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The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis.

Devisetty UK, Mayes K, Mayes S - BMC Res Notes (2010)

Bottom Line: All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues.There are differences in the levels of expression of the three homoeologues of TaRAD51 and TaDMC1 as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant and Crop sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough LE12 5RD, UK. sean.mayes@nottingham.ac.uk.

ABSTRACT

Background: Meiotic recombination in eukaryotes requires two homologues of the E. coli RecA proteins: Rad51 and Dmc1. Both proteins play important roles in the binding of single stranded DNA, homology search, strand invasion and strand exchange. Meiotic recombination has been well studied in Arabidopsis, rice, maize and the orthologues of RAD51 and DMC1 have been characterized. However genetic analysis of the RAD51 and DMC1 genes in bread wheat has been hampered due to the absence of complete sequence information and because of the existence of multiple copies of each gene in the hexaploid wheat genome.

Findings: In this study we have identified that TaRAD51 and TaDMC1 homoeologues are located on group 7 and group 5 chromosomes of hexaploid wheat, respectively. Comparative sequence analysis of cDNA derived from the TaRAD51 and TaDMC1 homoeologues revealed limited sequence divergence at both the nucleotide and the amino acid level. Indeed, comparisons between the predicted amino acid sequences of TaRAD51 and TaDMC1 and those of other eukaryotes reveal a high degree of evolutionary conservation. Despite the high degree of sequence conservation at the nucleotide level, genome-specific primers for cDNAs of TaRAD51 and TaDMC1 were developed to evaluate expression patterns of individual homoeologues during meiosis. QRT-PCR analysis showed that expression of the TaRAD51 and TaDMC1 cDNA homoeologues was largely restricted to meiotic tissue, with elevated levels observed during the stages of prophase I when meiotic recombination occurs. All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.

Conclusions: Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues. While differences were detected between the three cDNA homoeologues of TaRAD51 as well as the three homoeologues of TaDMC1, it is unlikely that the predicted amino acid substitutions would have an effect on the protein structure, based on our three-dimensional structure prediction analyses. There are differences in the levels of expression of the three homoeologues of TaRAD51 and TaDMC1 as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.

No MeSH data available.


Related in: MedlinePlus

The deduced amino acids alignments of TaRAD51 homoeologous proteins and their 3D models. (a) Multiple alignments of the three TaRAD51 sequences identified in the three bread wheat genomes (A, B and D). Conserved amino acids are indicated by black with a yellow background. The amino acid differences between the three cDNA homoeologous proteins are indicated by black with a grey background. (b) SIFT predictions for the amino acid substitutions for the three cDNA homoeologues of TaRAD51. (c) The 3D structure of TaRad51-7A is represented in red, TaRad51-7B in green and TaRad51-7D in yellow. Blue arrow shows the magnified image of the side chains of three TaRad51 homoeologue proteins, which indicates the only structural dissimilarity.
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Figure 4: The deduced amino acids alignments of TaRAD51 homoeologous proteins and their 3D models. (a) Multiple alignments of the three TaRAD51 sequences identified in the three bread wheat genomes (A, B and D). Conserved amino acids are indicated by black with a yellow background. The amino acid differences between the three cDNA homoeologous proteins are indicated by black with a grey background. (b) SIFT predictions for the amino acid substitutions for the three cDNA homoeologues of TaRAD51. (c) The 3D structure of TaRad51-7A is represented in red, TaRad51-7B in green and TaRad51-7D in yellow. Blue arrow shows the magnified image of the side chains of three TaRad51 homoeologue proteins, which indicates the only structural dissimilarity.

Mentions: Sequence alignment of the TaRAD51 cDNA homoeologues at nucleotide and amino acid level revealed 97% and 99% identity respectively. Two of the TaRAD51 cDNA homoeologues (TaRAD51-7A and -7B) have a continuous ORF of 1032 bp, capable of encoding a protein of 343 amino acids and the TaRAD51-7D ORF has a deletion of three nucleotides at the N-terminal end, thus putatively encoding a predicted protein of 342 amino acids residues. Further analysis of the three translated TaRAD51 cDNA homoeologues revealed that this leads to the expected deletion of one amino acid at position 17 corresponding to an 'E' residue (Glutamic acid) and single amino acid substitutions are also expected between the three homoeologues at positions 4 (A, compared with B&D), 31 (A&B, compared with D) and 115 (A&D, compared with B) (Figure 4a). SIFT predictions suggested that the amino acid substitutions are not expected to affect protein 3D structure for the three cDNA homoeologues of TaRAD51 (Figure 4b). We have also found that the previously reported [14]TaRAD51A1 is identical to the TaRAD51-7D homoeologue isolated here and that the reported TaRAD51A2 sequence appears to be a truncated version of the TaRAD51-7A homoeologue albeit with a few amino acid differences at the truncated end (listed in Additional file 1). However the paralogous nature of the reported TaRAD51A1/A2 [14] was not observed in this study. This supports the idea that there is only one copy of TaRAD51 per haploid wheat genome, at least for the wheat genotype studied here. The predicted 3D homoeologue overlays superimposed onto each other TaRad51 revealed there is a high level of predicted conservation for secondary and tertiary structures (Figure 4c). The only noticeable structural dissimilarity observed between the three cDNA homoeologues is in peptide loops seen between α-helix 13 and α-helix 14 (indicated by the white arrow in Figure 4c)


The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis.

Devisetty UK, Mayes K, Mayes S - BMC Res Notes (2010)

The deduced amino acids alignments of TaRAD51 homoeologous proteins and their 3D models. (a) Multiple alignments of the three TaRAD51 sequences identified in the three bread wheat genomes (A, B and D). Conserved amino acids are indicated by black with a yellow background. The amino acid differences between the three cDNA homoeologous proteins are indicated by black with a grey background. (b) SIFT predictions for the amino acid substitutions for the three cDNA homoeologues of TaRAD51. (c) The 3D structure of TaRad51-7A is represented in red, TaRad51-7B in green and TaRad51-7D in yellow. Blue arrow shows the magnified image of the side chains of three TaRad51 homoeologue proteins, which indicates the only structural dissimilarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962619&req=5

Figure 4: The deduced amino acids alignments of TaRAD51 homoeologous proteins and their 3D models. (a) Multiple alignments of the three TaRAD51 sequences identified in the three bread wheat genomes (A, B and D). Conserved amino acids are indicated by black with a yellow background. The amino acid differences between the three cDNA homoeologous proteins are indicated by black with a grey background. (b) SIFT predictions for the amino acid substitutions for the three cDNA homoeologues of TaRAD51. (c) The 3D structure of TaRad51-7A is represented in red, TaRad51-7B in green and TaRad51-7D in yellow. Blue arrow shows the magnified image of the side chains of three TaRad51 homoeologue proteins, which indicates the only structural dissimilarity.
Mentions: Sequence alignment of the TaRAD51 cDNA homoeologues at nucleotide and amino acid level revealed 97% and 99% identity respectively. Two of the TaRAD51 cDNA homoeologues (TaRAD51-7A and -7B) have a continuous ORF of 1032 bp, capable of encoding a protein of 343 amino acids and the TaRAD51-7D ORF has a deletion of three nucleotides at the N-terminal end, thus putatively encoding a predicted protein of 342 amino acids residues. Further analysis of the three translated TaRAD51 cDNA homoeologues revealed that this leads to the expected deletion of one amino acid at position 17 corresponding to an 'E' residue (Glutamic acid) and single amino acid substitutions are also expected between the three homoeologues at positions 4 (A, compared with B&D), 31 (A&B, compared with D) and 115 (A&D, compared with B) (Figure 4a). SIFT predictions suggested that the amino acid substitutions are not expected to affect protein 3D structure for the three cDNA homoeologues of TaRAD51 (Figure 4b). We have also found that the previously reported [14]TaRAD51A1 is identical to the TaRAD51-7D homoeologue isolated here and that the reported TaRAD51A2 sequence appears to be a truncated version of the TaRAD51-7A homoeologue albeit with a few amino acid differences at the truncated end (listed in Additional file 1). However the paralogous nature of the reported TaRAD51A1/A2 [14] was not observed in this study. This supports the idea that there is only one copy of TaRAD51 per haploid wheat genome, at least for the wheat genotype studied here. The predicted 3D homoeologue overlays superimposed onto each other TaRad51 revealed there is a high level of predicted conservation for secondary and tertiary structures (Figure 4c). The only noticeable structural dissimilarity observed between the three cDNA homoeologues is in peptide loops seen between α-helix 13 and α-helix 14 (indicated by the white arrow in Figure 4c)

Bottom Line: All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues.There are differences in the levels of expression of the three homoeologues of TaRAD51 and TaDMC1 as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant and Crop sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough LE12 5RD, UK. sean.mayes@nottingham.ac.uk.

ABSTRACT

Background: Meiotic recombination in eukaryotes requires two homologues of the E. coli RecA proteins: Rad51 and Dmc1. Both proteins play important roles in the binding of single stranded DNA, homology search, strand invasion and strand exchange. Meiotic recombination has been well studied in Arabidopsis, rice, maize and the orthologues of RAD51 and DMC1 have been characterized. However genetic analysis of the RAD51 and DMC1 genes in bread wheat has been hampered due to the absence of complete sequence information and because of the existence of multiple copies of each gene in the hexaploid wheat genome.

Findings: In this study we have identified that TaRAD51 and TaDMC1 homoeologues are located on group 7 and group 5 chromosomes of hexaploid wheat, respectively. Comparative sequence analysis of cDNA derived from the TaRAD51 and TaDMC1 homoeologues revealed limited sequence divergence at both the nucleotide and the amino acid level. Indeed, comparisons between the predicted amino acid sequences of TaRAD51 and TaDMC1 and those of other eukaryotes reveal a high degree of evolutionary conservation. Despite the high degree of sequence conservation at the nucleotide level, genome-specific primers for cDNAs of TaRAD51 and TaDMC1 were developed to evaluate expression patterns of individual homoeologues during meiosis. QRT-PCR analysis showed that expression of the TaRAD51 and TaDMC1 cDNA homoeologues was largely restricted to meiotic tissue, with elevated levels observed during the stages of prophase I when meiotic recombination occurs. All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.

Conclusions: Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues. While differences were detected between the three cDNA homoeologues of TaRAD51 as well as the three homoeologues of TaDMC1, it is unlikely that the predicted amino acid substitutions would have an effect on the protein structure, based on our three-dimensional structure prediction analyses. There are differences in the levels of expression of the three homoeologues of TaRAD51 and TaDMC1 as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.

No MeSH data available.


Related in: MedlinePlus