Jennifer Okonta

Shared Assignment

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

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 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)