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
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 duplication
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
Carry genes carrying seletive advantage
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
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.
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 performed
on agar plates with added
X(hemin) & V(NAD) factors.
But H.Haemolyticus 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
