Introduction to Comparative Genomics
tarix 05.05.2017 ölçüsü 445 b. #16828
Introduction to Comparative Genomics Basic biology of Haemophilus spp. Specific goals Unique genes Virulent Factors Surface proteins Strategy Introduction to Comparative Genomics Introduction to Comparative Genomics Basic biology of Haemophilus spp. Specific goals Unique genes Virulent Factors Surface proteins Strategy DNA sequences encoding proteins and RNA responsible for function conserved from last ancestor should be preserved in contemporary genome sequences. DNA sequences encoding proteins and RNA responsible for function conserved from last ancestor should be preserved in contemporary genome sequences. DNA sequences controlling expression of genes regulated similarly in two related species > also be conserved . Sequences that control gene expression, proteins and RNAs responsible for differences between species should be divergent. To understand the genomic basis of the present Differences in lifestyle pathogen vs. nonpathogenic obligate vs. free-living Host specificity In the case of emerging pathogens: this understanding should help us in fighting disease (drug discovery, vaccines) To understand the past How organisms evolved to be what they are now Rearrangements of gene structure Rearrangements of gene structure Gene/region loss Chromosome plasmid DNA exchange Vertical descent (speciation) Horizontal gene transfer (HGT) Genetic exchange between different evolutionary lineages. Genetic exchange between different evolutionary lineages. - Transformation, Transduction, Conjugation Acquire variable number of accessory genes encoding adaptive traits. Most of these accessory genes acquired by HGT form syntenic blocks recognized as genomic islands (GEIs) Large segments of DNA Large segments of DNA Different GC content Often inserted at tRNA genes Often flanked by 16-20kb direct repeats Harbour genes encoding factors involved in mobility -integrase, transposases and IS Homologs: Homologs: Genes sharing a common ancestor and generally retain same function Orthologs: Genes (homologs) in different species derived from a single ancestral gene in the last common ancestor (LCA) (arise from speciation) Paralogs: Homologs in same species related via duplication Duplication before speciation (ancient duplication) Out-paralogs; may not have the same function Duplication after speciation (recent duplication) In-paralogs; likely to have the same function Introduction to Comparative Genomics Introduction to Comparative Genomics Basic biology of Haemophilus spp. Specific goals Unique genes Virulent Factors Surface proteins Strategy Genus of gram negative, coccobaccili bacteria Belonging to the Pasteurellaceae family Either aerobic or facultative anaerobic Of the eight Haemophilus species residing as commensal organisms in the pharyngeal cavity of humans. H. influenzae is by far the most pathogenic - Hi Strains possessing a type b capsule are often associated with invasive diseases such as meningitis, sepsis and pneumonia. - and strains lacking a capsule (NTHi) are associated with localized mucosal diseases, such as otitis media, sinusitis, and bronchitis. H. haemolyticus emerging pathogen. As the name of the species implies, is generally hemolytic on blood agar plates. Beta-hemolytic phenotype routinely used in the clinical setting to distinguish H.h from NTHi. Non-hemolytic H. haemolyticus strains are being isolated > misidentified as NTHI. Genotyping assays include: DNA-DNA hybridization, 16S rRNA gene sequencing, MLST : internal fragment of seven housing keeping genes others: PCR, DNA blot Introduction to Comparative Genomics Introduction to Comparative Genomics Basic biology of Haemophilus spp. Specific goals Unique genes Virulent Factors Surface proteins Strategy They will assist in successful characterization and distinction of H. Haemolyticus and H. influenza which is still an open challenge to be addressed. They will assist in successful characterization and distinction of H. Haemolyticus and H. influenza which is still an open challenge to be addressed. Are there any methods tried or currently available to address this challenge? …..Yes!! Bacterial culture of Bacterial culture of H. influenzae is performedon agar plates with added X(hemin) & V(NAD) factors. But H.Haemolyticu s also require both X and V factors for their growth. MLST is highly unambiguous and portable technique to characterize isolates of bacterial species using multiple house keeping genes. MLST is highly unambiguous and portable technique to characterize isolates of bacterial species using multiple house keeping genes. The principle of MLST is simple: the technique involves PCR amplification followed by DNA sequencing of the house keeping genes. MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic profiles. Let us assume there are three strains in certain bacterial species , say Strain_1 , Strain_2 and Strain_3 Let us assume there are three strains in certain bacterial species, say Strain_1 , Strain_2 and Strain_3 The first step in MLST is identification of house keeping genes . Lets say we have 3 house keeping genes in this species. MLST exploits the possibility of occurring different (variable) sequences for each house keeping gene. All unique sequences for each house keeping genes are assigned allele numbers Now we have allele profile for all the strains. PCR Amplification and DNA Sequencing Seven isolates presumed to be H.influenza were subjected to multilocus sequence typing by a group of researchers. Seven isolates presumed to be H.influenza were subjected to multilocus sequence typing by a group of researchers. They were consistently unable to amplify fucK from one isolate. Failure to amplify the fucK gene fragment from presumptive H. influenzae isolates has been considered an indicator of a misidentified strain. However, failure to detect the fucK gene cannot be considered conclusive since some strains of H. influenzae have recently been shown to lack the fucose operon. There have been many methods in the past to characterize and distinguish H. Haemolyticus and H. influenza. None of those methods saw success due to the associated disadvantages There have been many methods in the past to characterize and distinguish H. Haemolyticus and H. influenza. None of those methods saw success due to the associated disadvantages So now, our challenge is to identify and characterize unique genes that are specific to H. Haemolyticus. Detecting the presence of these unique genes in unknown strain using PCR assays will help characterize the strain as H. Haemolyticus Introduction to Comparative Genomics Introduction to Comparative Genomics Basic biology of Haemophilus spp. Specific goals Unique genes Virulent Factors Surface proteins Strategy Dostları ilə paylaş:
