- Email: [email protected]
Exon skipping creates novel splice variants of DMC1 gene in ruminants
Accepted Manuscript Exon skipping creates novel splice variants of DMC1 gene in ruminants S. Ahlawat, Scientist, M. Chopra, L. Jaiswal, R. Sharma, R. Arora, B. Brahma, S.V. Lal, S. De PII:
S0890-8508(16)30021-4
DOI:
10.1016/j.mcp.2016.03.001
Reference:
YMCPR 1203
To appear in:
Molecular and Cellular Probes
Received Date: 31 July 2015 Revised Date:
2 March 2016
Accepted Date: 2 March 2016
Please cite this article as: Ahlawat S, Chopra M, Jaiswal L, Sharma R, Arora R, Brahma B, Lal SV, De S, Exon skipping creates novel splice variants of DMC1 gene in ruminants, Molecular and Cellular Probes (2016), doi: 10.1016/j.mcp.2016.03.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Exon skipping creates novel splice variants of DMC1 gene in ruminants
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S. Ahlawata, M. Choprab, L. Jaiswalb, R. Sharmaa, R. Aroraa, B. Brahmac, S.V. Lalb, S. Deb
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National Bureau of Animal Genetic Resources, Karnal, 132001, India
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National Dairy Research Institute, Karnal, 132001, India
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Krishi Vigyan Kendra, Bhaderwah, SKUAST-Jammu, 180016, India
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Sonika Ahlawat
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Scientist, Animal Biotechnology Division, NBAGR
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E-mail address: [email protected]
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Tel.: +919416161369; Fax: +91 184 2267654
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Corresponding author:
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Abstract Disrupted meiotic cDNA1 (DMC1) recombinase plays a pivotal role in homology search and
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strand exchange reactions during meiotic homologous recombination. In the present study, full
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length coding sequence of DMC1 gene was sequence characterized for the first time from four
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ruminant species (cattle, buffalo, sheep and goat) and phylogenetic relationship of ruminant
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DMC1 with other eukaryotes was analyzed. DMC1 gene encodes a putative protein of 340 amino
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acids in cattle, sheep and buffalo and 341 amino acids in goat. A high degree of evolutionary
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conservation at both nucleotide and amino acid level was observed for the four ruminant
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orthologs. In cattle and sheep, novel alternatively spliced mRNAs with skipping of exons 7 and 8
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(Transcript variant 1, TV1) were isolated in addition to the full length (FL) transcript. Novel
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transcript variants with partial skipping of exon 7 and complete skipping of exon 8 (Transcript
12
variant 2, TV2) were found in sheep and goat. The presence of these variants was validated by
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amplifying cDNA isolated from testis tissue of ruminants using two oligonucleotides flanking
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the deleted region. To accurately estimate their relative proportions, real-time PCR was
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performed using primers specific for each variant. Expression level of DMC1-FL was
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significantly higher than that of TV1 in cattle and TV2 in goat (P < 0.05). Relative ratio for
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expression of DMC1-FL: TV1: TV2 in sheep was 6.78: 1.43: 1. In-silico analysis revealed
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presence of splice variants of DMC1 gene across other mammalian species underpinning the role
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of alternative splicing in functional innovation.
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Keywords: DMC1; splice variants; exon skipping; ruminants; meiosis; recombination
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1. Introduction
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Sexually reproducing organisms have a specialized developmental pathway for
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gametogenesis in which diploid cells undergo meiosis to produce haploid germ cells. Meiotic
4
recombination is a highly conserved process that is critical for the accurate segregation of
5
homologous chromosomes to opposite poles at the first meiotic division [1]. Recombination
6
ensures that the damage to the homologs is repaired and from an evolutionary perspective, it
7
contributes to genetic variability of a species in order for it to withstand the pressure of
8
natural selection. Meiotic recombination is initiated by the introduction of programmed
9
double-strand breaks (DSBs) catalyzed by an evolutionarily conserved, meiosis-specific,
10
topoisomerase-like protein, SPO11 [2]. Resection of the 5’ ends of the breaks results in 3’
11
single stranded tails which are bound by two conserved RecA-like proteins, RAD51 and
12
DMC1 in many organisms, including yeast and mammals [3, 4]. RAD51/DMC1-coated
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nucleofilaments promote homology search and strand exchange, to form joint molecule
14
intermediates, and are then displaced from synapsing chromosomes as the recombination
15
intermediates are processed [5]. DMC1 is known to be required for proficient double-strand
16
break repair as well [6, 7]. Subsequent processing of the resulting recombination
17
intermediates results in formation of either crossovers or non-crossovers [8]. RAD51 is
18
required for homologous recombination pathways in both mitotic and meiotic cells [9]. In
19
contrast, the other recombinase, DMC1, is a meiosis-specific protein [3]. RAD51 and DMC1
20
have been shown to co-localize in side-by-side foci on meiotic chromatin, suggesting their
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cooperative action during meiotic recombination [10]. Recent study by Brown et al. [11]
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provided further insights into localization of RAD51 and DMC1 and proposed that these
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proteins co-occupy the 3′ termini of most double-strand breaks and the two ends of a DSB
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are separated by distances of up to 400 nm. DMC1 gene was originally identified in Saccharomyces cerevisiae by using a screen for
4
meiosis-specific prophase-induced genes that cause a meiotic defect when disrupted [3]. That is
5
how its name "disrupted meiotic cDNA" or DMC1 originated. Subsequently it was isolated from
6
higher eukaryotes, such as humans and mice [12]. Importance of DMC1 in homologous
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recombination in meiosis can be appreciated from the fact that targeted mutation of this gene
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results in sterility in mice and the signs of poorly repaired double-strand breaks are apparent in
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reproductive cells [6].
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Mutations in S. cerevisiae DMC1 lead to the accumulation of resected DSBs and prevent
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complete meiosis in some strains [3]. However, in fission yeast, Schizosaccharomyces pombe,
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deletion of DMC1 causes only a moderate reduction in crossing over and a slight reduction in
13
fertility, suggesting that RAD51 can partially substitute for DMC1 in inter-homolog
14
recombination [13]. Several organisms such as Drosophila melanogaster, Caenorhabditis
15
elegans, Neurospora crassa and Sordaria macrospora do not possess a DMC1 ortholog [14].
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Why such an apparently important protein is absent in these organisms remains elusive.
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Meiotic recombination has several unique features that distinguish it from the
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homologous recombination that occurs during mitosis. These include the strong preference for
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inter-homolog recombination, and the coordination of recombination with meiosis specific,
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higher-order chromosome structures, such as the synaptonemal complex which mediates the
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stable pairing (synapsis) of homologous chromosomes during prophase I [15]. Role of DMC1
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for these unique aspects of meiotic recombination has long been speculated.
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DMC1 gene has been identified and characterized in humans, mice, some aquatic
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animals, crustaceans and chinese crab [16]. DMC1 orthologs have also been identified in plants
3
such as rice, maize and Arabidopsis [17]. However, there are no reports of characterization of
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this gene in mammalian species other than humans and mice. Keeping in view the significance of
5
this gene in meiosis and homologous recombination, the present study aimed to clone and
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sequence characterize full length coding sequence of meiotic recombinase DMC1 from four
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ruminant species (cattle, buffalo, sheep and goat) and to analyze the diversity and phylogenetic
8
relationship of ruminant DMC1 with other eukaryotic species.
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2. Material and Methods 2.1 Sample collection
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Testis tissue samples of cattle (Bos indicus), buffalo (Bubalus bubalis), sheep (Ovis aries)
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and goat (Capra hircus) were collected from the Gazipur slaughter house, New Delhi, India,
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with the help of an on-site veterinary officer. The fresh tissue samples were dispensed in
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RNAlater (Sigma-Aldrich) and stored in deep freeze (-80 ͦC) until further use.
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2.2 RNA isolation and cDNA preparation Total RNA was isolated from tissue samples using TRIzol reagent (Sigma-Aldrich) and
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treated with DNAse I (Fermentas) following manufacturer’s instructions. RNA integrity was
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assessed by loading 2 ul RNA sample on 1.5 % agarose gel. The concentration of isolated RNA
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was determined by measuring optical density at 260 nm using NanoDrop 1000
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Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). About 1 ug of total
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mRNA was reverse transcribed with Superscript III cDNA synthesis kit (Invitrogen Canada Inc.)
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using hexamer primers according to manufacturer’s instructions and cDNA was stored at -80°C
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for further use.
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2.3 RT-PCR and cloning
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The full length coding sequence of DMC1 gene was amplified from cDNA of testicular
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tissue of cattle, buffalo, sheep and goat. The primers for this study were designed manually using
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the conserved sequences from Bos taurus mRNA sequence (NM_001191338). Primer sequences
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used for amplification are as follows: F1- ATGAAGGAGGATCAAGTTGTGCTGGAAGA and
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R1- CTTCAGCTCCTAATAAGCACTAAGAAGCA. PCR was carried out on an Veriti 96 well
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thermal cycler (Applied Biosystems) in 25 µL reaction mixture containing 2.5 µL of 10X Taq
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reaction buffer, 0.5 µl of 10mM dNTP mix (Fermentas), 0.5 µl of 10µM for each primer and 0.25
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units of Taq DNA polymerase (Sigma). The PCR reaction cycle was accomplished by
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denaturation for 3 min at 94°C; 30 cycles of denaturation at 94°C for 30 sec, annealing at 57°C
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for 30 sec, extension step at 72°C for 30 sec, with a final extension at 72°C for 5 min.
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products were electrophoresed alongside DNA molecular weight marker in 1.5% agarose gel and
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then stained with ethidium bromide. The amplified fragments were gel purified using PureLink
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Quick Gel Extraction Kit (Invitrogen Canada Inc.) and subcloned into pTZ57R/T vector
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(Thermo scientific). Recombinant clones were selected and plasmid DNA was extracted.
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Multiple clones were sequenced using the Sanger sequencing to obtain the coding DNA
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sequence of DMC1 in the four ruminant species.
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2.4 Real-time PCR
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Quantitative RT-PCR was carried out to determine the relative mRNA expression of DMC1
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splice variants in testis tissues (3 each) of cattle, sheep and goat using primers described in Table
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1. Housekeeping genes GAPDH and RPS15 were included as internal controls. Equal amount of
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RNA (quantified using NanoDrop spectrophotometer) was used for cDNA preparation
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(Superscript III cDNA synthesis kit; Invitrogen). All qRT-PCR reactions were carried out on
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Light Cycler 480 II Real-Time PCR machine (Roche Diagnostics, USA). Each 10 µl reaction
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mixture consisted of 2 µl cDNA template, 5 µl of 2X SYBR Green PCR Master Mix, 0.25 µl
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each of forward and reverse primers (10 pmol/µl) and nuclease free water. All samples were run
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in duplicate. Analysis of real-time PCR (qRT-PCR) was performed by delta-delta-Ct (∆∆Ct)
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method [18].
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2.4 In silico analysis
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2.4.1 Sequence retrieval
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A comprehensive search of the sequence database on the NCBI website was carried out in
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order to find and compare DMC1 gene orthologs among different species. Briefly, BLASTp [19]
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search was performed using protein sequence corresponding to DMC1 in our study to retrieve
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similar
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(http://smart.emblheidelberg.de) and Pfam (pfam.sanger.ac.uk/search) were used to predict the
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functional domains of the DMC1 protein.
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sequences
from
other
eukaryotes
(yeast,
plants
and
animals).
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2.4.2 Evolutionary analysis Multiple
protein
sequence
alignment
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performed
using
MAFFT
(http://mafft.cbrc.jp/alignment/server/) and Bioedit. Phylogenetic analysis was performed using 7
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Neighbour Joining and Maximum Likelihood algorithm with MEGA 6.0 package and in each
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case, branch confidence value was calculated with bootstrapping with 1000 iterations using
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‘‘pairwise deletion option’’ of amino acid sequences with gamma parameters. Here, the Jones,
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Taylor and Thorton (JTT) model for amino acid sequences and gamma parameters was used.
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2.4.3 Protein tertiary structure prediction
To predict the DMC1 protein structure in the four ruminant species, comparative
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modeling was performed using Modeller 9.15 (https://salilab.org/modeller/about_modeller.html)
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and PyMol was used for structure visualization. To identify the target structure, BlastP was
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performed with PDB database and protein structure with PDB ID 1V5W was taken as target
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structure for modeling. Protein model was generated through many iterations by Modeller, which
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produces homology models by satisfaction of spatial restraints.
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prepared for each of the ruminant species. DOPE (Discrete Optimized Protein Energy) score
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was calculated for all structures. Lower the energy score, higher is the stability of modeled
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structure. The models returning to the minimum molpdfs and minimum DOPE score were
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chosen as best probable structures. Z-score was calculated using ProSA-web server to recognize
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the errors in experimental and theoretical models of the protein. The degree of similarity of two
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protein structures was measured with the RMSD (Root-Mean-Square Distance).
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3. Results
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The complete open reading frame of DMC1 gene was amplified by RT-PCR from mRNA
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isolated from testis tissue of cattle, buffalo, sheep and goat. The amplified products were
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sequenced in both directions after cloning in pTZ57R/T vector and the DNA sequences from 8
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these four species were submitted to NCBI and are available under accession numbers:
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KR935226-KR935230, KT318875-KT318877. The complete open reading frame of DMC1 gene
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in cattle, buffalo and sheep was observed to be 1023 nucleotides in length encoding a putative
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protein of 340 amino acids. In goats, the complete ORF was found to be 1026 nucleotides in
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length encoding a putative protein of 341 amino acids. Comparative analysis of cDNA sequence
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of four ruminant orthologs revealed limited sequence divergence at both the nucleotide and the
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amino acid level indicating a high degree of evolutionary conservation. The sequence identity at
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nucleotide and amino acid level between cattle and buffalo was 97.3% whereas between sheep
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and goat, it was 95.3%. The homology of cattle and buffalo DMC1 amino acid sequence was
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more with that of sheep as compared to goat. In cattle and sheep, novel alternatively spliced
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mRNAs of 858 nucleotides were isolated in addition to the full length (FL) transcript.
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Interestingly, novel transcript variants of 897 nucleotides were found in sheep and goat (Fig. 1).
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However, in case of buffalo only full length transcript was observed even by increasing the
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sample size to three.
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Evolutionary analysis of ruminant DMC1 with other animals, humans, fish, plants and yeast clearly separated Saccharomyces cerevisiae (yeast) gene from all other sequences (Fig. 2).
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Plants such as Arabidopsis thaliana and Solanum lycopersicum formed a separate cluster which
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was quite distinct from all other animal species. As is expected, Mus musculus and Rattus
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norvegicus clustered together with 75% probability and Homo sapiens and Pan troglodytes with
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84% probability. All the mammals were clearly separated from Danio rerio (Zebra fish),
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Xenopus tropicalis (frog) and Gallus gallus (Junglefowl) with high confidence values. All the
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ruminants (our sequences as well as sequences retrieved from NCBI) clustered together
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indicating high sequence similarity.
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Functional feature prediction by SMART and PDBsum revealed significant identity of cattle,
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buffalo, sheep and goat DMC1 protein with humans as well as other animal species. Multiple
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sequence alignment of the ruminant DMC1 protein with other orthologs also revealed significant
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sequence homology (Fig. 3). DMC1 protein in humans and mice typically has 2 domains:
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Domain I (amino acid residues 27-95) and domain II (amino acid residues 96-319) [12]. We also
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observed these domains to be present in the DMC1 protein of ruminants. Domain I has a helix-
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hairpin-helix (HhH) motif of 54 amino acids and the sequence of this motif was found to be
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conserved in all orthologs. The domain II has Walker A and Walker B motifs which interact with
9
single and double stranded DNAs respectively. The sequence of these motifs were also found to
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be highly conserved in all DMC1 homologs except for one amino acid change in Walker A motif
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in Ovis aries sequences isolated in this study. The C-terminal region of DMC1 protein includes
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amino acid residues 319-340 and its sequence was conserved in all DMC1 orthologs. Sequence
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analysis of the full length transcripts and the two novel splice variants revealed that the transcript
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variant of 858 nucletides identified in cattle and sheep had a deletion of amino acids at residues
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142-196. This deletion lies in the domain II region of DMC1 protein. This deletion also altered
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the junctional amino acids in the putative protein. The junctional amino acid at the start of the
17
deletion was glycine instead of valine at position 142 and serine at the end of the deletion at
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position 196 was also replaced by glycine. As far as the second splice variant of 897 nucleotides
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is concerned, there is deletion of residues 154-196 within the domain II region of DMC1 protein.
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Here again, the junctional amino acid at position 196 is glycine in place of serine.
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DMC1 gene in cattle consists of 13 exons (AC_00162) whereas in other three ruminant species
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i.e. buffalo (NW_005785436), sheep (NC_019460) and goat (NC_022297), there are 14 exons,
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out of which 13 code for functional protein. Comparison of the sequence of transcripts with
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DMC1 gene sequence in the analyzed species indicated that there is skipping of coding exons 7
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and 8 in transcript variant 1 (TV1) and partial skipping of exon 7 and complete skipping of exon
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8 in transcript variant 2 (TV2) (Fig. 4). To confirm the presence and location of different spliced forms of mRNAs, two
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oligonucleotides flanking the deleted region were used to amplify cDNA isolated from testis
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tissue of ruminants such that the full length transcript would yield a product of 326 bp and the
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splice variants 1 and 2 would produce products of 161 and 200 bp respectively. The sequences of
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the primers employed were: F2: TAAGTTGCTAGGAGGTGGAATTGAAAGCAT and R2:
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TAAAGATGCCAGCTTCTTCATGGAACTTTGC. The position of these primers is depicted in
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Fig. 4. We observed amplified products of expected sizes from the different transcripts of DMC1
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gene in cattle, buffalo, sheep and goat. Sequencing of the products also confirmed that they were
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indeed the different splice variants of DMC1 and that there is loss of two exons in the variants
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(Fig. 5).
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Expression patterns of full length transcript and different splice variants were assessed by
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real-time PCR in testis tissue of cattle, sheep and goat using primers described in Table 1. The
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results revealed that the expression level of DMC1-FL was significantly higher than that of TV1
17
in cattle and TV2 in goat (P < 0.05). In sheep, DMC1-FL was most abundantly expressed
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followed by TV1 and TV2 (DMC1 FL: TV1: TV2 = 6.78: 1.43: 1) (Fig. 6).
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The tertiary structure for the different variants of DMC1 protein was predicted for the four
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ruminant species using Modeller 9.15 as the structure is not yet available in the PDB database.
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Errors in experimental and theoretical models of the predicted protein were recognized by
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calculating the z-score using ProSA-web server. The structure for DMC1 protein could be reliably
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predicted with satisfactory Z score and the protein was found to contain Walker A, Walker B and
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HhH motifs that are peculiar features of this protein. We found that tertiary structure of full length
2
sequences in cattle, buffalo and sheep had 9 α-helices and 8 β-sheets whereas goat protein
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structure was found to contain 9 α-helices and 7 β-sheets because of difference in sequence at the
4
C terminal end. Transcript variants revealed loss of some α-helices and β-sheets. Transcript
5
variant 1 had 7α-helices and 6 β-sheets whereas transcript variant 2 had 6 α-helices and 7 β-
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sheets. We found that few residues in ATP binding sites {128 (F), 132(K), 133(T), 158 (F),
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160(D), 162(E), 223(D)} and multimer BRC sites {157 (I), 159 (F) , 167(P), 170(L), 185 (L) ,
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188(V), 189 (L), 191(A), 202 (L), 203 (L), 204(D), 205(Y), 206(V), 207(A), 209(K), 251(S) , 254
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(Q), 255(K), 258(E), 259 (E)} were absent in both the variants because of deletion of 2 exons in
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the crucial domain II region of the protein (Supplement Fig. S1-S4). Both template and query
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structures were superimposed to calculate the similarity of target and template structure.
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Complete protein sequence had approximately 92% and partial sequences had 79% residues
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similarity, suggesting reliable 3D structure. Hence, the representative structure verified by
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PDBsum, PROCHEK, ProSA z-score was satisfactory and can thus be considered as a reliable
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source for further analyses. DOPE score, z score and Molpdf for different variants are depicted in
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Table 2.
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4. Discussion
Homologous recombination (HR) is an indispensable process for the maintenance of
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genomic integrity and for creation of genetic diversity [9]. During HR reaction, the double strand
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break ends introduced by the SPO11 complex undergo 5′ strand resection to generate 3′ single
22
stranded DNA tails that are bound by either of the two conserved recombinases, DMC1 and
23
RAD51. Both RAD51 and DMC1 are homologs of Escherichia coli RecA gene which is among
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the most studied recombination gene encoding a DNA-dependent ATPase that binds to single-
2
stranded DNA and promotes strand invasion and exchange between homologous DNA
3
molecules [4]. RAD51 and DMC1 are composed of two domains: the highly conserved ATPase
4
domain, which makes up the core oligomeric structure, and the N-terminal domain (NTD), which
5
is not conserved in the bacterial RecA protein. The ATPase domain is the core catalytic domain
6
important for DNA binding and ATP hydrolysis [20]. DNA binding is mediated by N-terminal
7
domain in RAD51 and DMC1 and by C-terminal domain in RecA. Specifically, it is the helix–
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hairpin–helix (HhH) motif in the NTDs of RAD51 and DMC1 which mediates dsDNA binding
9
[20, 21]. Two oligomeric DNA binding forms, an octameric ring and a helical filament have
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been reported for DMC1 whereas the binding form in case of RAD51 is the helical filament. In the present study, open reading frame of DMC1 gene was characterized for the first
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time in four ruminant species (cattle, buffalo, sheep and goat). The ORF of DMC1 was predicted
13
to encode a putative protein of 340 amino acids in cattle, buffalo and sheep and 341 amino acids
14
in goats. Two novel transcript variants of 858 and 897 nucleotides were also identified in these
15
ruminants which encode proteins of 285 and 298 amino acids respectively. Transcript variant 1
16
(858 nucleotides) which was observed in cattle and sheep has previously been reported in
17
humans and mice [12] whereas the other variant (897 nucleotides) which was isolated in sheep
18
and goat has been identified for the first time in mammals. Another study [7] also confirmed
19
alternative splicing of DMC1 mRNAs in mice where targeted disruption of DMC1 gene resulted
20
in an arrest of meiosis of germ cells in both sexes. Western blotting was performed using rabbit
21
anti-DMC1 antibody on the nuclear extracts of wild type and DMC1 deficient mouse testis. The
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nuclear extracts from wild-type and heterozygous mouse testes revealed two bands
23
corresponding to full length DMC1 and DMC1-D (a product from alternatively spliced allele)
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proteins, respectively as had been previously reported [12]. In contrast, the nuclear extract from
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the DMC1-deficient mouse testis did not show these bands. In silico analysis revealed that alternatively spliced variants with skipping of coding
4
exons 7 and 8 are predicted in a wide range of mammalian species such as Bos mutus, Pan
5
troglodytes and Pan paniscus in addition to those reported in Homo sapiens and Mus musculus.
6
Splice variants with skipping of some other exons have also been predicted in other species as
7
shown in Table 3. However, in some species such as Rattus norvegicus, Equus asinus and Canis
8
lupus familaris, there is no report available so far on alternate splicing. By real time PCR, we
9
found that mRNA expression levels of DMC1-FL were significantly higher than TV1 and TV2
10
in cattle, sheep and goat suggesting that DMC1-FL is the most abundantly expressed isoform. It
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is worth emphasizing that evolutionary conservation of splice variants of DMC1 gene across
12
different taxa possibly reinforces a major physiological role for these isoforms since alternative
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splicing is a major mechanism for augmentation of transcriptome and proteome diversity [22].
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Sequence analysis revealed that all the variants of DMC1 identified in the present study
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have intact N-terminal domain with highly conserved HhH motif. Researchers in the past have
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reported that high flexibility or multiple conformations of DMC1 are because of the N-terminal
17
domain [20]. It has been experimentally validated that deletion mutant of DMC1 which lacks N-
18
terminal 81 amino acid residues is completely defective in both single-stranded and double-
19
stranded DNA binding activities [20]. Since the variants identified in the present study have
20
intact N-terminal domain, it indicates that DNA binding activity is probably retained in the splice
21
variants. DMC1 transcript variant 1 does not contain the region at amino acids positions 142-196
22
and transcript variant 2 lacks region at amino acids positions 154-196 of ruminant DMC1. These
23
deletions lie in the critical domain II region that is conserved in the RecA-like gene family. Also
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the deleted region locates close to Walker A and Walker B motifs which are necessary for ATP
2
binding, therefore the DMC1 splice variants (1 and 2) may have a low binding affinity for
3
nucleotides and/or low ATPase activity. Domain II region also has multimer BRC interface that
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binds to BRCA2 tumour suppressor protein which is involved in maintaining the integrity of the
5
genome by participating in error-free double-strand break repair [23]. BRCA2 is a universal
6
regulator of RAD51/DMC1 recombinase actions. In the novel splice variants identified in the
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present study, some of the residues which are part of the multimer BRC interface have been
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deleted. So it is logical to speculate that these variants may have reduced binding affinity for
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BRCA2.
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Amino acid residues from 319-340 comprise the C-terminal region of DMC1 protein
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which was found to be conserved in all transcript variants of DMC1. Sequence conservation of
12
this region in all eukaryotic DMC1 orthologs suggests that this region may be important in
13
recombination by interacting with other components of the recombination machinery, one of
14
them being the tumor suppressor protein p53. It is a multifunctional molecule that regulates
15
meiotic cell-cycle and DNA repair. It binds to C-terminal domain of mammalian DMC1 and is
16
involved in homologous recombination and maintenance of genomic stability during meiosis
17
[24]. Therefore probably the novel transcript variants retain the ability to interact with other
18
proteins.
EP
AC C
19
TE D
10
It is interesting and intriguing to note that since the transcript variants are conserved in
20
different mammalian species such as humans, mice [12], cattle, sheep and goat (our study) and
21
also predicted in other animal species, it implies that these variants with the deletion in domain II
22
region which harbors the ATPase domain might have novel roles to play in recombination.
23
Kowalczykowski and Krupp [25] reported that ATPase activity is not required for the strand
15
ACCEPTED MANUSCRIPT
exchange reaction with bacterial RecA. On similar lines, DMC1 splice variants may have
2
attenuated ATPase activity but could still be capable of strand exchange. Put together, these
3
observations suggest that the novel transcript variants of DMC1 with intact N-terminal and C-
4
terminal domains represent proteins with diverse biological functions or with altered functional
5
activities and represent an interesting area to explore for researchers in future.
RI PT
1
6
8
SC
7
5. Conclusion
The present study for the first time determined the full length coding sequence of DMC1 gene in
10
four ruminant species (buffalo, cattle, sheep and goat). Evolutionary analysis between the
11
different orthologs suggested that these proteins are conserved from yeast to humans. Two novel
12
transcript variants with deletion in the ATPase domain were also characterized for the first time
13
in ruminants. Since these transcripts are also observed in other mammalian species such as
14
humans and mice and predicted in some other species, it indicates that they are evolutionarily
15
conserved and may have novel/diverse biological functions in recombination.
TE D
M AN U
9
EP
16
Conflict of interest statement
18
The authors declare that they have no competing interests.
19
AC C
17
20
Acknowledgements
21
This study was supported by the Indian Council of Agricultural Research, New Delhi, India.
22
23 16
ACCEPTED MANUSCRIPT
1
References [1] Henry JM, Camahort R, Rice DA, Florens L, Swanson SK, Washburn MP, et al.
3
Mnd1/Hop2 Facilitates DMC1-Dependent Interhomolog Crossover Formation in Meiosis
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of Budding Yeast. Mol Cell Biol 2006; 26(8):2913–2923.
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[2] Keeney S. SPO11 and the formation of DNA double-strand breaks in meiosis.
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Genome Dynam Stabil 2008; 2:81–123.
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[3] Bishop DK, Park D, Xu L, Kleckner N. DMC1: a meiosis-specific yeast homolog of
8
E. coli RecA required for recombination, synaptonemal complex formation, and cell cycle
9
progression. Cell 1992; 69:439–456.
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2
10
[4] Shinohara A, Ogawa H, Ogawa T. RAD51 protein involved in repair and
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recombination in S. cerevisiae is a RecA-like protein. Cell 1992; 69:457–470.
12
[5] Hong EL, Shinohara A,
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14
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[6] Pittman DL, Cobb J, Schimenti KJ, Wilson LA, Cooper DM, Brignull E, et al.
16
Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for
17
DMC1, a germ line specific RecA homolog. Mol Cell 1998; 1:697–705.
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AC C
18
Bishop DK. Saccharomyces cerevisiae DMC1 protein
[7] Yoshida K, Kondoh G, Matsuda Y, Habu T, Nishimune Y, Morita T. The mouse RecA-like gene DMC1 is required for homologous chromosome synapsis during meiosis. Mol Cell 1998; 1:707–718.
21
[8] de Massy B. Initiation of meiotic recombination: how and where? Annu Rev Genet
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2013; http://dx.doi. org/ 10.1146/annurev-genet-110711-155423.
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ACCEPTED MANUSCRIPT
[9] Krogh BO, Symington LS. Recombination proteins in yeast. Annu Rev Genet 2004;
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38:233–271.
3
[10] Bishop DK. RecA homologs DMC1 and RAD51 interact to form multiple nuclear
4
complexes prior to meiotic chromosome synapsis. Cell 1994; 79:1081–1092.
5
[11] Brown MS, Grubb J, Zhang A, Rust MJ, Bishop DK. Small RAD51 and DMC1
6
complexes often co-occupy both ends of a meiotic DNA double strand break. PLoS
7
Genet 2015; 11(12): e1005653.
8
[12] Habu T, Taki T, West A, Nishimune Y, Morita T. The mouse and human homologs
9
of DMC1, the yeast meiosis-specific homologous recombination gene, have a common
M AN U
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RI PT
1
unique form of exon-skipped transcript in meiosis. Nucleic Acid Res 1996; 24:470–477.
11
[13] Grishchuk AL, Kohli J. Five RecA-like proteins of Schizosaccharomyces pombe are
12
involved in meiotic recombination. Genetics 2003; 165:1031–1043.
13
[14] Neale MJ, Keeney S. Clarifying the mechanics of DNA strand exchange in meiotic
14
recombination. Nature 2006; 442:153–158.
15
[15] Roeder GS. Meiotic chromosomes: it takes two to tango. Genes Dev 1997; 11:2600–
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[16] Liu Y, Cui Z. Molecular Cloning and Characterization of DMC1 from the Chinese
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Mitten Crab (Eriocheir sinensis). Int J Aquacult Fish Sci 2015; 1(1):024–029.
20
EP
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TE D
10
[17] Etedali F, Baghban Kohnehrouz B, Valizadeh M, Gholizadeh A, Malboobi MA. Genome wide cloning of maize meiotic recombinase DMC1 and its functional structure
21
through molecular phylogeny. Genet Mol Res 2011; 10(3):1636–1649.
22
[18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
23
quantitative PCR and the 2−∆∆Ct method. Methods 2001; 25:402–408.
18
ACCEPTED MANUSCRIPT
[19] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment
2
search tool. J Mol Biol 1990; 215(3):403–10.
3
[20] Kinebuchi T, Kagawa W, Kurumizaka H, Yokoyama S. Role of the N-terminal
4
Domain of the Human DMC1 Protein in Octamer Formation and DNA Binding. J Biol
5
Chem 2005; 280(31):28382–28387.
6
[21] Aihara H, Ito Y, Kurumizaka H, Yokoyama S, Shibata T. The N-terminal domain of
7
the human RAD51 protein binds DNA: structure and a DNA binding surface as revealed
8
by NMR. J Mol Biol 1999; 290:495–504.
9
[22] Keren H, Lev-Maor G, Ast G. Alternative splicing and evolution: diversification,
M AN U
SC
RI PT
1
exon definition and function. Nat Rev Genet 2010; 11:345–355.
11
[23] Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2.
12
Cell 2002; 108:171–182.
13
[24] Habu T, Wakabayashi N, Yoshida K, Yomogida K, Nishimune Y, Morita T. p53
14
protein interacts specifically with the meiosis-specific mammalian RecA-like protein
15
DMC1 in meiosis. Carcinogenesis 2004; 25(6):889–893.
16
TE D
10
17
EP
[25] Kowalczykowski SC, Krupp RA. DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in proteinpromoted nucleic acid transactions. Proc Natl Acad Sci U S A 1995; 92(8):3478-3482.
AC C
18
19
ACCEPTED MANUSCRIPT
Table 1. Primers for Real-time PCR of DMC1 variants in ruminants Variant
Primer sequence
Product size
RI PT
(bp)
Wild type
F- ACAGAAGCTTTTGGAGAATTTCGAACTGG
151
F- TTTCCATATCACCACTGGGAGCCAGGA
152
cattle and sheep
R- CAAGTAGCTCCATCTGATGTTCACCACAG
Transcript variant 2:
F- GCTAGGAGGTGGAATTGAAAGCATGGC
152
sheep and goat
R- CAAGTAGCTCCATCTGATGTTCACCTCCT
AC C
EP
TE D
M AN U
Transcript variant 1:
SC
R- CCGAAGGCGATCTGGACGGAAAGTATT
ACCEPTED MANUSCRIPT
Table 2. Energy minimization score using different methods Molpdf
DOPE score
z-score
Cattle
2292.52222
28590.24805
-4.58
Cattle_TV1
2240.96851
RI PT
Structure
-23632.13086
-3.13
2093.68140
-29477.53125
-4.81
Sheep
2181.68262
-28506.82227
-4.07
Sheep_TV1
2018.29260
-21557.39453
-3.03
Sheep_TV2
2083.81421
-24983.40039
-3.35
Goat
2066.36719
-28991.46680
-4.49
Goat_TV2
2008.12634
-22084.85156
-3.95
AC C
EP
TE D
M AN U
SC
Buffalo
ACCEPTED MANUSCRIPT
Table 3. Alternative splicing of DMC1 gene in different species Accession
Total exon
Coding
Skipped
number
count
exon count
coding exon
RI PT
Species
in variant
Bos taurus
NM_001191338
13
Bos indicus_NDRI
KT318875
13
Bos indicus_NDRI_TV
KR935227
11
Bubalus bubalis
XM_006071328
14
Bubalus bubalis_NDRI
KR935226
13
13
Ovis aries
XM_012175647
14
13
Ovis aries_NDRI
KT318876
13
13
Ovis aries_NDRI_TV1
KR935228
11
11
Exons 7 and 8
12
12
Partial exon 7
Capra hircus
SC
13
M AN U
TE D KT318877
EP
Ovis aries_NDRI_TV2
13
11
13
and complete exon 8
XM_005681072
14
13
XM_005681073
11
11
Capra hircus_NDRI
KR935229
13
13
Capra hircus_NDRI_TV
KR935230
12
12
AC C
Capra hircus_TV
Exons 7 and 8
Exons 7 and 8
Partial exon 7 and complete exon 8
Bos mutus
XM_005899439
14
13
ACCEPTED MANUSCRIPT
XM_005899440
11
11
Homo sapiens
NM_007068
14
13
Homo sapiens_TV
NM_001278208
11
11
Mus musculus
NM_010059
14
13
Mus musculus_TV1
NM_001278226
12
Mus musculus_TV2
NR_103477
9
Sus scrofa
XM_005655492
14
Sus scrofa_TV
XM_005655493
12
Exons 7 and 8
Exons 7 and 8
RI PT
Bos mutus_TV
11
Exons 7 and 8
9
Exons 6-9, 11
M AN U
SC
13 11
Exons 12 and 13
XM_009438403
14
13
Pan troglodytes_TV1
XM_009438407
13
12
Exon 3
Pan troglodytes_TV2
XM_009438408
12
11
Exons 7 and 8
Pan paniscus
XM_003821593
14
13
XM_008975092
13
12
Exon 6
XM_008975093
12
11
Exons 7 and 8
XM_002914548
14
13
Pan paniscus_TV1 Pan paniscus_TV2
EP
Ailuropoda melanoleuca
TE D
Pan troglodytes
XM_011218836
13
12
Equus asinus
XM_014851322.1
13
13
Rattus norvegicus
NM_001130567.1
15
13
Canis lupus familaris
XM_005625878
14
13
AC C
Ailuropoda melanoleuca _TV
Exon 3
Lane 1: DMC1-FL (1023 bp) and TV1 (858 bp) in cattle
M AN U
Lane 2: DMC1-FL in buffalo
SC
Fig. 1. Gel image of RT-PCR product of DMC1 gene in ruminants
RI PT
ACCEPTED MANUSCRIPT
Lane 3: DMC1-FL (1026 bp), TV2 (897 bp) in goat
Lane 4: DMC1-FL, TV2 (897 bp) and TV1 (858 bp) in sheep
AC C
EP
TE D
M: 100 bp DNA marker, bright bands correspond to 500 bp and 1000 bp
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 2. Phylogenetic tree of DMC1 in eukaryotes constructed by neighbour joining method
AC C
(Poission correction with gamma parameters)
ACCEPTED MANUSCRIPT HhH motif
RI PT
MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLPEAKADKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTEFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTEFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEVKVDKIKEAANKHIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLPEAKADKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTEFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLAAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKQKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVAEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVAEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVQEESGFQDDEESLFQDIDLLQKHGINMADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVEKIKEAANKLIEPGFLTAFQYSERRKMV MK--EDQVVQEESGFQDDEESLFQDIDLLQKHGINMADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVEKIKEAANKLIEPGFLTAFQYSERRKMV MKVLEEQTLEEESGYQDDEESFFQDIELLQKHGINVADIKKLKSVGICTVKGIQMTTRRALCNIKGLSEAKVDKIKEAAGKLLTCGFQTASEYCIKRKQV MK--EDQVVQEESGFQDEEESLFQDIDLLQKHGINMADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVEKIKEAANKLIEPGFLTAFQYSEKRKMV MKAMEDQVVQEESGYHDDEESFFQDIDLLQKHGINVADIKKLKSVGICTIKGVQMTTRRALCNVKGLSEVKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVVEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MKSMEDQVVEEDVGFHDE-ESFFHDIEMLQKQGINVADIKKLKSVGICTIKGIQMTTRKALCNIKGLSEAKVEKIKEAANKVIEPGFLTAFEYSAKRRMV
Walker A
TE D
M AN U
FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHAPCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGGFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGGFRTGKTQLSHTLCVTAQLPGAGGYSG------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIYTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHAPCG-------------------------------------------------------EH FHITTGSQEFDKLLGGGIESMAITEAFGGFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSG------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCG-------------------------------------------------------EH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYPGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCG-------------------------------------------------------EH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGTGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHEAVLDNVLYARAYTSEH FHITTGSLEFDKLLGGGVESMAITEAFGEFRTGKTQLSHTLCVTAQLPGEYGYTGGKVIFIDTENTFRPERLKDIADRFNVDHEAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGADGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGPKGYTGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYPGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHISTGSQEFDKLLGGGIESMAITETFGEFRTGKTQLAHTLCVTAQLPGPNGYTGGKIIFIDTENTFRPDRLHDIADRFSVDHDAVLDNVLYARAYTSEH
EP
B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens_TV H_sapiens M.musculus_TV M.musculus D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
SC
B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens H_sapiens_TV M.musculus M.musculus_TV D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
Walker B
QMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQNLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGRHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMTLFRVDFSGRGESAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMTLFRVDFSGRGESAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQNLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGRHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMTLFRVDFSGRGESAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKILRGYNVAVFVTNQMTADPRAPMTFQADPKNPFGGHISGHHCF QMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKEPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDFVAAKFHEEGGVFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGAGMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIVDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPVGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEPGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHAS-
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B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens H_sapiens_TV M.musculus M.musculus_TV D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
TTRISLREGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISSRKGRGELRIAQIYGSPEMPENEATFAITAGGIGDAKE TTRISSRKGRGELRIAQIYGSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFTITAGGIGDAKE TTRISLREGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISSRKGRGELRIAQIYGSPEMPENEATFAITAGGIGDAKE TTRISLAKRKRKLEMPKIYDSPEIAENEANLAITAGGIGNAKR TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFTITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRAELRIAKIFDSPHMPENEATFAITAGGITDAKD TTRISLRKGRGELRIAKIYDSPEMPENEATFAITTGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITPGGIGDAKE TTRVSLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITSGGINDAKE
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B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens H_sapiens_TV M.musculus M.musculus_TV D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
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Fig. 3. Multiple sequence alignment of DMC1 gene in different eukaryotes. HhH motif in domain I and Walker A & Walker B motifs in domain II are highlighted by horizontal bars above the sequence alignment.
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ATP binding sites are marked by asterisks and BRCA2 interface by bolts.
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Fig. 4. Schematic representation of DMC1 gene and different splice variants
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The position of primers used for cloning (F1 and R1) and for confirmation of variants (F2 and
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Fig. 4. Gel image of RT-PCR product of DMC1 gene in ruminants confirming deletion of two exons
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Lane 1: DMC1-FL (326 bp) and TV1 (161 bp) in cattle Lane 2: DMC1-FL in buffalo
Lane 3: DMC1-FL, TV2 (200 bp) in goat
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Lane 4: DMC1-FL, TV2 (200 bp) and TV1 (161 bp) in sheep
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M: 100 bp DNA marker, bright band corresponds to 500 bp
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Fig. 6. Expression profiles of DMC1 splice variants in sheep, goat and cattle (P < 0.05)
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Highlights •
DMC1 recombinase was sequence characterized for the first time in four ruminant species. Comparative analysis of ruminant DMC1 orthologs revealed limited sequence
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•
divergence.
Novel alternatively spliced mRNAs with skipping of exons 7 and 8 were also isolated.
•
Presence of variants was validated by amplifying cDNA using primers flanking the
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deleted region.
Real-time PCR was performed to estimate relative proportion of each variant.
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Alternative splicing was observed to be an evolutionarily conserved process across
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S0890-8508(16)30021-4
DOI:
10.1016/j.mcp.2016.03.001
Reference:
YMCPR 1203
To appear in:
Molecular and Cellular Probes
Received Date: 31 July 2015 Revised Date:
2 March 2016
Accepted Date: 2 March 2016
Please cite this article as: Ahlawat S, Chopra M, Jaiswal L, Sharma R, Arora R, Brahma B, Lal SV, De S, Exon skipping creates novel splice variants of DMC1 gene in ruminants, Molecular and Cellular Probes (2016), doi: 10.1016/j.mcp.2016.03.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Exon skipping creates novel splice variants of DMC1 gene in ruminants
1
S. Ahlawata, M. Choprab, L. Jaiswalb, R. Sharmaa, R. Aroraa, B. Brahmac, S.V. Lalb, S. Deb
2 3
National Bureau of Animal Genetic Resources, Karnal, 132001, India
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b
National Dairy Research Institute, Karnal, 132001, India
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Krishi Vigyan Kendra, Bhaderwah, SKUAST-Jammu, 180016, India
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Sonika Ahlawat
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Scientist, Animal Biotechnology Division, NBAGR
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E-mail address: [email protected]
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Tel.: +919416161369; Fax: +91 184 2267654
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Corresponding author:
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Abstract Disrupted meiotic cDNA1 (DMC1) recombinase plays a pivotal role in homology search and
3
strand exchange reactions during meiotic homologous recombination. In the present study, full
4
length coding sequence of DMC1 gene was sequence characterized for the first time from four
5
ruminant species (cattle, buffalo, sheep and goat) and phylogenetic relationship of ruminant
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DMC1 with other eukaryotes was analyzed. DMC1 gene encodes a putative protein of 340 amino
7
acids in cattle, sheep and buffalo and 341 amino acids in goat. A high degree of evolutionary
8
conservation at both nucleotide and amino acid level was observed for the four ruminant
9
orthologs. In cattle and sheep, novel alternatively spliced mRNAs with skipping of exons 7 and 8
10
(Transcript variant 1, TV1) were isolated in addition to the full length (FL) transcript. Novel
11
transcript variants with partial skipping of exon 7 and complete skipping of exon 8 (Transcript
12
variant 2, TV2) were found in sheep and goat. The presence of these variants was validated by
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amplifying cDNA isolated from testis tissue of ruminants using two oligonucleotides flanking
14
the deleted region. To accurately estimate their relative proportions, real-time PCR was
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performed using primers specific for each variant. Expression level of DMC1-FL was
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significantly higher than that of TV1 in cattle and TV2 in goat (P < 0.05). Relative ratio for
17
expression of DMC1-FL: TV1: TV2 in sheep was 6.78: 1.43: 1. In-silico analysis revealed
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presence of splice variants of DMC1 gene across other mammalian species underpinning the role
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of alternative splicing in functional innovation.
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Keywords: DMC1; splice variants; exon skipping; ruminants; meiosis; recombination
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1. Introduction
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Sexually reproducing organisms have a specialized developmental pathway for
3
gametogenesis in which diploid cells undergo meiosis to produce haploid germ cells. Meiotic
4
recombination is a highly conserved process that is critical for the accurate segregation of
5
homologous chromosomes to opposite poles at the first meiotic division [1]. Recombination
6
ensures that the damage to the homologs is repaired and from an evolutionary perspective, it
7
contributes to genetic variability of a species in order for it to withstand the pressure of
8
natural selection. Meiotic recombination is initiated by the introduction of programmed
9
double-strand breaks (DSBs) catalyzed by an evolutionarily conserved, meiosis-specific,
10
topoisomerase-like protein, SPO11 [2]. Resection of the 5’ ends of the breaks results in 3’
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single stranded tails which are bound by two conserved RecA-like proteins, RAD51 and
12
DMC1 in many organisms, including yeast and mammals [3, 4]. RAD51/DMC1-coated
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nucleofilaments promote homology search and strand exchange, to form joint molecule
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intermediates, and are then displaced from synapsing chromosomes as the recombination
15
intermediates are processed [5]. DMC1 is known to be required for proficient double-strand
16
break repair as well [6, 7]. Subsequent processing of the resulting recombination
17
intermediates results in formation of either crossovers or non-crossovers [8]. RAD51 is
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required for homologous recombination pathways in both mitotic and meiotic cells [9]. In
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contrast, the other recombinase, DMC1, is a meiosis-specific protein [3]. RAD51 and DMC1
20
have been shown to co-localize in side-by-side foci on meiotic chromatin, suggesting their
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cooperative action during meiotic recombination [10]. Recent study by Brown et al. [11]
22
provided further insights into localization of RAD51 and DMC1 and proposed that these
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proteins co-occupy the 3′ termini of most double-strand breaks and the two ends of a DSB
2
are separated by distances of up to 400 nm. DMC1 gene was originally identified in Saccharomyces cerevisiae by using a screen for
4
meiosis-specific prophase-induced genes that cause a meiotic defect when disrupted [3]. That is
5
how its name "disrupted meiotic cDNA" or DMC1 originated. Subsequently it was isolated from
6
higher eukaryotes, such as humans and mice [12]. Importance of DMC1 in homologous
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recombination in meiosis can be appreciated from the fact that targeted mutation of this gene
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results in sterility in mice and the signs of poorly repaired double-strand breaks are apparent in
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reproductive cells [6].
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Mutations in S. cerevisiae DMC1 lead to the accumulation of resected DSBs and prevent
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complete meiosis in some strains [3]. However, in fission yeast, Schizosaccharomyces pombe,
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deletion of DMC1 causes only a moderate reduction in crossing over and a slight reduction in
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fertility, suggesting that RAD51 can partially substitute for DMC1 in inter-homolog
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recombination [13]. Several organisms such as Drosophila melanogaster, Caenorhabditis
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elegans, Neurospora crassa and Sordaria macrospora do not possess a DMC1 ortholog [14].
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Why such an apparently important protein is absent in these organisms remains elusive.
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Meiotic recombination has several unique features that distinguish it from the
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homologous recombination that occurs during mitosis. These include the strong preference for
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inter-homolog recombination, and the coordination of recombination with meiosis specific,
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higher-order chromosome structures, such as the synaptonemal complex which mediates the
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stable pairing (synapsis) of homologous chromosomes during prophase I [15]. Role of DMC1
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for these unique aspects of meiotic recombination has long been speculated.
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DMC1 gene has been identified and characterized in humans, mice, some aquatic
2
animals, crustaceans and chinese crab [16]. DMC1 orthologs have also been identified in plants
3
such as rice, maize and Arabidopsis [17]. However, there are no reports of characterization of
4
this gene in mammalian species other than humans and mice. Keeping in view the significance of
5
this gene in meiosis and homologous recombination, the present study aimed to clone and
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sequence characterize full length coding sequence of meiotic recombinase DMC1 from four
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ruminant species (cattle, buffalo, sheep and goat) and to analyze the diversity and phylogenetic
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relationship of ruminant DMC1 with other eukaryotic species.
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2. Material and Methods 2.1 Sample collection
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Testis tissue samples of cattle (Bos indicus), buffalo (Bubalus bubalis), sheep (Ovis aries)
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and goat (Capra hircus) were collected from the Gazipur slaughter house, New Delhi, India,
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with the help of an on-site veterinary officer. The fresh tissue samples were dispensed in
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RNAlater (Sigma-Aldrich) and stored in deep freeze (-80 ͦC) until further use.
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2.2 RNA isolation and cDNA preparation Total RNA was isolated from tissue samples using TRIzol reagent (Sigma-Aldrich) and
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treated with DNAse I (Fermentas) following manufacturer’s instructions. RNA integrity was
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assessed by loading 2 ul RNA sample on 1.5 % agarose gel. The concentration of isolated RNA
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was determined by measuring optical density at 260 nm using NanoDrop 1000
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Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). About 1 ug of total
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mRNA was reverse transcribed with Superscript III cDNA synthesis kit (Invitrogen Canada Inc.)
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using hexamer primers according to manufacturer’s instructions and cDNA was stored at -80°C
2
for further use.
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2.3 RT-PCR and cloning
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The full length coding sequence of DMC1 gene was amplified from cDNA of testicular
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tissue of cattle, buffalo, sheep and goat. The primers for this study were designed manually using
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the conserved sequences from Bos taurus mRNA sequence (NM_001191338). Primer sequences
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used for amplification are as follows: F1- ATGAAGGAGGATCAAGTTGTGCTGGAAGA and
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R1- CTTCAGCTCCTAATAAGCACTAAGAAGCA. PCR was carried out on an Veriti 96 well
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thermal cycler (Applied Biosystems) in 25 µL reaction mixture containing 2.5 µL of 10X Taq
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reaction buffer, 0.5 µl of 10mM dNTP mix (Fermentas), 0.5 µl of 10µM for each primer and 0.25
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units of Taq DNA polymerase (Sigma). The PCR reaction cycle was accomplished by
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denaturation for 3 min at 94°C; 30 cycles of denaturation at 94°C for 30 sec, annealing at 57°C
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for 30 sec, extension step at 72°C for 30 sec, with a final extension at 72°C for 5 min.
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products were electrophoresed alongside DNA molecular weight marker in 1.5% agarose gel and
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then stained with ethidium bromide. The amplified fragments were gel purified using PureLink
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Quick Gel Extraction Kit (Invitrogen Canada Inc.) and subcloned into pTZ57R/T vector
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(Thermo scientific). Recombinant clones were selected and plasmid DNA was extracted.
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Multiple clones were sequenced using the Sanger sequencing to obtain the coding DNA
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sequence of DMC1 in the four ruminant species.
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2.4 Real-time PCR
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Quantitative RT-PCR was carried out to determine the relative mRNA expression of DMC1
2
splice variants in testis tissues (3 each) of cattle, sheep and goat using primers described in Table
3
1. Housekeeping genes GAPDH and RPS15 were included as internal controls. Equal amount of
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RNA (quantified using NanoDrop spectrophotometer) was used for cDNA preparation
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(Superscript III cDNA synthesis kit; Invitrogen). All qRT-PCR reactions were carried out on
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Light Cycler 480 II Real-Time PCR machine (Roche Diagnostics, USA). Each 10 µl reaction
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mixture consisted of 2 µl cDNA template, 5 µl of 2X SYBR Green PCR Master Mix, 0.25 µl
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each of forward and reverse primers (10 pmol/µl) and nuclease free water. All samples were run
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in duplicate. Analysis of real-time PCR (qRT-PCR) was performed by delta-delta-Ct (∆∆Ct)
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method [18].
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2.4 In silico analysis
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2.4.1 Sequence retrieval
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A comprehensive search of the sequence database on the NCBI website was carried out in
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order to find and compare DMC1 gene orthologs among different species. Briefly, BLASTp [19]
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search was performed using protein sequence corresponding to DMC1 in our study to retrieve
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similar
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(http://smart.emblheidelberg.de) and Pfam (pfam.sanger.ac.uk/search) were used to predict the
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functional domains of the DMC1 protein.
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sequences
from
other
eukaryotes
(yeast,
plants
and
animals).
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2.4.2 Evolutionary analysis Multiple
protein
sequence
alignment
was
performed
using
MAFFT
(http://mafft.cbrc.jp/alignment/server/) and Bioedit. Phylogenetic analysis was performed using 7
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Neighbour Joining and Maximum Likelihood algorithm with MEGA 6.0 package and in each
2
case, branch confidence value was calculated with bootstrapping with 1000 iterations using
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‘‘pairwise deletion option’’ of amino acid sequences with gamma parameters. Here, the Jones,
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Taylor and Thorton (JTT) model for amino acid sequences and gamma parameters was used.
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2.4.3 Protein tertiary structure prediction
To predict the DMC1 protein structure in the four ruminant species, comparative
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modeling was performed using Modeller 9.15 (https://salilab.org/modeller/about_modeller.html)
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and PyMol was used for structure visualization. To identify the target structure, BlastP was
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performed with PDB database and protein structure with PDB ID 1V5W was taken as target
11
structure for modeling. Protein model was generated through many iterations by Modeller, which
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produces homology models by satisfaction of spatial restraints.
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prepared for each of the ruminant species. DOPE (Discrete Optimized Protein Energy) score
14
was calculated for all structures. Lower the energy score, higher is the stability of modeled
15
structure. The models returning to the minimum molpdfs and minimum DOPE score were
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chosen as best probable structures. Z-score was calculated using ProSA-web server to recognize
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the errors in experimental and theoretical models of the protein. The degree of similarity of two
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protein structures was measured with the RMSD (Root-Mean-Square Distance).
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Five protein models were
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3. Results
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The complete open reading frame of DMC1 gene was amplified by RT-PCR from mRNA
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isolated from testis tissue of cattle, buffalo, sheep and goat. The amplified products were
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sequenced in both directions after cloning in pTZ57R/T vector and the DNA sequences from 8
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these four species were submitted to NCBI and are available under accession numbers:
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KR935226-KR935230, KT318875-KT318877. The complete open reading frame of DMC1 gene
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in cattle, buffalo and sheep was observed to be 1023 nucleotides in length encoding a putative
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protein of 340 amino acids. In goats, the complete ORF was found to be 1026 nucleotides in
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length encoding a putative protein of 341 amino acids. Comparative analysis of cDNA sequence
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of four ruminant orthologs revealed limited sequence divergence at both the nucleotide and the
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amino acid level indicating a high degree of evolutionary conservation. The sequence identity at
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nucleotide and amino acid level between cattle and buffalo was 97.3% whereas between sheep
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and goat, it was 95.3%. The homology of cattle and buffalo DMC1 amino acid sequence was
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more with that of sheep as compared to goat. In cattle and sheep, novel alternatively spliced
11
mRNAs of 858 nucleotides were isolated in addition to the full length (FL) transcript.
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Interestingly, novel transcript variants of 897 nucleotides were found in sheep and goat (Fig. 1).
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However, in case of buffalo only full length transcript was observed even by increasing the
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sample size to three.
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Evolutionary analysis of ruminant DMC1 with other animals, humans, fish, plants and yeast clearly separated Saccharomyces cerevisiae (yeast) gene from all other sequences (Fig. 2).
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Plants such as Arabidopsis thaliana and Solanum lycopersicum formed a separate cluster which
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was quite distinct from all other animal species. As is expected, Mus musculus and Rattus
19
norvegicus clustered together with 75% probability and Homo sapiens and Pan troglodytes with
20
84% probability. All the mammals were clearly separated from Danio rerio (Zebra fish),
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Xenopus tropicalis (frog) and Gallus gallus (Junglefowl) with high confidence values. All the
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ruminants (our sequences as well as sequences retrieved from NCBI) clustered together
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indicating high sequence similarity.
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Functional feature prediction by SMART and PDBsum revealed significant identity of cattle,
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buffalo, sheep and goat DMC1 protein with humans as well as other animal species. Multiple
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sequence alignment of the ruminant DMC1 protein with other orthologs also revealed significant
4
sequence homology (Fig. 3). DMC1 protein in humans and mice typically has 2 domains:
5
Domain I (amino acid residues 27-95) and domain II (amino acid residues 96-319) [12]. We also
6
observed these domains to be present in the DMC1 protein of ruminants. Domain I has a helix-
7
hairpin-helix (HhH) motif of 54 amino acids and the sequence of this motif was found to be
8
conserved in all orthologs. The domain II has Walker A and Walker B motifs which interact with
9
single and double stranded DNAs respectively. The sequence of these motifs were also found to
10
be highly conserved in all DMC1 homologs except for one amino acid change in Walker A motif
11
in Ovis aries sequences isolated in this study. The C-terminal region of DMC1 protein includes
12
amino acid residues 319-340 and its sequence was conserved in all DMC1 orthologs. Sequence
13
analysis of the full length transcripts and the two novel splice variants revealed that the transcript
14
variant of 858 nucletides identified in cattle and sheep had a deletion of amino acids at residues
15
142-196. This deletion lies in the domain II region of DMC1 protein. This deletion also altered
16
the junctional amino acids in the putative protein. The junctional amino acid at the start of the
17
deletion was glycine instead of valine at position 142 and serine at the end of the deletion at
18
position 196 was also replaced by glycine. As far as the second splice variant of 897 nucleotides
19
is concerned, there is deletion of residues 154-196 within the domain II region of DMC1 protein.
20
Here again, the junctional amino acid at position 196 is glycine in place of serine.
21
DMC1 gene in cattle consists of 13 exons (AC_00162) whereas in other three ruminant species
22
i.e. buffalo (NW_005785436), sheep (NC_019460) and goat (NC_022297), there are 14 exons,
23
out of which 13 code for functional protein. Comparison of the sequence of transcripts with
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1
DMC1 gene sequence in the analyzed species indicated that there is skipping of coding exons 7
2
and 8 in transcript variant 1 (TV1) and partial skipping of exon 7 and complete skipping of exon
3
8 in transcript variant 2 (TV2) (Fig. 4). To confirm the presence and location of different spliced forms of mRNAs, two
5
oligonucleotides flanking the deleted region were used to amplify cDNA isolated from testis
6
tissue of ruminants such that the full length transcript would yield a product of 326 bp and the
7
splice variants 1 and 2 would produce products of 161 and 200 bp respectively. The sequences of
8
the primers employed were: F2: TAAGTTGCTAGGAGGTGGAATTGAAAGCAT and R2:
9
TAAAGATGCCAGCTTCTTCATGGAACTTTGC. The position of these primers is depicted in
10
Fig. 4. We observed amplified products of expected sizes from the different transcripts of DMC1
11
gene in cattle, buffalo, sheep and goat. Sequencing of the products also confirmed that they were
12
indeed the different splice variants of DMC1 and that there is loss of two exons in the variants
13
(Fig. 5).
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Expression patterns of full length transcript and different splice variants were assessed by
15
real-time PCR in testis tissue of cattle, sheep and goat using primers described in Table 1. The
16
results revealed that the expression level of DMC1-FL was significantly higher than that of TV1
17
in cattle and TV2 in goat (P < 0.05). In sheep, DMC1-FL was most abundantly expressed
18
followed by TV1 and TV2 (DMC1 FL: TV1: TV2 = 6.78: 1.43: 1) (Fig. 6).
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The tertiary structure for the different variants of DMC1 protein was predicted for the four
20
ruminant species using Modeller 9.15 as the structure is not yet available in the PDB database.
21
Errors in experimental and theoretical models of the predicted protein were recognized by
22
calculating the z-score using ProSA-web server. The structure for DMC1 protein could be reliably
23
predicted with satisfactory Z score and the protein was found to contain Walker A, Walker B and
11
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HhH motifs that are peculiar features of this protein. We found that tertiary structure of full length
2
sequences in cattle, buffalo and sheep had 9 α-helices and 8 β-sheets whereas goat protein
3
structure was found to contain 9 α-helices and 7 β-sheets because of difference in sequence at the
4
C terminal end. Transcript variants revealed loss of some α-helices and β-sheets. Transcript
5
variant 1 had 7α-helices and 6 β-sheets whereas transcript variant 2 had 6 α-helices and 7 β-
6
sheets. We found that few residues in ATP binding sites {128 (F), 132(K), 133(T), 158 (F),
7
160(D), 162(E), 223(D)} and multimer BRC sites {157 (I), 159 (F) , 167(P), 170(L), 185 (L) ,
8
188(V), 189 (L), 191(A), 202 (L), 203 (L), 204(D), 205(Y), 206(V), 207(A), 209(K), 251(S) , 254
9
(Q), 255(K), 258(E), 259 (E)} were absent in both the variants because of deletion of 2 exons in
10
the crucial domain II region of the protein (Supplement Fig. S1-S4). Both template and query
11
structures were superimposed to calculate the similarity of target and template structure.
12
Complete protein sequence had approximately 92% and partial sequences had 79% residues
13
similarity, suggesting reliable 3D structure. Hence, the representative structure verified by
14
PDBsum, PROCHEK, ProSA z-score was satisfactory and can thus be considered as a reliable
15
source for further analyses. DOPE score, z score and Molpdf for different variants are depicted in
16
Table 2.
18 19
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4. Discussion
Homologous recombination (HR) is an indispensable process for the maintenance of
20
genomic integrity and for creation of genetic diversity [9]. During HR reaction, the double strand
21
break ends introduced by the SPO11 complex undergo 5′ strand resection to generate 3′ single
22
stranded DNA tails that are bound by either of the two conserved recombinases, DMC1 and
23
RAD51. Both RAD51 and DMC1 are homologs of Escherichia coli RecA gene which is among
12
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the most studied recombination gene encoding a DNA-dependent ATPase that binds to single-
2
stranded DNA and promotes strand invasion and exchange between homologous DNA
3
molecules [4]. RAD51 and DMC1 are composed of two domains: the highly conserved ATPase
4
domain, which makes up the core oligomeric structure, and the N-terminal domain (NTD), which
5
is not conserved in the bacterial RecA protein. The ATPase domain is the core catalytic domain
6
important for DNA binding and ATP hydrolysis [20]. DNA binding is mediated by N-terminal
7
domain in RAD51 and DMC1 and by C-terminal domain in RecA. Specifically, it is the helix–
8
hairpin–helix (HhH) motif in the NTDs of RAD51 and DMC1 which mediates dsDNA binding
9
[20, 21]. Two oligomeric DNA binding forms, an octameric ring and a helical filament have
SC
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been reported for DMC1 whereas the binding form in case of RAD51 is the helical filament. In the present study, open reading frame of DMC1 gene was characterized for the first
12
time in four ruminant species (cattle, buffalo, sheep and goat). The ORF of DMC1 was predicted
13
to encode a putative protein of 340 amino acids in cattle, buffalo and sheep and 341 amino acids
14
in goats. Two novel transcript variants of 858 and 897 nucleotides were also identified in these
15
ruminants which encode proteins of 285 and 298 amino acids respectively. Transcript variant 1
16
(858 nucleotides) which was observed in cattle and sheep has previously been reported in
17
humans and mice [12] whereas the other variant (897 nucleotides) which was isolated in sheep
18
and goat has been identified for the first time in mammals. Another study [7] also confirmed
19
alternative splicing of DMC1 mRNAs in mice where targeted disruption of DMC1 gene resulted
20
in an arrest of meiosis of germ cells in both sexes. Western blotting was performed using rabbit
21
anti-DMC1 antibody on the nuclear extracts of wild type and DMC1 deficient mouse testis. The
22
nuclear extracts from wild-type and heterozygous mouse testes revealed two bands
23
corresponding to full length DMC1 and DMC1-D (a product from alternatively spliced allele)
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1
proteins, respectively as had been previously reported [12]. In contrast, the nuclear extract from
2
the DMC1-deficient mouse testis did not show these bands. In silico analysis revealed that alternatively spliced variants with skipping of coding
4
exons 7 and 8 are predicted in a wide range of mammalian species such as Bos mutus, Pan
5
troglodytes and Pan paniscus in addition to those reported in Homo sapiens and Mus musculus.
6
Splice variants with skipping of some other exons have also been predicted in other species as
7
shown in Table 3. However, in some species such as Rattus norvegicus, Equus asinus and Canis
8
lupus familaris, there is no report available so far on alternate splicing. By real time PCR, we
9
found that mRNA expression levels of DMC1-FL were significantly higher than TV1 and TV2
10
in cattle, sheep and goat suggesting that DMC1-FL is the most abundantly expressed isoform. It
11
is worth emphasizing that evolutionary conservation of splice variants of DMC1 gene across
12
different taxa possibly reinforces a major physiological role for these isoforms since alternative
13
splicing is a major mechanism for augmentation of transcriptome and proteome diversity [22].
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3
Sequence analysis revealed that all the variants of DMC1 identified in the present study
15
have intact N-terminal domain with highly conserved HhH motif. Researchers in the past have
16
reported that high flexibility or multiple conformations of DMC1 are because of the N-terminal
17
domain [20]. It has been experimentally validated that deletion mutant of DMC1 which lacks N-
18
terminal 81 amino acid residues is completely defective in both single-stranded and double-
19
stranded DNA binding activities [20]. Since the variants identified in the present study have
20
intact N-terminal domain, it indicates that DNA binding activity is probably retained in the splice
21
variants. DMC1 transcript variant 1 does not contain the region at amino acids positions 142-196
22
and transcript variant 2 lacks region at amino acids positions 154-196 of ruminant DMC1. These
23
deletions lie in the critical domain II region that is conserved in the RecA-like gene family. Also
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the deleted region locates close to Walker A and Walker B motifs which are necessary for ATP
2
binding, therefore the DMC1 splice variants (1 and 2) may have a low binding affinity for
3
nucleotides and/or low ATPase activity. Domain II region also has multimer BRC interface that
4
binds to BRCA2 tumour suppressor protein which is involved in maintaining the integrity of the
5
genome by participating in error-free double-strand break repair [23]. BRCA2 is a universal
6
regulator of RAD51/DMC1 recombinase actions. In the novel splice variants identified in the
7
present study, some of the residues which are part of the multimer BRC interface have been
8
deleted. So it is logical to speculate that these variants may have reduced binding affinity for
9
BRCA2.
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Amino acid residues from 319-340 comprise the C-terminal region of DMC1 protein
11
which was found to be conserved in all transcript variants of DMC1. Sequence conservation of
12
this region in all eukaryotic DMC1 orthologs suggests that this region may be important in
13
recombination by interacting with other components of the recombination machinery, one of
14
them being the tumor suppressor protein p53. It is a multifunctional molecule that regulates
15
meiotic cell-cycle and DNA repair. It binds to C-terminal domain of mammalian DMC1 and is
16
involved in homologous recombination and maintenance of genomic stability during meiosis
17
[24]. Therefore probably the novel transcript variants retain the ability to interact with other
18
proteins.
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It is interesting and intriguing to note that since the transcript variants are conserved in
20
different mammalian species such as humans, mice [12], cattle, sheep and goat (our study) and
21
also predicted in other animal species, it implies that these variants with the deletion in domain II
22
region which harbors the ATPase domain might have novel roles to play in recombination.
23
Kowalczykowski and Krupp [25] reported that ATPase activity is not required for the strand
15
ACCEPTED MANUSCRIPT
exchange reaction with bacterial RecA. On similar lines, DMC1 splice variants may have
2
attenuated ATPase activity but could still be capable of strand exchange. Put together, these
3
observations suggest that the novel transcript variants of DMC1 with intact N-terminal and C-
4
terminal domains represent proteins with diverse biological functions or with altered functional
5
activities and represent an interesting area to explore for researchers in future.
RI PT
1
6
8
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7
5. Conclusion
The present study for the first time determined the full length coding sequence of DMC1 gene in
10
four ruminant species (buffalo, cattle, sheep and goat). Evolutionary analysis between the
11
different orthologs suggested that these proteins are conserved from yeast to humans. Two novel
12
transcript variants with deletion in the ATPase domain were also characterized for the first time
13
in ruminants. Since these transcripts are also observed in other mammalian species such as
14
humans and mice and predicted in some other species, it indicates that they are evolutionarily
15
conserved and may have novel/diverse biological functions in recombination.
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Conflict of interest statement
18
The authors declare that they have no competing interests.
19
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Acknowledgements
21
This study was supported by the Indian Council of Agricultural Research, New Delhi, India.
22
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Mnd1/Hop2 Facilitates DMC1-Dependent Interhomolog Crossover Formation in Meiosis
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of Budding Yeast. Mol Cell Biol 2006; 26(8):2913–2923.
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[2] Keeney S. SPO11 and the formation of DNA double-strand breaks in meiosis.
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Genome Dynam Stabil 2008; 2:81–123.
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[3] Bishop DK, Park D, Xu L, Kleckner N. DMC1: a meiosis-specific yeast homolog of
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9
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[4] Shinohara A, Ogawa H, Ogawa T. RAD51 protein involved in repair and
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[5] Hong EL, Shinohara A,
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promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into
14
homologous super-coiled duplex DNA. J Biol Chem 2001; 276:41906–41912.
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[6] Pittman DL, Cobb J, Schimenti KJ, Wilson LA, Cooper DM, Brignull E, et al.
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Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for
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DMC1, a germ line specific RecA homolog. Mol Cell 1998; 1:697–705.
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Bishop DK. Saccharomyces cerevisiae DMC1 protein
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21
[8] de Massy B. Initiation of meiotic recombination: how and where? Annu Rev Genet
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2013; http://dx.doi. org/ 10.1146/annurev-genet-110711-155423.
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[9] Krogh BO, Symington LS. Recombination proteins in yeast. Annu Rev Genet 2004;
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38:233–271.
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[10] Bishop DK. RecA homologs DMC1 and RAD51 interact to form multiple nuclear
4
complexes prior to meiotic chromosome synapsis. Cell 1994; 79:1081–1092.
5
[11] Brown MS, Grubb J, Zhang A, Rust MJ, Bishop DK. Small RAD51 and DMC1
6
complexes often co-occupy both ends of a meiotic DNA double strand break. PLoS
7
Genet 2015; 11(12): e1005653.
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[12] Habu T, Taki T, West A, Nishimune Y, Morita T. The mouse and human homologs
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of DMC1, the yeast meiosis-specific homologous recombination gene, have a common
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unique form of exon-skipped transcript in meiosis. Nucleic Acid Res 1996; 24:470–477.
11
[13] Grishchuk AL, Kohli J. Five RecA-like proteins of Schizosaccharomyces pombe are
12
involved in meiotic recombination. Genetics 2003; 165:1031–1043.
13
[14] Neale MJ, Keeney S. Clarifying the mechanics of DNA strand exchange in meiotic
14
recombination. Nature 2006; 442:153–158.
15
[15] Roeder GS. Meiotic chromosomes: it takes two to tango. Genes Dev 1997; 11:2600–
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2621.
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[16] Liu Y, Cui Z. Molecular Cloning and Characterization of DMC1 from the Chinese
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Mitten Crab (Eriocheir sinensis). Int J Aquacult Fish Sci 2015; 1(1):024–029.
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[17] Etedali F, Baghban Kohnehrouz B, Valizadeh M, Gholizadeh A, Malboobi MA. Genome wide cloning of maize meiotic recombinase DMC1 and its functional structure
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through molecular phylogeny. Genet Mol Res 2011; 10(3):1636–1649.
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[18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
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quantitative PCR and the 2−∆∆Ct method. Methods 2001; 25:402–408.
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[19] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment
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search tool. J Mol Biol 1990; 215(3):403–10.
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[20] Kinebuchi T, Kagawa W, Kurumizaka H, Yokoyama S. Role of the N-terminal
4
Domain of the Human DMC1 Protein in Octamer Formation and DNA Binding. J Biol
5
Chem 2005; 280(31):28382–28387.
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[21] Aihara H, Ito Y, Kurumizaka H, Yokoyama S, Shibata T. The N-terminal domain of
7
the human RAD51 protein binds DNA: structure and a DNA binding surface as revealed
8
by NMR. J Mol Biol 1999; 290:495–504.
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[22] Keren H, Lev-Maor G, Ast G. Alternative splicing and evolution: diversification,
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exon definition and function. Nat Rev Genet 2010; 11:345–355.
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[23] Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2.
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Cell 2002; 108:171–182.
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[24] Habu T, Wakabayashi N, Yoshida K, Yomogida K, Nishimune Y, Morita T. p53
14
protein interacts specifically with the meiosis-specific mammalian RecA-like protein
15
DMC1 in meiosis. Carcinogenesis 2004; 25(6):889–893.
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[25] Kowalczykowski SC, Krupp RA. DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in proteinpromoted nucleic acid transactions. Proc Natl Acad Sci U S A 1995; 92(8):3478-3482.
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Table 1. Primers for Real-time PCR of DMC1 variants in ruminants Variant
Primer sequence
Product size
RI PT
(bp)
Wild type
F- ACAGAAGCTTTTGGAGAATTTCGAACTGG
151
F- TTTCCATATCACCACTGGGAGCCAGGA
152
cattle and sheep
R- CAAGTAGCTCCATCTGATGTTCACCACAG
Transcript variant 2:
F- GCTAGGAGGTGGAATTGAAAGCATGGC
152
sheep and goat
R- CAAGTAGCTCCATCTGATGTTCACCTCCT
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Transcript variant 1:
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R- CCGAAGGCGATCTGGACGGAAAGTATT
ACCEPTED MANUSCRIPT
Table 2. Energy minimization score using different methods Molpdf
DOPE score
z-score
Cattle
2292.52222
28590.24805
-4.58
Cattle_TV1
2240.96851
RI PT
Structure
-23632.13086
-3.13
2093.68140
-29477.53125
-4.81
Sheep
2181.68262
-28506.82227
-4.07
Sheep_TV1
2018.29260
-21557.39453
-3.03
Sheep_TV2
2083.81421
-24983.40039
-3.35
Goat
2066.36719
-28991.46680
-4.49
Goat_TV2
2008.12634
-22084.85156
-3.95
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Buffalo
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Table 3. Alternative splicing of DMC1 gene in different species Accession
Total exon
Coding
Skipped
number
count
exon count
coding exon
RI PT
Species
in variant
Bos taurus
NM_001191338
13
Bos indicus_NDRI
KT318875
13
Bos indicus_NDRI_TV
KR935227
11
Bubalus bubalis
XM_006071328
14
Bubalus bubalis_NDRI
KR935226
13
13
Ovis aries
XM_012175647
14
13
Ovis aries_NDRI
KT318876
13
13
Ovis aries_NDRI_TV1
KR935228
11
11
Exons 7 and 8
12
12
Partial exon 7
Capra hircus
SC
13
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EP
Ovis aries_NDRI_TV2
13
11
13
and complete exon 8
XM_005681072
14
13
XM_005681073
11
11
Capra hircus_NDRI
KR935229
13
13
Capra hircus_NDRI_TV
KR935230
12
12
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Capra hircus_TV
Exons 7 and 8
Exons 7 and 8
Partial exon 7 and complete exon 8
Bos mutus
XM_005899439
14
13
ACCEPTED MANUSCRIPT
XM_005899440
11
11
Homo sapiens
NM_007068
14
13
Homo sapiens_TV
NM_001278208
11
11
Mus musculus
NM_010059
14
13
Mus musculus_TV1
NM_001278226
12
Mus musculus_TV2
NR_103477
9
Sus scrofa
XM_005655492
14
Sus scrofa_TV
XM_005655493
12
Exons 7 and 8
Exons 7 and 8
RI PT
Bos mutus_TV
11
Exons 7 and 8
9
Exons 6-9, 11
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Exons 12 and 13
XM_009438403
14
13
Pan troglodytes_TV1
XM_009438407
13
12
Exon 3
Pan troglodytes_TV2
XM_009438408
12
11
Exons 7 and 8
Pan paniscus
XM_003821593
14
13
XM_008975092
13
12
Exon 6
XM_008975093
12
11
Exons 7 and 8
XM_002914548
14
13
Pan paniscus_TV1 Pan paniscus_TV2
EP
Ailuropoda melanoleuca
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Pan troglodytes
XM_011218836
13
12
Equus asinus
XM_014851322.1
13
13
Rattus norvegicus
NM_001130567.1
15
13
Canis lupus familaris
XM_005625878
14
13
AC C
Ailuropoda melanoleuca _TV
Exon 3
Lane 1: DMC1-FL (1023 bp) and TV1 (858 bp) in cattle
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SC
Fig. 1. Gel image of RT-PCR product of DMC1 gene in ruminants
RI PT
ACCEPTED MANUSCRIPT
Lane 3: DMC1-FL (1026 bp), TV2 (897 bp) in goat
Lane 4: DMC1-FL, TV2 (897 bp) and TV1 (858 bp) in sheep
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M: 100 bp DNA marker, bright bands correspond to 500 bp and 1000 bp
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Fig. 2. Phylogenetic tree of DMC1 in eukaryotes constructed by neighbour joining method
AC C
(Poission correction with gamma parameters)
ACCEPTED MANUSCRIPT HhH motif
RI PT
MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLPEAKADKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTEFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTEFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEVKVDKIKEAANKHIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLPEAKADKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTEFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLAAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKQKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEDESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVAEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVAEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVQEESGFQDDEESLFQDIDLLQKHGINMADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVEKIKEAANKLIEPGFLTAFQYSERRKMV MK--EDQVVQEESGFQDDEESLFQDIDLLQKHGINMADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVEKIKEAANKLIEPGFLTAFQYSERRKMV MKVLEEQTLEEESGYQDDEESFFQDIELLQKHGINVADIKKLKSVGICTVKGIQMTTRRALCNIKGLSEAKVDKIKEAAGKLLTCGFQTASEYCIKRKQV MK--EDQVVQEESGFQDEEESLFQDIDLLQKHGINMADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVEKIKEAANKLIEPGFLTAFQYSEKRKMV MKAMEDQVVQEESGYHDDEESFFQDIDLLQKHGINVADIKKLKSVGICTIKGVQMTTRRALCNVKGLSEVKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVLEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MK--EDQVVVEEPGFQDEEESLFQDIDLLQKHGINVADIKKLKSVGICTIKGIQMTTRRALCNVKGLSEAKVDKIKEAANKLIEPGFLTAFEYSEKRKMV MKSMEDQVVEEDVGFHDE-ESFFHDIEMLQKQGINVADIKKLKSVGICTIKGIQMTTRKALCNIKGLSEAKVEKIKEAANKVIEPGFLTAFEYSAKRRMV
Walker A
TE D
M AN U
FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHAPCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGGFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGGFRTGKTQLSHTLCVTAQLPGAGGYSG------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIYTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHAPCG-------------------------------------------------------EH FHITTGSQEFDKLLGGGIESMAITEAFGGFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSG------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHNAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLC-------------------------------------------------------GEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCG-------------------------------------------------------EH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYPGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCG-------------------------------------------------------EH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGTGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHEAVLDNVLYARAYTSEH FHITTGSLEFDKLLGGGVESMAITEAFGEFRTGKTQLSHTLCVTAQLPGEYGYTGGKVIFIDTENTFRPERLKDIADRFNVDHEAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGADGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGPKGYTGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYSGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHITTGSQEFDKLLGGGIESMAITEAFGEFRTGKTQLSHTLCVTAQLPGAGGYPGGKIIFIDTENTFRPDRLRDIADRFNVDHDAVLDNVLYARAYTSEH FHISTGSQEFDKLLGGGIESMAITETFGEFRTGKTQLAHTLCVTAQLPGPNGYTGGKIIFIDTENTFRPDRLHDIADRFSVDHDAVLDNVLYARAYTSEH
EP
B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens_TV H_sapiens M.musculus_TV M.musculus D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
SC
B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens H_sapiens_TV M.musculus M.musculus_TV D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
Walker B
QMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQNLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGRHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMTLFRVDFSGRGESAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMTLFRVDFSGRGESAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQNLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGRHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMTLFRVDFSGRGESAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKILRGYNVAVFVTNQMTADPRAPMTFQADPKNPFGGHISGHHCF QMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKEPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDFVAAKFHEEGGVFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGAGMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIVDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPVGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEAGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHASQMELLDYVAAKFHEEPGIFKLLIIDSIMALFRVDFSGRGELAERQQKLAQMLSRLQKISEEYNVAVFVTNQMTADPGATMTFQADPKKPIGGHILAHAS-
AC C
B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens H_sapiens_TV M.musculus M.musculus_TV D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
TTRISLREGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISSRKGRGELRIAQIYGSPEMPENEATFAITAGGIGDAKE TTRISSRKGRGELRIAQIYGSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFTITAGGIGDAKE TTRISLREGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISSRKGRGELRIAQIYGSPEMPENEATFAITAGGIGDAKE TTRISLAKRKRKLEMPKIYDSPEIAENEANLAITAGGIGNAKR TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFTITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRAELRIAKIFDSPHMPENEATFAITAGGITDAKD TTRISLRKGRGELRIAKIYDSPEMPENEATFAITTGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITPGGIGDAKE TTRVSLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITAGGIGDAKE TTRISLRKGRGELRIAKIYDSPEMPENEATFAITSGGINDAKE
SC
B.indicus_NDRI O.aries_NDRI O.aries_NDRI_TV2 B.Bubalis_NDRI B.indicus_NDRI_TV1 O.aries_NDRI_TV1 C.hircus_NDRI C.hircus_NDRI_TV2 B.taurus B.bubalis B.mutus B.mutus_TV C.hircus C.hircus_TV O.aries O.aries_TV H.sapiens H_sapiens_TV M.musculus M.musculus_TV D.rerio R.norvegicus G.gallus C.L.familiaris P.troglodytes X.tropicalis
RI PT
ACCEPTED MANUSCRIPT
M AN U
Fig. 3. Multiple sequence alignment of DMC1 gene in different eukaryotes. HhH motif in domain I and Walker A & Walker B motifs in domain II are highlighted by horizontal bars above the sequence alignment.
AC C
EP
TE D
ATP binding sites are marked by asterisks and BRCA2 interface by bolts.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 4. Schematic representation of DMC1 gene and different splice variants
TE D
The position of primers used for cloning (F1 and R1) and for confirmation of variants (F2 and
AC C
EP
R2) has been indicated with arrows.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 4. Gel image of RT-PCR product of DMC1 gene in ruminants confirming deletion of two exons
TE D
Lane 1: DMC1-FL (326 bp) and TV1 (161 bp) in cattle Lane 2: DMC1-FL in buffalo
Lane 3: DMC1-FL, TV2 (200 bp) in goat
EP
Lane 4: DMC1-FL, TV2 (200 bp) and TV1 (161 bp) in sheep
AC C
M: 100 bp DNA marker, bright band corresponds to 500 bp
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Fig. 6. Expression profiles of DMC1 splice variants in sheep, goat and cattle (P < 0.05)
ACCEPTED MANUSCRIPT
Highlights •
DMC1 recombinase was sequence characterized for the first time in four ruminant species. Comparative analysis of ruminant DMC1 orthologs revealed limited sequence
RI PT
•
divergence.
Novel alternatively spliced mRNAs with skipping of exons 7 and 8 were also isolated.
•
Presence of variants was validated by amplifying cDNA using primers flanking the
SC
•
deleted region.
Real-time PCR was performed to estimate relative proportion of each variant.
•
Alternative splicing was observed to be an evolutionarily conserved process across
M AN U
•
AC C
EP
TE D
species.