Instructor:  David Kirchman (kirchman@udel.edu
 
September 4, 1997                         MAST 634                  Lecture 1:  Overview 

Molecular Taxonomy 
             Classical methods don't work; inadequate 
             What organism is it? 
             Microbes only way 

      Other problems 
        Stock Identification 
        Invading species 
        Aquaculture 
        General population biology
            What is molecular taxonomy? 
                Using molecules to identify organisms, specifically the sequence 
                       DNA sequence 
                       Deduced amino acid sequence for protein 

                Several molecules used; see homework problem 
                Concentrate here on small sub unit RNA 

Review 
            Prokaryotes = bacteria and archaea 
            Eukaryotes = everything else 

            See table for differences in cellular organization 

Brief review of ribosomes 
           -know role of ribosome in protein synthesis 
           p 982 and 989 for details 

           Complex structures 
                   several proteins 
                                         SS 70 S               21 polypeptides 
                                         LS 80 S               49 polypeptides 

Miscellaneous information about ribosomes           See Table 
     40% of ribosome is protein 

       ribosomal protein   15% of total protein 
                    protein is   60% of cell mass 

ribosomal RNA = rRNA; genes for rRNA = rDNA 

rRNA up to 30% of cell mass 

    not always true for eukaryotes 
 
Remember: 80% of RNA is rRNA 
 
 
Fundamental problem in Marine Biology and Oceanography: determining growth   rates in the field 

Two major ribosome types  

70S
80S
Organisms Bacteria 
Organelles
Eukaryotes
Small Subunit 30S 
RNA 16S 1542 nt) 
40S 
RNA 18S (1874 nt)
Large Subunit 50S 
RNA 23S (2904   nt) 
5S (120 nt) 
60S 
RNA 285(4718 nt) 
5.8 
5S
Sensitivity chloramphenicol  cycloheximide
 
           Page 1002 in textbook for others 

Antibiotics useful experimental tool 

plants have different rRNA 
 
Some work on 5S, but mostly 16S and 18S rRNA 
 

Why 16S? 
           1. All organisms have it 
           2. Enough data, but not too much 
                                 5S ~ 120 nt 
                                16S   1542 
                                23     2904 
           3. Conserved regions       but some variable 
                                all organisms 
                                only Eucarya, bacteria 
                                selected groups of organisms 
           - gets fuzzy on whether you can determine species 

Why conserved? Determined by 3-D structure. 

See picture of SS rRNA in textbook; good test of sequence is to reproduce structure 

Why is it important to have conserved regions? 
          1. Have to have variable because that's how you determine what's different 
          2. Conserved regions important in finding variable (different) regions 

                                                               Variable 
                          5' conserved...............................................conserved 3' 
 
What have we learned from 16S? 
         - not really good for stock ID 

I. Origin of eukaryotic cell 
         - look at rootless tree 
         - chloroplasts similar to cyanobacteria 
                   same type of P.S. 
         - mitochondria similar to heterotrophic 
                   bacteria, i.e. [CH2O] + O2 H2O + CO2 

Endosymbiotic Theory 
        Eukaryotic cell = collection of prokaryotic cells 
        organelles 
                       mitochondria = heterobacteria 
                       chloroplast = cyanobacteria 
        Other evidence 
                       1. Modern day symbiosis 
                       2. Presence of some genes, but not all in organelles see tables 

II. Recognition of 3rd Domain: Archaea 
                      Carol Woese 
         - very different based on 16S RNA, although they "look" like bacteria 
         - thought to be ancient   found in extreme environments, at first. 

                      High temperature > ~ 80 C 
                      weird metabolism (more later) 
         But now found everywhere Table showing difference between 3 domains 

Not so ancient 
         closer to eukarya? 
         Controversy pt. 

Some evidence for similarity to Eukarya 
Archaean               Methanococcus jannaschii            completely sequenced 
 

              Hydrothermal vents 2600 m 
              Grows high pressure 
              Temperature optimum = 85 C 
              Methanogen = makes methane 
              CO2 + H2 CH4 + cell biomass 
      Unique to archaea 

Compare sequence with known genes 
  - Identify open-reading frames ORF 
         long stretches of sequence that can be translated to a protein 
           without hitting a stop 
         DNA   AA sequence 

Ribosomal proteins in M. jannaschii  
 

                  Present 
            All                      ++ 
            Archaea-only      ++ 
            Bacteria only       - 
Elongation factors     more similar to Eukarya than bacteria 

III. Origin of Life: 3rd major finding from 16S data 
           Deeply-branched microbes are Hyperthermophiles 
           Organisms grow with optimum temperature 80-110 C 
                        unable to grow <60 C 
           Record temperature ~ 160 C ; have to have high pressure for T >100 C 
           How can they survive? No simple answer 
                        some have high G-C 
                              G  C stronger than A=T 
                        ether linkage in  archaea membranes 

Where are they found? 
          Hot springs    Yellowstone National Park 
          Hydrothermal vents 
Very difficult to culture these organisms 
In fact, > 99% of all microbes cannot be cultured 

How do we determine what species are present? 
How do we find 16S rRNA genes = (rDNA) in complex genome? 
 
PCR = polymerase chain reaction 

1.  
 
     Known sequence = primers of ~ 20 nt 
     

2. Any other DNA can be present 
3. Amplifies =   makes many copies of unknown sequence, substantial above other 
DNA 
4. Key: heat to high temperature to separate strands. Originally used heat-labile     enzyme 

Original DNA polymerase: 
          Taq: Thermus aquaticus 
          Found by Thomas Brock in Yellowstone 

Kary Mullis: 
         method presented 1985 
         Nobel Prize 1993 
See description in textbook 
         Can be used 
         Single organisms 
         Single cells 
         Mixed microbial assemblages