pubmed article of crotalus horridus
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MITOGENOME ANNOUNCEMENT
The complete mitochondrial DNA sequence of Crotalus horridus
(timber rattlesnake)
JACOB B. HALL*, VINCENT A. COBB, & A. BRUCE CAHOON
Department of Biology, Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA
(Received 2 August 2012; accepted 17 August 2012)
AbstractThe complete mitogenome of the timber rattlesnake (Crotalus horridus) was completed using Sanger sequencing. It is17,260 bp with 13 protein-coding genes, 21 tRNAs, two rRNAs and two control regions. Gene synteny is consistent with othersnakes with the exception of a missing redundant tRNA Ser. This mitogenome should prove to be a useful addition of awell-known member of the Viperidae snake family.
Keywords: Crotalus horridus, Viperidae, timber rattlesnake, mitogenome
Report
The mitogenome of the timber rattlesnake (Crotalus
horridus; order: Squamata, suborder: Serpentes,
family: Viperidae) has been sequenced using oral
tissue taken from a specimen collected in Rutherford
County, Tennessee, USA (358 510N, 868 180 W) on
20 October 2007. This specimen (APSU 19297) is
deposited in the Museum of Zoology, Austin
Peay State University, Clarksville, TN, USA. The
sequence is deposited in GenBank as accession
number HM641837.
Primers were initially designed based on members
of the snake subfamily Crotalinae: Agkistrodon
piscivorus (DQ523161), Deinagkistrodon acutus
(DQ343647), Gloydis blomhoffi (EU913477) and
Ovophis okinavensis (AB175670). Additional primers
were made using the newly collected C. horridus
sequences. The primer set used to complete this
mitogenome should be a useful starting point for the
sequencing of other members of the family Viperidae,
and is available upon request. Overall, 247 successful
sequencing runs were collected for a total of
.123,500 bases for 7X average depth of coverage.
The Dual Organellar Genome Annotator (DOGMA;
Wyman et al. 2004) was used to begin the annotation
process. Start and stop codons of all protein-
coding genes were located and/or confirmed using
Sequencher (Gene Codes Corporation, Ann Arbor,
MI, USA) and Virtual Ribosome (Wernersson 2006).
tRNAs were initially identified using DOGMA and
tRNAscan-SE (Lowe and Eddy 1997) and checked
individually using Sequencher’s alignment features
or by predicting secondary structure using mFold
(Zuker 2003). Other mitochondria-specific features
such as control regions and origin of light-strand
replication were found using existing snake mitochon-
drial genomes and Sequencher’s alignment features.
The C. horridus mitochondrial genome (GenBank
accession no. HM641837) is 17,260 bp with
13 protein-coding genes, 21 tRNAs, two rRNAs and
two control regions (Figure 1). One exceptional
feature is a missing tRNA Ser gene typically between
tRNA His and tRNA Leu. This region was screened
ISSN 1940-1736 print/ISSN 1940-1744 online q 2012 Informa UK, Ltd.
DOI: 10.3109/19401736.2012.722999
*Current address: Center for Human Genetics Research, 519 Light Hall, Vanderbilt University, Nashville, TN 37232, USA.
Correspondence: A. Bruce Cahoon, Department of Biology, Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA.Tel: þ 615 494 8792. Fax: þ 615 898 5093. E-mail: [email protected]
Mitochondrial DNA, 2013; 24(2): 94–96
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using DOGMA and tRNAscan-SE, neither of which
recognized a tRNA. Also, secondary structure, as
predicted by mFold, suggested that no potential tRNA
structures were possible from the sequence. The gene
synteny of vertebrate mitochondrial genomes, as a
group, is relatively conserved, especially when
compared with other metazoans (Gissi et al. 2008).
In spite of this, it is not unprecedented for a single
redundant tRNA to differ between taxonomically
related species. For example, the tRNA pro found
between tRNA Iso and control region 2 in C. horridus
and A. piscivorus is not consistently present in all snake
mtDNA ( Jiang et al. 2007). Other features include
four genes with possible translation anomalies. The
gene nad2 lacks a clear start codon. cox3 and nad5 have
single-base insertions, and cob has a two-base insertion
that would create frameshifts. All the anomalies were
found in multiple runs (five or more repeats from
more than one clone). These could be the result of
mitochondrial heteroplasmy in the individual and/or
tissue that we sampled. It is also possible that these are
the actual coding regions, in which case ribosome
slippage (Farabaugh and Bjork 1999) could produce
functional protein.
Acknowledgements
Reagents were provided by Middle Tennessee State
University’s Department of Biology.
Declaration of interest: The Applied Biosystems
Genetic Analyzer 3130xl used for sequencing was
purchased with a US National Science Foundation
major instrumentation grant awarded to A.B.C. The
authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
References
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Gissi C, Iannelli F, Pesole G. 2008. Evolution of the mitochon-
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Figure 1. Gene content and order of the C. horridus mitochondrial genome. C. horridus is represented as a linear array of labeled boxes
(genes) that are not drawn to scale. Boxes above the central line represent genes expressed from the heavy-strand promoters (CR1 and CR2,
left-facing arrows) while boxes below the line represent those expressed from the light strand. The stem-loop structure (right-facing arrow)
represents a secondary structure important for light-strand replication. The mtDNA of C. horridus’ closest taxonomic relative with a
completed genome, A. piscivorus (Jiang et al. 2007) as well as the composite typical snake and typical vertebrate genomes (Dong and
Kumazawa 2005) are shown for comparison. Snake mitogenomes differ from the typical vertebrate by the addition of a control region and the
absence of a tRNA Leu. C. horridus differs from the typical snake mitogenome due to the absence of a tRNA Ser found in the HSL cluster
between the nad4 and nad5 protein-coding genes. Both C. horridus and A. piscivorus have an additional tRNAPro not found in the typical
vertebrate or typical snake synteny.
Mitogenome of timber rattlesnake 95
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J.B. Hall et al.96
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