Jennifer Okonta

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Contents

Defined Words

  1. transposases-Transposase is an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. en.wikipedia.org/wiki/Transposases
  2. chemontaxis-movement by a cell or organism in reaction to a chemical stimulus. wordnetweb.princeton.edu/perl/webwn
  3. prophage-A prophage is a phage genome inserted as part of the linear structure of the DNA chromosome of a bacterium. en.wikipedia.org/wiki/Prophage
  4. integron-A genetic unit that, among others, encodes proteins that splice gene cassettes into chromosomes, where the cassettes can become functional. www.nature.com/nrg/journal/v5/n3/glossary/nrg1292_glossary.html
  5. aetiological- the cause of a disease. wordnetweb.princeton.edu/perl/webwn
  6. heterotrophic- obtaining nourishment from organic substances, not from food produced within the organism.wordnetweb.princeton.edu/perl/webwn
  7. Transposon- a segment of DNA that can become integrated at many different sites along a chromosome (especially a segment of bacterial DNA that can be translocated as a whole).wordnetweb.princeton.edu/perl/webwn
  8. pathogenesis- the origination and development of a disease. wordnetweb.princeton.edu/perl/webwn
  9. Permease - The permeases are membrane transport proteins, a class of multipass transmembrane proteins that facilitate the diffusion of a specific molecule in or out of the cell.en.wikipedia.org/wiki/Permease
  10. Rho-Independent terminator- The rho-independent signal is found on the DNA template strand and consists of a region that contains a section that is then repeated a few base pairs away in the inverted sequence. http://www.sparknotes.com/biology/molecular/dnatranscription/section2.rhtml


Outline

What is Vibrio Cholerae

  • the aetiological agent of cholera
  • includes pathognenic and non pathognenic strains that vary in gene content
  • contains a wide variety of strains and biotypes
  • receives and transfers genes for toxins
  • has colonization factors
  • is resistant to antibiotics
  • has capsular polysaccharides that provide resistance to chlorine and new surface antigens
  • it represents a significant portion of the culturable heterotrophic bacteria of oceans, coastal waters and estuaries
  • these bacteria strongly influence nutrient cycling in the marine environment

Purpose of the Work

  • To determine and analyze the genome sequence of Vibrio Cholerae.
  • It is an important step toward the complete molecular description of how this free-living environmental organism became a human pathogen by horizontal gene transfer.

Genome Analysis & Comparative Genome Analysis

  • Sequenced by the whole genome random sequencing method.
  • Two circular chromosomes make up this genome
  • 3,885 predicted open reading frames (ORFs) and 792 predicted Rho-independent terminators
  • Most genes required for growth and viability are located on chromosome 1
  • two-chromosome structure of V. cholerae allows for comparisons
  • There is pronounced asymmetry in the distribution of genes known to be essential for growth and virulence between the two chromosomes
  • Chromosome 2 contains a larger fraction (59%) of hypothetical genes and genes of unknown function, compared with chromosome
  • Most genes known to be essential in bacterial pathogenicityare located on chromosome 1

Figure Analysis

Figure 1

Linear representation of the V. cholerae chromosomes

  • Shows the location of the predicted coding regions, colour-coded by biological role, RNA genes, tRNAs, other RNAs, Rho-independent terminators and Vibrio cholerae repeats.
  • Arrows represent the direction of transcription for each predicted coding region
Figure 2

Circular representation of the V. cholerae genome

  • 2 chromosomes
  • the first and second circles show predicted protein-coding regions on the plus and minus strand
  • The third circle shows recently duplicated genes on the same chromosome and on different chromosomes
  • The fourth circle shows transposon-related (black), phage-related (blue), VCRs (pink) and pathogenesis genes (red
  • The fifth circle shows regions with significant values for trinucleotide composition in a 2,000-bp window
  • The sixth circle shows percentage G+C in relation to mean G+C for the chromosome.
  • The seventh and eighth circles are tRNAs and rRNAs, respectively.
Figure 3
  • Pathways for energy production and the metabolism of organic compounds, acids and aldehydes are shown.
  • Transporters are grouped by substrate specificity.
  • Question marks associated with transporters indicate a putative gene, uncertainty in substrate specificity, or direction of transport
  • Export or import of solutes is designated by the direction of the arrow through the transporter
  • Gene location on the two chromosomes, for both transporters and metabolic steps, is indicated by arrow color.
Table 1

General features of the Vibrio cholerae genome.

  • self explanatory
  • the size, number of ORF, the number of tRNA, the number rRNA, the number of significant and unsignificant proteins and other things are listed.
Figure 4

Percentage of total Vibrio cholerae open reading frames (ORFs) in biological roles compared with other -Proteobacteria

  • V. cholerae, chromosome 1 (blue)
  • V. cholerae, chromosome 2 (red)
  • Escherichia coli (yellow)
  • Haemophilus influenzae (pale blue)
Figure 5

Comparison of the V. cholerae ORFs with those of other completely sequenced genomes

  • All V. cholerae ORFs (large chromosome, blue; small chromosome, red) were searched against all other genomes with FASTA
Figure 6

Phylogenetic tree of methyl-accepting chemotactic proteins (MCP) homologues in completed genomes

  • Amino-acid sequences of the proteins were aligned using CLUSTALW
  • Neighbor-joining phylogenetic tree was generated from the alignment using the PAUP program

Methods

  • Grown from a single isolated colony
  • Cloning, sequencing and assembly were as described for genomes sequenced by TIGR
  • Sequences from both ends of served as a genome make up to show the orientation, order and integrity of the contigs
  • Sequence gaps were closed by editing the end sequences and/or primer
  • The initial set of ORFs was identified with GLIMMER and those shorter than 30 codons were eliminated
  • ORFs were searched against a non-redundant protein database
  • Frameshifts and point mutations were detected and corrected when needed
  • Paralogous gene families were made by searching the ORFs against themselves using the program BLASTX
  • Probability values for this analysis are based on the assumption that the DNA composition is relatively uniform throughout the genome
  • Homologues of the genes of interest were identified using the BLASTP and FASTA3 search programs
  • All homologues were then aligned to each other using the CLUSTALW program with default settings
  • Phylogenetic trees were generated from the alignments using the neighbour-joining algorithm

Conclusions

  • New starting point for the study of V. Cholerae environmental and pathobiological characteristics
  • The genomic sequence of V. cholerae should facilitate the study of this model multi-chromosomal prokaryotic organism
  • Might provide important clues to understanding the metabolic and regulatory networks that link genes on the two chromosomes
  • Represents a promising genetic system for studying how several horizontally acquired loci located on separate chromosomes can still efficiently interact at the regulatory, cell biology and biochemical levels

Jennifer Okonta 13:32, 17 October 2010 (EDT)

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