Key for Midterm #1 - MB 409 - Feb 17, 2003

1. What are the three primary evolutionary branches of life? (5 points)

Bacteria, Archaea, and Eukarya (note that these must be spelled correctly!)

2. List 5 ways in which all known organisms are alike. (5 points)


3. List one of the typical distinctions between prokaryotes and eukaryotes, and then describe why this is not such a fundamental difference at all. (5 points)

e.g. Prokaryotes have one circular chromosome, eukaryotes have lots of linear chromosomes.

But, many prokaryotes have multiple chromosomes. In some cases, these are linear. On the other hand, most eukaryotes have a small number of chromosomes. Large genomes in lots of chromosomes are rare in eukaryotes, limited essentially to vascular plants and animals.

4. Why does the similarity between two sequences underestimate their evolutionary distance? Does this effect incease or decrease as sequences become less related? Why? (10 points)

Counting differences between two sequences underestimates the number of changes that occured between them, because more than one evolutionary change at a single position (e.g. A -> G -> U) counts as only one difference between two sequences, and in the case of reversion counts as no change at all (e.g. A -> G -> A). This underestimation increases as sequences diverge, because the probability of such multiple mutations at individual sites increase as the total number of observed changes increases.

5. Why is the ssu-rRNA gene sequence such a good molecular chronometer for molecular phylogenetic analysis?. (10 points)

6. Describe one method other than ssu-rRNA molecular phylogenetics used to determine relationships between microbial organisms. (10 points)

e.g. Serology is used primarily to identify very closely-related clinical isolates, usually different strains of a single species. This method uses antisera developed from various strains of Bacteria to identify which strain a new isolate is. For example, when Salmonella is isolated from a patient, a bank of antisera is used to determine which of the hundreds of serotypes that particular isolate is. This is an old but still widely used method, since the antisera are easy to make and the assay is very quick, easily automated, & reliable.

7. Align the following ssequences on the basis of sequence similarity. (10 points)

	Sequence A :	  C U C G A G U U A A C C C G G C A C C C G 
	Sequence B :	  G C U C G G G U U A A C A C G G A C C C G 
	Sequence C :	  U C G A G C C A A C U C G G A C C C G

	Sequence A :	  - C U C G A G U U A A C C C G G C A C C C G 
	Sequence B :	  G C U C G G G U U A A C A C G G - A C C C G 
	Sequence C :	  - - U C G A G C C A A C U C G G - A C C C G

8. Draw the secondary structure of RNA B based on this sequence alignment and the secondary structure of RNA A. (10 points)

	RNA A : A C G U C G C G A G G U U C G C C A - C G C A -
	RNA B : - - G C C C C G U G G C U U G C C A A C G G A U

                                     U U
                    U C             C   G
          RNA A    U   G             G-C
                    G-C              G-C
                    G-C             U   A
                   A   A                A
                    G-C              G-C
                    C-G              C-G
          A C G U C G-C A      G C C C-G A U

9. Attached to this test is the secondary structure of the E. coli ssu-rRNA and an unknown ssu-rRNA. Below is a short signature table designed to discriminate between bacterial, archaeal, and eukaryotic ssu-rRNAs. The nucleotide numbering on the signature table is based on the E. coli ssu-rRNA (i.e. the E. coli RNA is the reference sequence). Perform a signature analysis, and identify which kind of organism the unknown is. Show your work - no credit will be awarded without it! (10 points)

     Position    Bacteria   Archaea   Eukarya    unknown
     537         A or G +   C X       C X        G
     551         U +        A or G X  U +        U
     585         G X        C +       U X        C
     675         A +        U X       U X        A
     716         A +        C X       C or U X   A
     756         C X        G +       A X        G
     912         C +        U X       U X        C
     923         A X        G +       A X        G
     930         C +        A X       G X        C
     931         C +        G X       G X        C
     952         U +        C X       C X        U
     962         C +        G X       U X        C
     966         G +        U X       U X        G
     973         G +        C X       G +        G
     975         A +        G X       G X        A
     1060        U X        C +       C +        C
     1086        U +        C X       C or U +   U
     1087        G +        C X       U X        G
     1109        C +        A X       A X        C
     1110        A +        G X       G X        A

                16/20       4/20      4/20     

This sequence seem to be from a bacterium (in fact, it is Aquifex pyrophilus)

10. Redraw the phenogram below as a dendrogram. Keep approximately the same scale. (10 points)


11. Answer the following questions about this ssu-rRNA tree: (10 points)

a. Which organism is probably the outgroup? Methanosarcina barkeri

b. Which 2 organisms are apparently the most closely related? Halobacterium cutirubrum and Halobacterium sp NRC-1

c. Which ssu-rRNA sequence is most similar to that of Natronobacterium gregoryi? Haloferax volcanii

d. Which ssu-rRNA sequence is least similar to that of Methanosarcina barkeri? Halococcus morrhuae

c. Other than giving a formal species name to Halobacterium sp. NRC-1, what problem do you see in the taxonomy of the species shown in this tree?

The genus Halobacterium is split - one specie (H. trapanicum) in one part of the tree, the rest (H.cutirubrum & NRC-1) elsewhere. One of these groups needs to be renamed.

12. In an episode of the X-files, Agent Scully sequences some alien DNA and finds 'missing bands' in the sequences that she interprets to correspond to bases that are unique to aliens (not found in earthling DNA). Why is this not technically feasible? (5 points)

DNA sequencing reactions contain all 4 "earthling" dNTPs (dATP, dGTP, dCTP, and dTTP) for DNA synthesis, and 4 chain-terminating ddNTPs (ddATP, ddGTP, ddCTP, ddTTP). If alien DNA contained additional bases, and even if DNA polymerase could use these bases for DNA synthesis, there are no dNTPs or ddNTPs to match these extra bases and so the sequencing reactions would most likely just fail to work at all, stopping at the first occurence of one of these extra bases, because there would be no dNTP to basepair to the alien base. If the alien bases could basepair to the standard 'earthling' bases, the sequencing reactions might work, but there would be no gaps - the alien bases would appear in the sequencing reactions to be replaced by standard earthling bases.