CPN60 Gene Conservancy Via Protein-Sequence Comparison Across
Extremophilic Microorganisms
Richard Barrera
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692.
Abstract
Microorganisms can survive and flourish in
environments which are detrimental to the
majority of life on the planet. Research
focused on final polypeptide modification in
extremophilic microorganisms, comparing
protein production as in more advanced,
eukaryotic cells. I hypothesize that effective
protein modification-management is an
integral step in early cellular ; therefore the
sample organisms will demonstrate a high
level of cpn60 gene converservancy. This will
provide evidence for the shared ancestry
and interrelatedness of organisms separated
by millions of years of evolution; the data
from this study and recently published
literature also hint at how prion and
neurodegenerative
diseases
may
be
propagated. Specifically, this study searched
for the presence of the groEL protein across
twelve sub-classes of extremophiles. The
groEL protein is a 60 kilo-dalton protein,
which makes up the large subunit of the
groES/L complex and is the primary Type I
chaperonin used for final polypeptide
modification and maintenance in thousands
of organisms. I searched for the presence of
the groEL protein, and its exon: cpn60, via
amino acid comparison using the National
Center for Biotechnology Information’s
(NCBI) Basic Local Alignment Search Tool
(BLAST) server. Homo sapiens groEL amino
acid sequence was used as target query in
order to demonstrate similarity between
divergent species. N = 24 extremophiles were
identified as candidates for protein
comparison, selecting one to three members
per sub-class; twenty were sequenced and
obtainable via public record; seventeen
organisms demonstrated ≥ 40% positive
match, fourteen demonstrated ≥ 70%
positive match, and three organisms used a
separate Type I chaperonin. Data suggests
homologous toroid chambers for facilitation
of
post-translational
polypeptide
modification are extremely prevalent among
the majority of organisms on the planet.
Introduction
It is now known that molecular chaperones
participate in a large variety of cellular
functions. They assist in de novo protein
folding, stabilize proteins under duress and
maintain polypeptide chain components in a
loosely folded state for translocation across
organelle membranes (Kumarevel, et al. 1998).
Research focused on the presence of the groEL
protein, contained in the cpn60 exon; the 60 kD
protein is the large subunit of the groEL/S
complex, in extremophilic microorganisms. The
groEL/S complex is one of the primary Type I
chaperonins used for final polypeptide
modification, and among other responsibilities,
handles
the
folding
of
monomeric
mitochondrial rhodanese (Mendoza, et al.
1991). The target query was the five hundred
and four character sequence of the groEL
protein in Homo sapiens. The target region is
believed to be a universal target of about five
hundred and fifty five bp, and has been found to
be a robust target for species-level
characterization of bacteria, archaea, and
eukaryotes (Hill, et al. 2012). The presence of
the protein sequence among examined
microorganisms illustrates a shared requirement
among divergent species for post-translational
polypeptide modification in order to sustain
basic cellular function. This is significant
because in recent years the scientific
community has discovered life across a
multitude of environments that were heretofore
believed to be uninhabitable: these chaperones,
in conjunction with stress-induced shock
proteins, act as an efficient protein management
system, preventing the aggregation of denatured
proteins within the cell and programmed cell
death. This has led researchers to reconsider the
pervasiveness of life and its ability to adapt,
colonize, and thrive in extraordinarily
demanding environments. Conversely, prion
and neurodegenerative diseases are often the
result of malfunctioning chaperones within the
cytoplasm or intermitochondrial matrix
respectively, which results in protein
aggregation and cell death (Schon and
Manfredi, 2003).
Materials and Methods
Research was conducted via the following:
Thirty microorganisms across twelve subclasses of extremophiles were identified using
publicly available online databases. Identified
microorganisms were vetted using the Kyoto
Encyclopedia of Genes & Genomes and
GenBank in order to identify whether or not the
complete genome had been sequenced and
published; incomplete sequences are not
available for comparison and were eliminated
from the population. Genome identification
continued until a population of N ≥ 20 was
reached. Amino acid examination was
conducted via protein comparison using the
National
Center
for
Biotechnology
Information’s (NCBI) Basic Local Alignment
Search Tool (BLAST) server. Percentage
conversancy of the amino acid sequence was
calculated between the species using the NCBI
Graphic Representation Tool. A cladogram was
constructed using the NCBI Phylogenetic Tool
in order to visualize the divergence between
species.
Results
Twenty four species of extremophiles across
twelve sub-classes were identified as candidates
for comparison. Of the twenty four species
identified, twenty microorganisms representing
ten groups are sequenced and available on the
National
Center
for
Biotechnology
Information’s database. I could not locate
sequenced organisms belonging to piezophiles
or xerophiles. The targeted amino acid
sequence of the groEL protein was obtained
from Homo sapiens, and contains five hundred
and four characters:
MLRLPTVFRQMRPVSRVLAPHLTRAYAKD
VKFGADARALMLQGVDLLADAVAVTMGP
KGRTVIIEQSWGSPKVTKDGVTVAKSIDLK
DKYKNIGAKLVQDVANNTNEEAGDGTTT
ATVLARSIAKEGFEKISKGANPVEIRRGVM
LAVDAVIAELKKQSKPVTTPEEIAQVATISA
NGDKEIGNIISDAMKKVGRKGVITVKDGK
TLNDELEIIEGMKFDRGYISPYFINTSKGQ
KCEFQDAYVLLSEKKISSIQSIVPALEIANA
HRKPLVIIAEDVDGEALSTLVLNRLKVGLQ
VVAVKAPGFGDNRKNQLKDMAIATGGAV
FGEEGLTLNLEDVQPHDLGKVGEVIVTKD
DAMLLKGKGDKAQIEKRIQEIIEQLDVTTS
EYEKEKLNERLAKLSDGVAVLKVGGTSD
VEVNEKKDRVTDALNATRAAVEEGIVLGG
GCALLRCIPALDSLTPANEDQKIGIEIIKRTL
KIPAMTIAKNAGVEGSLIVEKIMQSSSEVG
YDAMAGDFVNMVEKGIIDPTKVVRTALL
DAAGVASLLTTAEVVVTEIPKEEKDPGMG
AMGGMGGGMGGGMF
Homo sapiens amino acid sequence was
selected for comparison in order to punctuate
evolutionary conservancy between divergent
species. Species were entered into the database,
and seventeen organisms were found to contain
a similar target sequence. The remaining three
organisms, members of the archaea domain,
utilize the DnaJ Type I chaperonin in order to
complete
post-translational
polypeptide
modification. The seventeen species of
extremophiles containing the cpn60 protein
sequence demonstrated a ≥ 40% positive match,
fourteen demonstrated a ≥ 70% positive match,
as shown in Figure 1. Figure 1A gives a graphic
representation of the matching sections of
genome, generated by the NCBI. A visual
representation of genetic divergence is depicted
in Figure 2. The expected value (E-value) was ≤
3.00x10-11.
Discussion
The E-Value represents background noise, or
the percent likelihood that a false positive will
be encountered in the query sequence. The subclasses included in the research were:
acidophiles,
alkaliphiles,
cryptoendoliths,
osmophiles, lithoautotrophs, metallophiles,
oligotrophs,
piezophiles,
psychrophiles,
radiophiles, thermophiles, and xerophiles. This
data is significant because it confirms that life
is predicated on the proper function,
maintenance, and destruction of proteins. Cells
cannot function without a form of intermediary
Identical
Match %
chamber which allows polypeptide chains to
assume , resume, or degrade their tertiary
structures. As such, the evolution of
chaperonins was an integral and promethean
step in the evolution of life on Earth., any
chaperonin mutation which alters its interaction
with hydrolysable ATP binding, or alters the
protein-modification chamber in such a way as
to produce a renegade protein, may result in
significant havoc and ultimately cell death
(Walters et al. 2002). For example, research has
found that the
malfunctioning of oxidative
phosphorylation pathways in mitochondria
leads to the excess generation of reactive
oxygen species. These species decimate the
mitochondria, altering the structure of whatever
they come in contact with, including
chaperonins. These altered chaperonins can no
longer
fulfill their duties of protein
maintenance, and as a result, the mitochondria
self-destructs (Mukherjee and Chakrabarti,
2013).
Positive
Match %
Identical
Match
Expect value
Sphingopyxis alaskensis
56.16
78.36
233
0
Saccharomyces cerevisiae
56.98
75.23
224
0
Wallemia ichthyophaga
56.5
72.74
235
0
Debaryomyces hansenii
56.91
76
231
0
54.7
76.69
238
0
Nitrosomonas sp. AL212
52.37
74.19
250
0
Cupriavidus metallidurans
53.77
74.15
244
0
Acidithiobacillus ferrooxidans
51.04
74.38
257
0
Thiobacillus denitrificans
53.02
73.4
247
1.00E-180
Pelagibacter ubique
Flavobacterium psychrophilum JIP02/86
51.7
73.3
251
1.00E-179
Bacillus subtilis
51.33
73.19
253
1.00E-179
Amphibacillus xylanus
50.57
72.62
257
9.00E-179
48.7
70.26
264
1.00E-163
Pyrolobus fumarii 1A
23.36
44.86
340
1.00E-017
Methanopyrus kandleri AV19
23.23
42.47
323
5.00E-017
Methanococcoides burtonii DSM 6242
23.03
43.07
340
2.00E-015
Sulfolobus solfataricus P2
22.02
41.74
352
3.00E-011
Deinococcus radiodurans
Figure 1. Hit table generated by BLAST data analysis. Percent match is in relation to Homo sapiens template code.
Identical match is number of correct chemical and spatial amino acid matches. Chart is ordered by E Value (most certain
match to least).
Figure 1A. Graphical representation of hit table from Figure 1. Red areas indicate identical matches, grey areas indicate
positive matches. Organisms are listed in the same order as Figure 1.
Figure 2. A phylogenetic tree based on genetic divergence of 504 character amino acid sequence; present are the seventeen
microorganisms sampled which contain the cpn60 gene.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s):_Richard Barrera____
Title: CPN60 Gene Conservancy Via Protein-Sequence Comparison Across Extremophilic
Microorganisms
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
This study focuses on the relationship between protein modification-management and the
CPN60 gene. The researchers used NCBI to do a protein comparison between twenty four
microorganisms (extremophiles) whose complete genomes had been sequenced. Homo sapiens amino
acid sequence was also used as a comparison with the microorganisms. When they were compared to
Homo sapiens, seventeen species showed a match that was greater than 40%, and fourteen showed a
match greater than 70%. Any statistics that were run for this study were unclear or absent, so the
significance of the data was difficult to determine. According to the study, the data is significant, and
shows that protein modification-management is essential to survival in extreme environments, and thus
conservancy of the CPN60 gene is high in these organisms. The significance of the data in relation to
Homo sapiens was unclear.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of
knowledge.
The study seems very interesting, but the overall goal and results of the study were a little
unclear. The hypothesis states only that organisms in the sample would have “high CPN60 gene
conservancy” but it’s difficult to discern which organisms are being referred to. The introduction and
abstract discuss microorganisms (extremophiles), but the results and discussion involve a comparison
with Homo sapiens. I also was a bit confused about the statistics that were run for this study. Maybe the
program they used ran a different type of statistical analysis than we learned in class, but I didn’t see
any statistical values that show that the data is significant. There was a sentence in the results about the
percent match that some of the organisms had with Homo sapiens. Is this the statistical analysis?
Overall it was difficult to tell exactly what this study was about and what experiment was carried out.
The paper was also missing a Literature Cited section and the figures were not placed in the
results section (they were at the end of the paper/in the discussion section). Not sure if this is some kind
of formatting error occurred, but it made the paper look unfinished (like a rough draft).
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor
textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
This paper was a final version
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published
This paper was a rough draft