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Lecture 2.1

Overview of Phylogeny

I.  Phylogeny - refers to a hypothesis for the evolutionary history of a group of species (or molecule)

    A.  Zuckerkandl & Pauling (1965  ``semantides") Molecules as documents of evolutionary information

    1. More information available by directly studying the molecule than in its morphological or biochemical effects

      2. Evolutionary chronometers - compare homologous macromolecules  (note difference between homologous and analogous)

      3. Requirements

        a. universally distributed

        b. functionally homologous

        c. appropriate level of conservation - must be able to align yet also need change in order to see evolutionary relationships

        d. no lateral gene transfer- gene history must reflect organismal history

        e. sufficient information, readily available

      4. Phylogeny - finds the best fit tree, not necessarily correct tree  - gives a hypothesis!

    B.  Ribosome (Prokaryotic) - 23S, 16S, & 5S rRNAs + ribosomal proteins

      1. 5S rRNA - used at first, small, about 120 nucleotides (nt)

      2. 16S rRNA - ca. 1500 nt, widely used, pioneered by Carl Woese 1970s, >1,300,000 sequences in databases

        a. 18S in eukaryote cytoplasmic ribosomes

        b. mitochondria and chloroplasts have ribosomes with 16S-like rRNA

        c. Phylogenetic analysis of small subunit (SSU) rRNA (both 16S & 18S) by Carl Woese led to the 3 domain tree

        Bacteria                  Archaea             Eukarya

                      

         

        Note: Members of the domain Bacteria and Archaea are prokaryotes, members of the domain Eukarya are eukaryotes

      3. 23S rRNA - more information, harder to sequence, evolving more rapidly

C.  Evolutionary relatedness is a useful way to classify prokaryotes, which lack many of
the obvious phenotypic characteristics of eukaryotes, and it can be used with organisms that we have been unable to culture in the laboratory.  Thanks to phylogeny, there is an awareness of prokaryotic diversity and the number of divisions has exploded

II.  Identification using SSU (small subunit - 16S or 18S) rRNA

A.  Has highly conserved and highly variable regions, allowing for identification to different levels

    B. Signature sequences - nucleotides at specific positions or more generally short oligonucleotides unique to certain groups of organisms

    CAA-AGGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATAAAAACGGAG
    CAA-AGGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATAAAAAGGGAG
    CAATAGGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATAAAAACGGAG
    TAG- AGGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATAAAAACGGAG
    CAA-AGGAAC CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATAAAAACGGGG
    CGA-AGGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAGTAAAAACGGCG
    YRR- AGGAAS CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TARTAAAAASGGRG 

         So the region from 11-42 (if unique to this group of organisms) is a good signature sequence region and can be used to design a probe for this group of organisms

    C. Probes - short oligonucleotides complementary to signature sequences

      1. Can be tagged with a label (radioactive or fluorescent typically) for use in hybridizations to single cells (FISH - fluorescent in situ hybridization) or extracted nucleic acids

      2. Can be used as one of the set of primers for polymerase chain reaction (PCR)

    D. Useful in microbial ecology due to cloning - identify organisms you can't grow!

      1. Many sequences found in environment represent unknown organisms

      2. Helps overcome some culture technique biases - in order to grow something you need to know its growth requirements, so how do you grow an unknown organism?

 

III.   Antarctic Carnobacteria example

 
   
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