Final exam - MB409 - May 7, 2003 Name _____Key_____

All questions are worth 5 points

1. What are the 3 primary evolutionary branches of life?

Archaea, Bacteria, and Eukarya

2. List the three potential origins for viruses.

  1. genetic offshoots of their hosts
  2. remnants of precellular life
  3. degenerate parasites

3. __B__ Why aren't viruses included in ssu-rRNA molecular phylogenetic trees?

A. because they aren't organisms
B. because they don't have ssu-rRNA genes
C. because most of them have DNA and not RNA
D. because their ssu-rRNAs are so rapidly evolving that they cannot be aligned with the others

4. Describe how one virus (chose one) can be "linked" to the genes/genome of it's host.

For example:

Retroviruses are essentially transmissible retroposons (transposons with reverse transcriptase genes, e.g. Ty in yeast, Copia and P-element in flys.

5. Anna-Louis Reysenbach obtained 3 different bacterial ssu-rDNA sequences from the Octopus Springs pink filamentous microbial community: EM3, EM17, and EM19. How did she determine which of these sequences (EM17) was really the pink filamentous organism?

By fluorescent in situ hybridization using an EM17-specific probe against a sample of the pink filamentous community.

6. List two methods that can be used to sort all of the ssu-rRNA clones in an community analysis into groups so that you don't have to do a complete sequence analysis of every clone.

For example:

RFLP & T-tracts

7. How did Robert Huber use the information from the ssu-rRNA analysis of the Octopus Spring pink filaments to get a pure culture of the organism in captivity?

He used the phenotypic properties of the relatives of EM17 - Aquifex - to hypothesize that maybe EM17 was a hydrogen oxidizer and so supplied enrichment cultures with small amounts of oxygen (i.e. microaerophilic conditions) and hydrogen. He also used the EM17-specific probes to monitor the enrichment cultures - i.e. he probed the enrichments in FISH experiments using the EM17 probe to find those that contained the EM17 organism.

8. What is a bootstrap analysis? What is its purpose?

Bootstrapping is a meethod to assess the reliability of a tree. In a bootstrap analysis, a sequence alignment is randomly sampled over-and-over-again, typically 100 or 1000 times, and trees are generated from each random sampling. The reliability of a particular branching arrangement in a tree is judged by the frequency that the branch appears in the resulting trees.

9. Describe one method you could use to determine whether or not an ssu-rRNA sequence cloned after PCR amplification of DNA from a natural microbial population was a chimera.

For example:

By using the CHECK_CHIMERA function in the Ribosomal Database Project. This program compares the similarity of a sequence along it's length to other sequences in the database - a "break" in this similarity, where the sequence begins to look less and less like one sequence and more like another, indicates that the sequence is probably a chimera.

10. __C__ Why did Sue Barns use primers for her PCR amplifications that would amplify both eukaryotic (nuclear) and archaeal ssu-rRNAs in attempt to get sequences from deep branches of eukaryotes?

A. because she also wanted to know what Archaea were in the environment
B. because it is impossible to design primers that are eukaryotic (nuclear) specific
C. because primers for known eukaryotes might exclude unknown eukaryotes from deeper branches
D. because she screwed up
E. because the eukaryotic-specific primers failed to yeild any PCR product

11. __B__ Mike Manfield used 13C-containing phenol to label the ssu-rRNA of organisms that could degrade phenol in an industrial wastewater bioreactor. How did he separate the labeled ssu-rRNAs of the phenol-degraders from the non-labeled ssu-rRNAs of organisms that could not degrade phenol?

A. by sucrose gradient rate-zonal centrifugation
B. by cesium tetrafluoroacetate density gradient centrifugation
C. by denaturing gradient gel electrophoresis
D. by electrospray mass spectroscopy
E. by flame ionization atomic absorption spectroscopy

12. __A__After separating the labeled vs non-labeled ssu-rRNAs, he used RT-PCR to convert them into rDNA. The rDNAs are all the same size - how did he get them to separate into distinct bands on a gel for each species?

A. by denaturing gradient gel electrophoresis
B. by low-melting agarose gel electrophoresis
C. by SDS-polyacrylamide gel electrophoresis
D. by MALDI-TOF mass spectroscopy
E. by standard agarose gel electrophoresis after separation on cesium-TFA gradients

13. Oded Beja, in Ed DeLong's lab, described the "proteorhodopsin" gene found in a fragment of DNA from an uncultured gamma-proteobacterium of the SAR86 group. What evidence did he present to argue that this was a proton-pumping rhodopsin, rather than a sensory rhodopsin?

1) the light reaction cycle of the proteorhodopsin (expressed in E.coli) was 15msec, similar to ion-pumping rhodopsins but an order of magnitude faster than sensorry opsins, and...

2) suspensions of E.coli expressing proteorhodopsin and given retinal acidify the media when exposed to light - protons are pumped from insdie the cells to the outside. This depends completely on the presence of proteorhodopsin, retinal, and light.

14. Given that it looks like the proteorhodopsin really is a functional light-driven proton pump, that the organisms use to harvest energy for phototrophy, how would you go about determining if the organisms are photosynthetic?

Given that autotrophic gamma proteobacteria fix carbon using the Calvin cycle, you would probably search for the presence of the key enzyme in this pathway, ribulose-bis-phosphate carboxylase/oxidase (RuBPCO).

15. Mike Drancourt isolated DNA from the unerupted teeeth of children that were presumed to have died of bubonic plague, and showed that he could detect, by PCR, both the virulence gene pla and the housekeeping gene rpoB from Yersinia pestis in this DNA. Why did he use unerupted teeth for this?

Unerupted teeth were thought to be the best source for finding the DNA of septicemia bacteria because it is probably the part of a skeletal remain containing organisms in an infected person that is most isolated from the decay process.

16. Raul Cano described the isolation of ssu-rDNA sequences from Bacillus symbionts of stingless bees preserved in 25-40 Mya amber. How did he do the experiment to convince you that the DNA he amplifed by PCR really comes from bacteria trapped with the insect and not more recent (even laboratory) comtamination?

The head, thorax and abdomens of the amberized insects were separated and DNA was isolated from each. The rational here is that in living bees, Bacillus is found predominantly or exclusively in the abdomens, and so they expected only to get a PCR product from the abdomen samples, and this was the case.

17. __E__ What is a massive sulfide deposit?

A. a common sulfide deposit, only bigger
B. the remains of an anerobic, sulfate-reducing sediment
C. an extinct obsidian spring
D. the remains of a volcanic cinder cone
E. the remains of a deepsea hydrothermal vent

18. __A__ Why didn't Birger Rasmussen find any fossil tubeworms or other vent creatures along with the filamentous microfossils in the massive sulfide deposit?

A. because the deposit is precambrian
B. because these creatures are not thermophilic enough to live in this environment
C. because these creatures don't have hard body parts to be fossilized
D. Birger did find such fossils - Calyptogena, the giant vent clam
E. the deposit is too heavily transformed to retain such fossils

19. Describe the origin & travels of the rock that makes up the meteorite ALH84001.

The rock originated on Mars. It is igneous, and solidified 4.5 Bya. The rock went through two shocks on Mars, probably nearby impacts, about 4 Mya. The carbonates that the authors interpret to be fossils originated later, about 3.6 Bya, in fissures in the rock. The rock was ejected 16 Mya from Mars by an asteroid impact. The rock drifted in space for most of the time after that, then descended to Earth 13,000 years ago, landing in what is now called the Allen Hills ice field in Antarctica.

20. List the three things found in the meteorite ALH84001 that McKay & collegues considered evidence for life on Mars Describe one of them.

  1. Polyaromaatic hydrocarbons (PAHs)
  2. Carbonate microfossils
  3. Magnetite bead fossils

For example:

Probably the best evidence that these really are fossils is the presence of magnetite beads. These look exactly like the magnetite beads in magnetotactic Bacteria in size and composition, and if found on Earth would be considered to be unquestionably "magnetofossils". Magnetite can be produced synthetically, but not in this form and it is produced naturally, as far as we know, only biologically. The larger granules (20-100nm) are single-domain magnets, like the internal magnetosome granules, and the smaller ones are supermagnetic, like the periplasmic crystals produced by some magnetotactic species.