Transcript S. solfataricus - York College of Pennsylvania

Comparing rRNA sequences in Korarchaeota and unclassified Methanogen species
with modern day phylogenies
Michael Coco
York College of Pennsylvania, Department of Biological Sciences
Project Summary
There is debate over the conditions surrounding the first
organisms and the divergence of eukaryotes and archaea. Eukaryotes
are structurally and molecularly similar to certain archaea; uncertainty
still surrounds the idea of which archaea were closest to the common
ancestor and where newly found archaea fit phylogenetically. Newer
phylogenic interpretations are currently provided using morphological
and molecular evidence, in tandem. Much work has been done using
small ribosomal subunits for classification; using an SSU and LSU
combined comparison could yield more reliable results. Deriving the
phylogenies of new species may add important information about a
common ancestor. In this project early eukaryotes, such as Giardia
lamblia, will be compared to Sulfolobus solfataricus, Korarchaeote
pJP27, and methanogenic archaeon “E” series. The experiment will be
conducted using PRC and automated sequencing. Universal SSU and
LSU subunit primers may be used to amplify DNA, which would be
sequenced using a MegaBACE 1000 computer or by
dideoxyribonucleotides and gelelctrophoresis. Differences in base pair
distances and compositions will help us better understand the phylogeny
of modern life. Greater knowledge in this field could facilitate future
experiments exploring endosymbiosis and the evolution of the first
eukaryotes.
Introduction
Early earth theories suggest that life may have begun in extreme
temperatures, which were believed to be bacteria because they were the
oldest known fossils. When the first themophillic archeae was found,
scientists believed that this may have been related to a common ancestor
(archeae were first categorized as bacteria). It is now believed that
archeae do share a common ancestor with modern day eukaryotes.
Archeae, like eukaryotes: possess cellular organelles, have a membranebound nucleus and do not have introns in their genetic code. Do to
archeae’s “abdaptation” to extreme environments, it is believed that they
may have been the first forms of life on earth.
Methanogens are of a great deal of interest in this area because
they offer a less haphazard theory of endosymbiosis. The first bacteria
and methanogens are believed to have come together to live off of each
other. In this proposed relationship, methanogens were able to fix
methane in the early oxygen-poor atmosphere and bacteria were able to
expel their waste without toxic side affects.
Many modern phylogenies are based on molecular data, which
examines specific parts of the genome. Because thermophillic organisms
appeared to have very high G-C content, which produces stronger
intermolecular bonds, these organisms are able to survive in extreme
heat; it is now believed that a high G-C content may correspond with the
age of the species. Certain eukaryotic species, such as Giardia lamblia,
have a high G-C content (75%) and are believed to be closely related to a
common eukaryote-archeae ancestor. The archeae Sulfolobus solfataricus
is thought to be a more recent species with only 67 percent of its genome
containing G-C bonds. Newly discovered families of archeae, such as
Korarchaeote and Anaerobic methanogenic archaeon “E” series, are
believed to be close to the common ancestor, but more research is
needed. Most of the recent research on these organisms has focused on
comparing G-C contents, and in some cases, comparing the SSU’s of
certain representative species; but there has been criticism that the SSU
does not provide enough distinguishing data. I propose to jointly compare
small subunits and large sub-units of rRNA using current techniques.
Comparisons of the Giardia, Korarcaeotea, S. solfataricus and the
methanogen series will help us establish their age. The general
hypothesis is, because Korarchaeotes are believed to be the most
primitive, they should have a higher percent of base pair similarities in
rRNA code as compared to that of Giardia. S. solfataricus should be more
compatible with the Korarchaeote and Methanogen than G. lamblia.
Methanogen should be closer to lamblia than S. solfataricus (vs. lamblia).
Proposed Methods
Literature Review
Dr. Carl Woese is usually given credit as the founder of molecular
evolution in microbes. In the 1970’s Woese constructed a
comprehensive phylogenetic tree, which determined that archaea and
bacteria were two distinct groups. He used G-C content comparisons
and small ribosomal subunits to develop his theory. Other researchers,
such as Galtier, analyzes G-C content in SSU and discovered that high GC correlated with high growth temperatures (66.3%). These studies
helped conclude that archeae are, in fact, their own domain and are
closer to modern-day eukaryotes than bacteria. More recent research
has used rRNA primes that focus on archeae to amplify and sequence
small ribosomal subunit DNA. Researchers were attempting to place
marine archeae within the phylogenetic tree. They concluded that there
was either a rapid separation event after the divergence for the common
ancestor, or the small rRNA subunit did not provide sufficient information
to clarify their origin.
Previous research has sequenced various proteins and rRNA
subunits from humans and a few representative prokaryotes. Data
below indicate some of these differences (Olsen, G., 2000). The data
suggest that differences in proteins and rRNA correspond with
evolutionary divergence. They can also help identify when certain
branches broke off and where they lead. Comparisons of these
sequences support the idea that archeae and eukaryotes may share a
common ancestor.
Obtain samples of G. lamblia, S. solfataricus, archaeon
“E” series and Korarchaeote pJ27. Disrupt cells and use
100ml cellular sample, centrifuge at 8000 X g for 5 min
Resuspend pellate in 2 ml 0.1 EDTA (PH 8.0) and Add
200 microliters of lysozyme, Incubate at 37 C for 1
hour
Add 500 microliters 20% SDS and leave at room
temp, then Centrifuge 10,000 X g for 10 min
Extract with alcohol (isoamyl). Use 120 ng of
primer with 20 ng target DNA and 1.0 U of
Taq polymerase
Conclusion
The results of this experiment will help us obtain a better
understanding of how the first life evolved on earth. The data should
indicate that the newly discovered methanogen and Korarchaeote have
a close relationship to Giardia lamblia. “Molecular distance” between
these species will help us identify when a common ancestor may have
branched off and the characteristics of that microbe. Understanding
what characteristics the common ancestor possessed will facilitate
experiments on endosybiosis and the formation of the first eukaryote.
Determining new phylogenies will also expand our knowledge of
evolution and how new species may be formed.
Literature Cited
•Alonso, R., Elwood, H., Gunderson, M. and Peattie, D. 1997
“Phylogenetic meaning of the kingdom concept: an unusual ribosomal
RNA from Giardia lamblia.” Science. 243: 75-78.
•Cohen, B, and Smith, T. 1998. “Molecular phylogeny of brachiopods
and phoronids based on nuclear-encoded small subunit ribosomal RNA
gene sequences.” Biological Sciences 353: 2039-2052.
•Cho, G., Da-Fei, R., and Doolittle, R. 1999. “Determining divergence
times with protein clocks.” The Biological Bulletin. 196: 356-3.
•DeLong, E. 1998. “Archael means and extremes.” Science 280:5424.
Use Universal Primer SSU:
A large study using small ribosomal subunits was conducted to
clarify the phylogeny of brachiopods and phronids. Researches
sequenced over thirty-three representative species and were able to
verify where certain families belong; before this researcher they were all
grouped in one phyla because they contained a lophophore. The results
were used to create a large cladogram, which classified almost every
genus phylogenetically and revealed which groups were closely related
to each other. The researchers were also able to predict that the groups
diverged from a common ancestor deep in the Precambrian period. The
molecular results were consistent with morphological differences
between the groups.
5’-ACGGGWACCTTGTTACGAGTT-3’
W= A or T
LSU Primer:
5’-ATCTTGGTGGACGAGT-3’
G. lamblia
PJ27
S. solfataricus
Archaeon
G. Lambli
100%
45%
37%
40%
S. solfataricus
37%
80%
100 %
85%
Archaeon “E”
40%
88%
85%
100%
PJ27
45%
100%
80%
88%
•Freeman, S. and Herron, Jon. Evolutionary Analysis. Prentice Hall:
1998.
•Galtier, N., Gouy, M., and Tourasse, N. “A Nonhyperthermophilic
common ancestor to extant life forms.” Science 283: 220-1.
•McInerney, J., Mullarkey, M., Powell, R., and Wernecke, M. 1997.
“Phylogenetic analysis of Group I marine archaeal rRNA sequences
emphasize the hidden diversity within the primary group Archaea.”
Biological Science. 264: 1663-1670
Expected Results
Table 1. Percent alike between PJ27, G. lamblia, S. solfataricus and archaeon “E”
•Embden, D., Gaastra, W., Jansen, R., and Schouls, L. 2002.
“Identification of a novel family of sequence repeats among
prokaryotes.” Science 6: 23-34.
Analysis by machine: Amersham Pharmacia Biotech
MegaBACE 1000. Data can alternatively be
determined using dideoxyribonucleotides and
gelelctrophoresis
•Olsen, G., 2000 “Major groups within the Archaea.” University of
Illinois. www.bact.wisc.edu/microtextbook/ClassAndPhylo/archaea.html
•Vogel, G. 1998 “Did the first complex cell eat hydrogen?”
279: 1633-35.
Science