Flashcards
phylogeny and taxonomy
Cards in this deck (25)
- 1What is ‘Phylogeny’?Phylogeny – The evolutionary history of organisms.
Phylogenetics - Studies that investigate the evolutionary relatedness of organisms - 2What is ‘Taxonomy?• Taxonomy – Is the science of classification, including the sub-disciplines of identification and nomenclature.
• Traditionally the study of organismal diversity (Systematics) has focused upon naming and classifying organisms (Taxonomy)
• In microbiology this has relied upon phenotypic analyses – morphological, metabolic characteristics….to group and name organisms. - 3Evolutionary Chronometers• Certain macromolecules can be used as ‘Evolutionary Chronometers’ – actual measures of evolutionary change
• Should contain highly conserved regions that allow the sequences to be aligned – but also contain regions of variability that allow an estimation of evolutionary divergence.
• Macromolecular sequencing used in mid 60’s to infer eukaryotic phylogenies – e.g. cytochromes - 4Essential to Choose the Right Chronometer• The molecule should be universally distributed across the group chosen for study.
• It must be functionally homologous in each organism.
• Phylogenetic comparisons must start with molecules of identical function.
• To compare sequences must be able to align – identification of sequence homology and sequence heterogeneity - 5-The sequence of the molecule should change at a rate commensurate with the evolutionary distance measured (broader the distance…. Ideally the slower the rate of change required).
- 6rRna• Ubiquitous. • Exhibits functional constancy. • Changes slowly in sequence (stable mutations).
• Easily isolated experimentally
• Limited influence of horizontal gene transfer (therefore an evaluation of vertically derived variation – over time) - 7rRNA• In eukaryotes ribosomes are classified as 80S (Svedberg units – a non-SI unit for sedimentation rate).
• In prokaryotes ribosomes are classified as 70S.
• In prokaryotes the ribosomes consist of 3 molecules of rRNA and 52 proteins.
– 5S rRNA subunit
– 16S rRNA subunit
– 23S rRNA subunit - 85S rRNAhas been used but because it is small (~120 bases) yields limited information
- 923S rRNA (~2900 bases) & 16S (~1500 bases)have very conserved regions – allows alignment of the sequences for comparative purposes.
• Also have sufficient variability in other regions – so serving as a phylogenetic chronometer.
• Most work carried out with 16S rRNA – due to ease of handling (18S SSU used from eukaryotes). - 10Sequencing of 16S rRNADNA is isolated from the organism and Polymerase Chain Reaction Amplification (PCR) is used to specifically amplify the 16S rRNA gene.
- 11Sequencing of 16S rRNADNA is isolated from the organism and Polymerase Chain Reaction Amplification (PCR) is used to specifically amplify the 16S rRNA gene
- 12Sequencing of 16S rRNAThe PCR method uses primers targeted at the conserved regions of the sequence – so the rRNA gene can be amplified from a wide diversity of organisms – isolated and cloned then sequenced
- 13PCR Basic Protocol• Isolate DNA.
• Heat to separate strands.
• PCR - Primer extension with DNA polymerase.
• Repeat – yields multiple copies of 16S rRNA gene target.
• Run agarose gel check and purify product. • Sequence product.
• Computer analysis and comparison.
• Can be done with mixed community DNA isolated from environmental samples – DGGE analysis. - 14Methodology for Total Community Analysis:• Microbial Community.
• Extract DNA (SDS Lysis/Bead Beating).
• Total Community DNA.
• PCR 16S rRNA genes.
• Ribosomal DNA clones or DGGE analysis.
• Rapid Sequencing and use this data to generate phylogenetic tree - 15Phylogenetic Analysis • Fluorescent in situ hybridisation –FISH• Allows direct identification and quantification of specific and/or general taxonomic groups in situ.
• Cells are fixed – 16SS or 23S rRNA genes are hybridised with fluorescently labeled taxon specific oligonucleotide probes (often 15-20 bases long).
• Viewed using a scanning confocal laser microscope (SCLM). - 16Phylogenetic AnalysisPhylogenetic Analysis
• Avoids artefacts arising from biases in DNA extraction, PCR amplification and cloning. • FISH is very sensitive and can detect microorganisms across all phylogenetic levels. • SCLM can generate images in 3-D of several taxonomic groups simultaneously – using distinctive fluorescent dyes. Therefore yields important information regarding spatial organisation of microbes in situ. - 17Conventional and molecular taxonomyDistinguish between bacterial taxonomy & phylogeny.
• Bacterial taxonomy has traditionally relied upon – phenotypic characteristics – morphology, metabolic features
• Bacteria were/are classified on the basis of phenotypic similarity. A PHENETIC SYSTEM - 18Phenetic systemThis is a natural classification system – that arranges organisms into groups whose members share many characteristics and reflects the biological nature of the organisms.
- 19Conventional and molecular taxonomyphenotypic analysis still plays an important role in classification and identification – especially where identification is the end point – e.g. in a clinical environment or identification of spoilage organisms
- 20Conventional and Molecular Taxonomy NUMERICAL TAXONOMYIf you require to classify a bacterium using phenotypic tests - lots and lots (100’s) of tests are carried out each yielding a definitive score – usually either +ve or –ve for that characteristic. Data is grouped as 0/1 data.
• This quantitative approach is known as numerical taxonomy – the grouping of taxonomic units (phenotypic characteristics) into taxa on the basis of their character states. - 21TAXAOrganisms sharing many characteristics make up a single group or taxon.
- 22Conventional and Molecular Taxonomy COMPUTITIONAL ANALYSISComputational analysis measures the agreement between two organisms and the characteristics possessed – this is carried out for the whole group. • Generates a SIMPLE MATCHING COEFFICIENT (Ssm)
- 23The Calculation of Association Coefficients for Two organisms
- 24The Calculation of Association Coefficients for Two organismsSsm are arranged to form a similarity matrix. • Rows and columns represent organisms and the Ssm between each is indicated in the table. • Often presented as a - DENDROGRAM
- 25-Once a group has been phenetically classified unknowns can be identified – as in % similarity to member of the various phenon.
Because it has not been possible to classify bacteria on the basis of phylogenetic relationships (until the advent of appropriate methodologies) – the system used is primarily PHENETIC
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- microbio Flashcards on phylogeny and taxonomy, created by Fionnuala O'Brien on 05/04/2017.
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- Fionnuala O'Brien
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