What have we learned from Unicellular Genomes?

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What have we learned from Unicellular Genomes?
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Propionibacterium acnes

• Responsible for acne, its genome sequenced in
  2004.
• It lives on human skin in sebaceous follicles
  feeds on sebum and this stimulates immune
  response of inflammation.
• Can we understand pimples?

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Anatomy of acne
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Propionibacterium acnes genome

• Sequenced by three different groups.
• 32 190 sequencing reactions
• 8.7-fold coverage of 2 560 265 bp genome
• Error rate of 0.0001
• Genome contains a single circular chromosome and
  no additional plasmids.
• Annotation of 2333 putative genes, allowed for
  construction of the metabolism.

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Propionibacterium acnes genome

• 12 encoded RNA products (rRNA and tRNA).
• 1578 (68) is orthologous with other organisms
  and 20 does not match with anything.

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GC skewing

• A non-uniform distribution of guanine and
  cytosine bases on the two strands of DNA.
• Origin of replication have the lowest GC skew
  (even distribution)
• Terminus of replication have higher GC skewing.

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Horizontal Transfer

• Genes appeared in genome through an unknown
  mechanism.
• To find alien genes, scan the genome with a
  sliding window for segments that have an abnormal
  GC content (either higher or lower than the
  species average) and evaluate the codon bias.
• Which codon is used more often than other codons
  for a particular amino acid.

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Transcriptional Phase Variation

• Variation in the Gs is used to produce
  transcriptional variation.
• Initiation of transcription depends on the number
  of consecutive guanines on a particular strand at
  a critical location upstream of the coding
  region.
• Regions of replicating bases are difficult to
  accurately replicate which will affect the
  transcriptional efficiency.

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Which genes cause pimples?

• Metabolic reconstruction
• Can grow anaerobically and aerobically.
• Has many enzymes to degrade lipids, esters and
  amino acids.
• P. acnes digestive enzymes have LPXTG motif that
  targets proteins to the extracellular wall these
  enzymes chew away on your cells.
• cell-wall sorting signal LPXTG responsible for
  covalently anchoring proteins to the cell-wall
  peptidoglycan
• LPxTG, the target for cleavage and covalent
  coupling to the peptidoglycan by enzymes called
  sortases

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Which genes cause pimples?

• Cells exterior is decorated with hyaluronate
  lyase that destroys the extracellular matrix
  binding your skin cells together and thus
  facilitates further tissue invasion and digestion.

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LPxTG Database Sortase substrates
http//bamics3.cmbi.kun.nl/cgi-bin/jos/sortase_sub
strates/index.py
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Stimulation of immune response

• Genome encodes five CAMP (Christie, Atkins,
  Munch-Peterson) factors. CAMP factors are
  secreted proteins that bind to antibodies (IgG
  and IgM) and can form pores in eukaryotic cell
  membranes.
• Lysis of our cells trigger an immune response.

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CAMP factors

• Proteins from BACTERIA and FUNGI that are soluble
  enough to be secreted to target ERYTHROCYTES and
  insert into the membrane to form beta-barrel
  pores. Biosynthesis may be regulated by HEMOLYSIN
  FACTORS

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Quorum Sensing

• Many bacteria have evolved the ability to
  condition culture medium by secreting
  low-molecular-weight signaling pheromones in
  association with growth phase to control
  expression of specific genes, a process termed
  quorum sensing
• Bioluminescence
• antibiotic biosynthesis
• Pathogenicity
• plasmid conjugal transfer

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Quorum Sensing

• LuxS produces the precursor of autoinducer-2
  (AI-2), 4,5,-dihydroxy-2,3-pentanedione (DPD),
  whilst converting S-ribosylhomocysteine to
  homocysteine.

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Are all bacteria Living in Us Bad for Us?

• An average adult body is composed of about 10
  trillion human cells.
• Every milliliter of your large intestines
  content is estimated to contain 10 billion
  microbes and our intestines contain about 1 L..
• There are about 500 to 1000 different species
  living in an adults intestines.

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Bacteroides thetaiotaomicron

• 31 million bases
• Assembly of 867 contigs with many gaps.
• Finished assembly by PCR
• 67 938 sequencing runs into a single 6 260 361 bp
  circular contig.
• Annotated 4779 predicted ORFs with 58 orthologs
  of known function, 18 orthologs of proteins with
  no known function and 24 with no recognizable
  sequence similarity.

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COGs

• Clusters of orthologous group are functional
  categories of genes.
• They are phylogenetic classiciation of proteins
  encoded in complete genomes.
• Transcription
• Energy production, etc.

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http//www.ncbi.nlm.nih.gov/COG/
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Eukaryotic Clusters
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ADH
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CDH
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Bacteroides thetaiotamicron

• It can metabolize sugars.
• 170 genes for polysaccharide metabolism paralogs
  of 23 genes.
• E. coli has only 8 of them.
• It can also import sugars into its own cytoplasm.
• Has two genes SusC and SusD represented by 163
  paralogs.

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Transposable Elements

• 63 TEs contain ORFs (open reading frames) that
  help spread tetracycline and erythromycin
  resistance between individual cells and between
  species in the microbiota of the gut.

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Coding Capacity

• Gene density for B. thetaiotaomicron is 89.
• Average size of a gene is 1170 bp-largest among
  bacteria.
• M. genitalium 1100 bp
• H. pylori 1000 bp
• E. coli 950 bp

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Can Microbial Genomes Become Dependent upon Human
Genes?

• Second smallest bacterial genome of a
  self-replicating species (589 070 bp).
• A team in TIGR (The Institute for Genomic
  Research)
• 5 people, 8 weeks assembled 8472 high-quality
  sequencing reactions.
• Overall GC content is 32
• GC skew reveals the origin of replication as DnaA
  and DnaN genes.
• Right to the OR transcribed from plus strand
• Left to the OR transcribed from minus strand
• tRNA and rRNA genes have higher GC content, 52
  and 44.

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Genome Map

• 470 ORFs 88 coding capacity average gene is
  1040bp.
• Retained genes for energy metabolism, fatty acid
  and PL metabolism, replication, transcription,
  and protein transport.
• Lost DNA when no need for it.
• aa synthesis
• Cofactors
• Cell envelope
• Regulatory factors

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Synteny

• When a series of genes are conserved in order and
  orientation between two or more species, the
  genes are described as syntenic.
• M. genitalium and H. influenzae has similar gene
  orders with respect to two clusters of ribosomal
  proteins.

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Minimum Number of Genes

• Synthetic biology to synthesize de novo (from
  scratch) a functioning genome with as few genes
  as possible.
• Bacillus subtilis 190 genes
• M. genitalium 260 genes

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Bacteria vs. Viruses

• Smallest genome is an Archean N. equitans (490
  kb)
• HIV-9200 nt
• SARS-29797 nt
• Lambda-48502 nt
• Acanthamoeba polyphaga-Mimivirus infects amoeba
• dsDNA-1 181 404 bp with 1262 ORFs linear
  chromosome

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Mimivirus Genome

• 28GC rich
• 90 coding capacity
• Uses biased codons-lacking G or C uses the least
  common codon in amoeba the least.
• It has proteins used for translation,
  posttranslation modification, DNA repair-sounds
  more like a eukaryote.
• Encodes topoisomerases
• Has a self-splicing intron

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Is Mimivirus Alive?

• Mimivirus is most closely associated with
  Eurkaryota
• Infectious after 1 year of incubation at 4 C.
• Survived 48 hours of desiccation and 1 survived
  55 C.
• Mimivirus can participate in all major steps of
  translation.
• A life form
• Highly modified virus?

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Malaria

• 3 billion people in the world in tropical and
  subtropical climates affected.
• Malaria causing ekaryotic parasite genus
  Plasmodium
• 2.7 million people die each year.

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Plasmodium

• Plasmodium falciparum is the most lethal form
  transmitted to humans by Anopheles mosquito.
• Infected mosquito bites, parasite leaves salivary
  glands move to liver and infects hepatocytes.
  They mature in hepatocytes and hatch out into
  RBCs.
• A new parasite emerges from RBCs by bursting it,
  release progeny and metabolic waste causing fever
  followed by chills.
• A few cells differentiate into gametes move
  through blood can be ingested by new mosquito and
  gatmetes form zygotes and meiosis and to salivary
  glands.

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Infection of RBCs

• RBC 6 micron plasmodium 1.2 micron
• Plasmodium enters RBC by evading immune system by
  sticking to RBCs.
• Apicoblast organnelle that is made up of a
  remnant internalized alga retaining its small
  genome needed for plasmodium survival.

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Plasmodium Genome

• Three genomes
• Nuclear chromosomes separated through
  pulse-field gel electrophoresis before random
  fragmentation and cloning 22 853 764 with 5268
  ORFs 19.4 GC 52.6 coding capacity average
  gene length 2283 bp.
• Mitochondrial 5967 bp encodes 3 proteins
• Apicoplastic 29 422 bp encodes 30 proteins

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Plasmodium is a eukaryote

• 54 of its genes contains one or more intron with
  an average 13.5GC (exons have higher GC).
• 60 of ORFs have no known function

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rRNA genes

• In many species rRNA genes appear in linear
  clusters
• In Plasmodium, rRNA gene distribution var, their
  expression is host specific some are expressed
  in human the other set is active in mosquito

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Centromeres and telomeres

• Centromeres are AT rich (97) and contain short
  tandem repeats.
• Telomeres have repeated sequences that vary in
  length some genes located nearby telomeres are
  replicated many times therefore genes have
  paralogs.
• Highly variable gene families, var, rif and
  stevor (polymorphic) and may add variation to the
  extracellular surface of the Plasmodium.

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Hydropathy plot
http//expasy.org/cgi-bin/protscale.pl
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Hydropathy plot
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Plasmodium

• 31 of the encoded polypeptides are predicted to
  be integral proteins.
• 1 cell-to-cell adhesion
• 4 evasion of immune system

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Apicoblast

• Derived chloroplast
• Synthesizes fatty acids, isoprenoids, and heme
  groups
• 10 of all proteins help apicoblast DNA
  replication and repair, transcription,
  translation, posttranslational glycosylation etc.

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Food

• Plasmodium feeds on hemoglobin, digests it in
  food vacuole
• It has no genes for aa synthesis no trehalose
  (storage sugar in yeast) storage nor glycogen
  lives at the moment

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Is there a model eukaryote genome?

• yeast

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Yeast Genome

• Published in October 1996
• 12 068 kb genome of 16 chromosomes
• 6272 ORFs
• 38.3 GC with a coding capacity of 70.3
• GC content for eukaryotes generally higher for
  the coding portions.
• Coding capacity is much lower than bacteria
• Yeast has a gene every 2 kb
• Worm has a gene every 6 kb
• Humans have a gene every 30 kb

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Genome structure

• S. cereviciae experienced genome duplication
  events.
• Chromosomes V and X, IV and II, and III and XIV
  are have paralogous regions.
• Duplicated region on chr III contains four genes
  one of which is citrate synthase (cit2).
• Cit2(chrIII) targets peroxisome and cit1(chrXIV)
  targets the mitochondrion.