CPN60 Gene Conservancy Via Protein-Sequence Comparison Across
Extremophilic Microorganisms
Richard Barrera
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692.
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 evolution and
that 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; my data and recently published
literature also hint at how prion and
neurodegenerative
diseases
may
be
propagated. Specifically, I 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 are sequenced and
obtainable via public record; seventeen
organisms demonstrate ≥ 40% positive
match, fourteen demonstrate ≥ 70% positive
match, and three organisms use 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, we are
beginning to understand that 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: (1)
Thirty microorganisms across twelve subclasses of extremophiles were identified using
publicly available online databases. (2)
Identified microorganisms were vetted using
the Kyoto Encyclopedia of Genes & Genomes
and GenBank in order to identify whether or
not the complete genome has been sequenced
and published; incomplete sequences are not
available for comparison and were eliminated
from the population. (3) Genome identification
continued until a population of N ≥ 20 was
reached. (4) Amino acid examination was
conducted via protein comparison using the
National
Center
for
Biotechnology
Information’s (NCBI) Basic Local Alignment
Search Tool (BLAST) server. (5) Percentage
conversancy of the amino acid sequence was
calculated between the species using the NCBI
Graphic Representation Tool. (6) 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 expect 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 the
chance 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.
Additionally, 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 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 paper discussed research based on amino acid sequencing, specifically the cpn60 protein
sequence, and its conservation across many species. The researcher focused their area of study on the
540 character protein sequence in humans and how this related to other organisms. By using public
databases and comparing the provided genome sequence for specific organisms to the one found in
humans, this study was able to determine that many of these sequences do in fact positively match the
one found in humans. This is an important find and the author definitely goes into discussion about the
importance of the hypothesis, especially in the introduction. Overall it was an interesting topic and the
importance of the study, and the fact that the hypothesis was shown to be true, is demonstrated.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of
knowledge.
This paper contained numerous grammatical and formatting errors. Its greatest weakness is how
it’s formatted and the fact that this research is missing critical information as well, including a literature
cited section. Despite these flaws, the research itself was interesting and relevant to current studies and
knowledge about this field. Although it took some reading to understand what this research was going
to do and how, the relevance of the findings is important. However, the lack of correct citations and the
incorrect formatting errors undermines the integrity of this research.
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
This paper was a rough draft
CPN60 Gene Conservancy Via Protein-Sequence Comparison Across Extremophilic
Microorganisms
Richard Barrera
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692.
Microorganisms can survive and flourish in environments which are detrimental to the
majority of life on the planet. (Whose research – yours?) 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 evolution and that 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. This data and recently
published literature also hint at how prion and neurodegenerative diseases may be propagated.
Specifically, I 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 are (which tense are you using?)
sequenced and obtainable via public record; seventeen organisms demonstrate ≥ 40% positive
match, fourteen demonstrate ≥ 70% positive match, and three organisms use 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).The first time you
reference a source, all authors should be included in the
citation. After that you can use et al. 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 posttranslational 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, we are beginning to
understand that 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
(Whose research – yours?)Research was
conducted via the following: (1) Thirty microorganisms
across twelve sub-classes of extremophiles were
identified using publicly available online databases. (2)
Identified microorganisms were vetted using the Kyoto
Encyclopedia of Genes & Genomes and GenBank in
order to identify whether or not the complete genome has
been sequenced and published; incomplete sequences are
not available for comparison and were eliminated from
the population. (3) Genome identification continued until
a population of N ≥ 20 was reached. (4) Amino acid
examination was conducted via protein comparison using
the National Center for Biotechnology Information’s
(NCBI) Basic Local Alignment Search Tool (BLAST)
server. (5) Percentage conversancy of the amino acid
sequence was calculated between the species using the
NCBI Graphic Representation Tool. (6) A cladogram
was constructed using the NCBI Phylogenetic Tool in
order to visualize the divergence between species.
(Possibly list as sequence instead? First this was done,
then this, lastly this..)
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:
MLRLPTVFRQMRPVSRVLAPHLTRAYAKDVKFGA
DARALMLQGVDLLADAVAVTMGPKGRTVIIEQSW
GSPKVTKDGVTVAKSIDLKDKYKNIGAKLVQDVA
NNTNEEAGDGTTTATVLARSIAKEGFEKISKGANP
VEIRRGVMLAVDAVIAELKKQSKPVTTPEEIAQVAT
ISANGDKEIGNIISDAMKKVGRKGVITVKDGKTLN
DELEIIEGMKFDRGYISPYFINTSKGQKCEFQDAYV
LLSEKKISSIQSIVPALEIANAHRKPLVIIAEDVDGEA
LSTLVLNRLKVGLQVVAVKAPGFGDNRKNQLKDM
AIATGGAVFGEEGLTLNLEDVQPHDLGKVGEVIVT
KDDAMLLKGKGDKAQIEKRIQEIIEQLDVTTSEYE
KEKLNERLAKLSDGVAVLKVGGTSDVEVNEKKDR
VTDALNATRAAVEEGIVLGGGCALLRCIPALDSLTP
ANEDQKIGIEIIKRTLKIPAMTIAKNAGVEGSLIVEKI
MQSSSEVGYDAMAGDFVNMVEKGIIDPTKVVRTA
LLDAAGVASLLTTAEVVVTEIPKEEKDPGMGAMG
GMGGGMGGGMF
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 Table 1. Figure 1 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 expect
value (E-value) was ≤ 3.00x10-11.
Identical Positive Identical Expect
Match % Match % Match value
Sphingopyxis
alaskensis
56.16
78.36
233
0
Saccharomyce
s 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
Acidithiobacillu
s ferrooxidans
51.04
74.38
257
0
Thiobacillus
denitrificans
53.02
73.4
247
1.00E180
51.7
73.3
251
1.00E179
Pelagibacter
ubique
Flavobacterium
psychrophilum
JIP02/86
Bacillus subtilis
51.33
73.19
253
1.00E179
Amphibacillus
xylanus
50.57
72.62
257
9.00E179
Deinococcus
radiodurans
48.7
70.26
264
1.00E163
Pyrolobus
fumarii 1A
23.36
44.86
340
1.00E017
Methanopyrus
kandleri AV19
23.23
42.47
323
5.00E017
Methanococcoi
des burtonii
DSM 6242
23.03
43.07
340
2.00E015
352
3.00E011
Sulfolobus
solfataricus P2
22.02
41.74
Table 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 1. Graphical representation of hit table from
Table 1. Red areas indicate identical matches, grey areas
indicate positive matches. Organisms are listed in the
same order as Table 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.
Discussion
The E-Value represents background noise, or
the percent likelihood that a false positive will be
encountered in the query sequence. The sub-classes
included in the research were: acidophiles, alkaliphiles,
cryptoendoliths,
osmophiles,
lithoautotrophs,
metallophiles, oligotrophs, piezophiles, psychrophiles,
radiophiles, thermophiles, and xerophiles.(Shouldn’t this
be in materials and methods?) 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 chamber
which allows polypeptide chains the chance 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.
Additionally, 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,
Chakraborty,
2013).
2013).
*Abstract- the abstract was not the correct format; it should not be 2 columns. Also, it was
lengthy with too much background information and run-on sentences.
*Headings - all headings should be centered in the middle of the column.
*Indentations - all paragraphs should begin with an indentation.
*Citations - the first citation should have all authors listed not et al. The researcher does not
appear to have a total of 10 citations.
*Grammar - numerous run-on sentences, incorrect tense used in Materials and Methods;
fluctuation between tenses within a paragraph are confusing. All sections appear to be one large
paragraph and the incorrect font size is used (text should be size 10; size 12 for headings and
abstract).
*Figures and Tables - these should be included after results not in the discussion section. They
are also the incorrect format, the text should be italicized and these should be within the column
formatting. Also, figure 1 looks more like a table?
**NO LITERATURE CITED!!!
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published