved more than primary or secondary
    • genes grouped into functional categories
  12. Why is it difficult to define essential genes?
    • find by knocking out every gene and seeing which are essential
    • done under lab conditions
    • find which are essential by exposing bacteria to different environments and conditions
  13. What does genome tell us about biology of M. tuberculosis H36Rv?
    • cell envelope of gram + contains an additional layer rich in unusual lipids, glycolipids and polysaccharides
    • large proportion of ORFs devoted to lipogenesis and lipolysis
    • - elucidated particular pathways and requirements for lipids from host
    • - elucidated particular enzymes/pathways atypical for bacteria

    • retains full virulence in animal models of tuberculosis
    • - 10% of ORFs are PE and PPE family members
    • - these are novel proteins likely to be of immunological significance as they are predicted to be abundant and potentially polymorphic antigens
    • - confirmed experimentally that PPEs do induce immune cell responses
  14. OMICS
    • provides "global" information
    • "all" of whichever information
  15. microarrays and the transcriptome
    • genes or gene fragments attached to a solid support in a known pattern
    • can be used to hydridize to mRNA and analyzed to determine patterns of gene expression

    microarrays are large and dense enough that the transcription pattern of the entire genome (the transcriptome) can be analyzed
  16. proteomics
    • the proteome encompasses all the proteins present in an organism at any one time
    • the aim of proteomics is to study these proteins to learn their structure, function and regulation

    protein profiling - assesses all expressed proteins at a given time under given conditions

    common experimental approach - 2D gel electrophoresis
  17. 2D electrophoresis
    • run under denaturing conditions
    • each spot is a protein
    • audioradiogram made using radiolabeled methionine

    1st dimension - separated by pH, isoelectric point where net charge of protein is 0

    2nd dimension - mass/size
  18. interactome
    • complete set of interactions among macromolecules within a cell
    • circles (nodes) depict protein
    • lines depict an interaction
  19. metabolomics
    the identification and qualification of all metabolites in a given biological situation

    • need to extract, separate and identify
    • technically difficult due to:
    • number of different compounds
    • variety of compounds (with different chemical properties)
    • low abundance for many compounds

    • common experimental approaches:
    • NMR
    • mass spectrometry