Human Molecular Genetics, 2002, Vol. 11, No. 7 841-851
© 2002 Oxford University Press
Structural analysis of the chicken BRCA2 gene facilitates identification of functional domains and disease causing mutations
CRC Gene Function and Regulation Group, The Breakthrough Toby Robins Breast Cancer Research Centre, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK and 1Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, UK
Received January 14, 2002; Revised and Accepted February 6, 2002.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Carriers of mutations in the BRCA2 gene have a high risk of developing breast and other cancers. The BRCA2 gene, which is located on human chromosome 13, encodes a very large protein of only poorly understood function. To define regions of sequence conservation and highlight potentially functionally important domains, we have cloned and characterized the chicken BRCA2 gene, the first non-mammalian BRCA2 gene to be described. The gene is organized similarly to the human BRCA2 gene, but is more compact and is localized to the subtelomeric region of chicken chromosome 1q, within a region that contains other genes from human chromosome 13. The chicken BRCA2 gene encodes a protein of 3399 amino acids, which is poorly conserved with mammalian BRCA2 proteins, having only 37% amino acid identity overall with human BRCA2. However, certain domains are much more highly conserved, indicating functional significance. We describe genes with some of these conserved domains in organisms as diverse as intracellular parasites, mosquitoes and plants. The evolutionarily divergent chicken BRCA2 sequence may also be useful in assigning the large number of sequence variants that have been described in the human BRCA2 gene which are of unknown significance in disease causation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Five to ten percent of breast cancers are thought to result from a hereditary predisposition to the disease (1) and two highly penetrant breast cancer associated genes, BRCA1 and BRCA2, have been mapped and cloned (2,3). Inheritance of a germline mutation in the BRCA2 gene is associated with a lifetime risk of 37–84% for breast cancer, and up to 27% for ovarian cancer. There is also an increased risk of male breast cancer, prostate cancer and pancreatic cancer (1). The BRCA genes appear to act as tumour suppressor genes, in that carriers show loss or mutation of the wild-type allele within the tumours.
The human BRCA2 gene, located on chromosome 13q12.3, spans >70 kb and consists of 27 exons (1). The gene encodes a large protein of 3418 amino acids with a molecular weight of 384 kDa showing no significant sequence similarity to other known human proteins, thus offering few clues as to possible functional domains. Furthermore, BRCA2 is poorly conserved in mammalian evolution; the mouse Brca2 protein shows only 59% amino acid identity with human BRCA2 (4). However, certain regions are more highly conserved and investigation of these regions has revealed functionally important motifs, including the BRC repeats in exon 11 implicated in binding RAD51 (5,6), nuclear localization signals (7,8) as well as potential interaction sites for various other proteins (9–14).
The BRCA2 protein is located in the cell nucleus and is most highly expressed in the S phase of the cell cycle (15). BRCA2 is thought to play a role in double strand DNA break repair, though there is also some evidence for a role in transcriptional regulation (16). Specifically, BRCA2 is implicated in DNA repair by homologous recombination (17) and loss of BRCA2 has been shown to lead to a switch from conservative gene conversion to the error-prone single strand annealing pathway (18).
Many independent sequence variants have now been identified within the BRCA2 gene (3,19,20). The Breast Cancer Information Core (BIC) database (http:www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic) provides a centralized source of BRCA1 and BRCA2 sequence variation data (21,22). Of the variants recorded, 90% are unique and they are distributed widely throughout the gene. Just over half are frameshift and nonsense changes resulting in truncation of the encoded protein; the majority of these are known to be disease associated. The remaining DNA sequence variants are either silent or result in an amino acid change in the BRCA2 protein. While the silent variants are likely to be polymorphisms, data is lacking on whether the majority of the missense variants are disease associated. This is due to the large size and relatively poor functional characterization of the BRCA2 protein so far (16). Some of these missense variants are found at relatively high frequency in the population and have multiple entries in the BIC database. For screening purposes and patient counselling it is important to determine whether inheritance of these potentially pathogenic variants poses an increased risk of developing cancer.
Here we describe the cloning and sequence analysis of the chicken BRCA2 gene. This is the most divergent BRCA2 gene isolated thus far and analysis of the sequence will be valuable in identifying and validating functionally important domains. Furthermore, the sequence should provide valuable information to aid the classification of unassigned, potentially disease-causing, variants in the human BRCA2 gene.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cloning of chicken BRCA2
A chicken genomic library was screened by hybridization with a cDNA probe encompassing exons 12–27 of the human BRCA2 gene. Hybridizing fragments were subcloned into pBluescript and sequenced. The derived genomic sequence showed similarity to human BRCA2. Oligonucleotides designed from putative chicken BRCA2 exons were used to amplify cDNA fragments, which were also then sequenced. Sequence corresponding to the extreme 5' terminus of the chicken BRCA2 mRNA was amplified using 5'-rapid amplification of cDNA ends (5'-RACE). Using a combination of these methods, sequence was derived covering all of the coding region of chicken BRCA2 (GenBank accession no. AY083934). The sizes of the intervening introns were also determined. The chicken BRCA2 gene spans
40 kb of genomic DNA, smaller than its human counterpart (>70 kb), the difference arising largely as a result of smaller introns (data not shown). Individual exon sizes are very similar between human and chicken BRCA2. The chicken BRCA2 gene has 26 coding exons producing an open reading frame (ORF) of 10 197 bp. A putative initiation codon was identified at nucleotide position 76 of the complete cDNA, with an in-frame stop codon present 6 bp upstream, suggesting that the entire ORF had been isolated (data not shown).
Chromosomal location of the chicken BRCA2 gene
A BAC clone containing the chicken BRCA2 gene was isolated by filter hybridization using chicken BRCA2 cDNA as a probe. Single colour fluorescence in situ hybridization (FISH) analysis using the bacterial artificial chromosome (BAC) on metaphase spreads of the chicken pre-B cell lymphoma cell line DT40 revealed that the chicken BRCA2 gene was located on one of the chicken macrochromosomes, probably chromosome 1 (Fig. 1). To confirm the chromosomal location of the gene, dual colour FISH was then performed using the BAC clone and a specific chromosomal paint for chicken chromosome 1 (23). This revealed that the chicken BRCA2 gene is located on the subtelomeric region of chromosome 1q (Fig. 1). The Retinoblastoma (Rb1) gene and LAMP1, two genes located on human chromosome 13 (present at 13q14.2 and 13q34, respectively), are also present on chicken chromosome 1q and it appears therefore that BRCA2 may form part of a conserved linkage group (for more details, see the chicken linkage map at http://www.zod.wau.nl/vf/chickensite/chicken.html).
|
Phylogenetic analysis of BRCA2 genes
Phylogenetic analysis was undertaken to confirm the orthologous relationship between the chicken BRCA2 gene and the previously described mammalian BRCA2 genes. Using the cDNA sequence of chicken BRCA2 and the three fully sequenced BRCA2 mammalian cDNAs (human, mouse and rat), a gene tree was constructed using a maximum parsimony method from the PHYLIP package (see Materials and Methods) (Fig. 2). This is a character state method using an algorithm to identify a topology of the rooted tree that requires the smallest number of evolutionary changes (24). A weighted method was used to minimize the effect of long branch attraction. The topology of the tree confirms that, as expected, there appears to be greater evolutionary divergence between chicken and mammalian lineages compared with rodent and human lineages. In addition, the branch lengths correspond to the known evolutionary distances between the species, assuming that the mammalian divergence occurred 60–85 million years ago, and that the vertebrate divergence occurred 230–300 million years ago (24). Thus both the intron/exon organization and the sequence divergence are consistent with the gene we isolated being the chicken orthologue of BRCA2.
|
Structure of the chicken BRCA2 protein
The full length chicken BRCA2 protein is composed of 3399 amino acids, slightly smaller than the human protein (3418 amino acids) but larger than the mouse protein (3329 amino acids). The predicted molecular weight of the protein encoded by the chicken BRCA2 gene is 378 kDa. Alignment of the human and chicken BRCA2 proteins demonstrates overall amino acid identity of only 37%, with 45% similarity (Fig. 3). This is a relatively low level of conservation and, as expected, it is considerably less than that between human and mouse BRCA2 (4). However, some parts of the protein are much more highly conserved. This is illustrated by graphical pairwise sequence alignment of the human and chicken BRCA2 amino acid sequences (Fig. 4). Only the more highly conserved regions—the N-terminal region encoded by exons 2/3 and sequence encoded by exons 16–20—are present on the diagonal indicating high sequence conservation. Exon 11 sequence is notably poorly conserved.
|
|
High sequence conservation within the context of overall poor conservation should reveal functionally important domains. Four distinct areas within the chicken BRCA2 sequence show a high degree of sequence conservation (Figs 3–5). One region of known high conservation between human and mouse BRCA2 which is of functional significance is the BRC repeat region encoded by exon 11, which is involved in RAD51 binding (5,6). Overall, exon 11 encoded sequence in chicken BRCA2 is poorly conserved (26% identity and 45% similarity with human BRCA2), though the BRC repeats show much higher conservation (Fig. 5). The most highly conserved repeats, as seen across other species, are BRC1, -7 and -8 (70% identity and 84% similarity with human BRCA2). Interestingly, individual repeats tend to be more similar to each other, even across large evolutionary distances, than to other repeats. This raises the possibility that individual BRC may have distinct functions. Two other regions encoded by exon 11 (residues 1058–1096 and 2175–2196 of chicken BRCA2) are also highly conserved but do not match the BRC repeat consensus sequence. These are located at the beginning and end of sequence encoded by the large exon 11 and their function is unknown. A non-BRC repeat RAD51 binding site in the C-terminus of mouse Brca2 (amino acids 3196–3232) has been reported (9). This region shows 95% identity with human BRCA2 but the chicken BRCA2 shows only 40% identity with both mouse and human BRCA2, significantly less than the most conserved BRC repeats in exon 11.
|
Three potential nuclear localization sequences (NLSs) have been described within the C-terminal region of human BRCA2 encoded by exon 27 (NLS1, K3263NCKKRR; NLS2, P3311IKKKEL and NLS3, R3381LKRR); NLS1 and NLS3 have been suggested to be functional (7). Overall, amino acid sequence encoded by exon 27 is poorly conserved, with 29% identity and 42% similarity between human and chicken (Fig. 3). Only one of the postulated NLSs, NLS1, is significantly conserved in chicken. Furthermore, nuclear localization assays suggest that the sequence conservation of NLS1 is reflected in functional activity (unpublished data). A human polymorphism (Lys3326Ter) has been reported which results in termination of the BRCA2 ORF 93 amino acids prematurely, but this carries no increased breast cancer risk (25). Interestingly, the extreme C-terminus of the protein beyond the Lys3326Ter polymorphism is highly divergent, supporting the suggestion that this domain is not functionally important.
A further conserved region close to the N-terminus of the protein (residues 8–42) (Fig. 3) spans the region encoded by exons 2 and 3 of BRCA2, a region previously reported to have a role in transcriptional activation (26). Amino acid sequence encoded by exon 7 (74% identity with human BRCA2) and exons 16–20 (72% identity with human BRCA2) are also well conserved but while they have been reported as binding sites for several proteins their function is unknown (10,12–14).
Identification of BRCA2-related genes in other species
The chicken BRCA2 sequence was used as a query sequence in BLAST searches of GenBank. This revealed several genes present in diverse non-mammalian species showing significant sequence similarity to the BRCA2 cDNA and encoded protein sequence. Sequence of genomic DNA (GenBank accession no. AL315381) from the Indonesian river puffer fish, Tetraodon nigroviridis, showed significant similarity to exons 15, 16 and 17 of chicken and human BRCA2. The presence of intron/exon junctions at identical positions in chicken, human and Tetraodon DNA indicates that the sequence in the database is very likely to represent a fish orthologue of BRCA2. Thus, the presence of BRCA2 genes in both avian and fish species suggests that the gene evolved before the divergence of vertebrates 300 million years ago (24).
Further BLAST searching of GenBank databases revealed the presence of several genes from diverse species showing significant similarity to vertebrate BRCA2 genes (Fig. 6). These species were the intracellular parasites Leishmania major (AAK2828.1), Trypanosoma (species brucei, CAB95357.1 and species cruzi, AZ050787) and Encephalitozoon cuniculi (NC_00324.1), the mosquito Anopheles gambiae (AJ283016) and the plants Arabidopsis thaliana (thale cress; two proteins CAB80760.1 and CAB82279.1) and Oryza sativa (rice; BAB64792.1) (Fig. 6 and data not shown). Only the Arabidopsis genes are annotated as BRCA2-related genes in GenBank. Although considerably smaller, the Leishmania, Trypanosoma and Arabidopsis proteins share two distinct regions of sequence similarity with vertebrate BRCA2 proteins. At the N-terminus these proteins have BRC-like repeats varying in copy number from 1 to 15 and therefore may bind RAD51. In addition each protein contains highly significant sequence similarity to a region encoded by exons 17–20 of vertebrate BRCA2 genes (Fig. 6B). The sequence conservation in very divergent organisms strongly suggests some conserved function for this region which we have named the BLAT (BRCA2-motif in Leishmania, Arabidopsis and Trypanosoma; these were the organisms in which we first noted the similarity) domain. The conservation of two domains, BRC and BLAT, in these novel proteins suggests that they may have function(s) in common with vertebrate BRCA2 proteins. The BLAT domain is also present in a partial expressed sequence tag (EST) cDNA derived from the mosquito A.gambiae but it is not known if this gene also contains BRC repeats (Fig. 6B). We detected a distantly related BLAT domain in the complete genome sequence of E.cuniculi (27), a member of the Microspiridia group of obligate intracellular parasites. This finding is remarkable in that the organism is a primitive eukaryote which lacks mitochondria and has a very small genome. The BLAT domain is contained within an ORF of 495 amino acids which lacks BRC repeats or significant similarity to other proteins.
|
Use of the chicken BRCA2 protein sequence in assignment of unclassified variants in human BRCA2
The BIC database (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic) (21,22) contains over 2000 entries for BRCA2 genetic variants. Of the unique sequence variants within the BIC database, 47% (308) are non-synonymous codon changes resulting in missense changes. Of these only 2.2% (7/308) are known to be disease associated, and only 6.1% (19/308) are thought to be neutral polymorphisms. The remaining 91.6% (282/308) are described as unassigned variants and, for these, data is lacking on whether they are disease associated.
Sequencing of the chicken BRCA2 gene allows comparative analysis of possible disease-causing missense variants in human BRCA2 compiled in the BIC database. Using a multiple alignment of human, chicken and mouse BRCA2 sequences we analysed the sequence conservation at amino acid residues corresponding to all 308 missense variants in the BIC database. For these residues, the equivalent amino acid in mouse and chicken BRCA2 was classified as being identical or of a similar chemical character [using the evolutionarily weighted Dayhoff amino acid substitution matrix (24,28)] or as being non-conserved.
These criteria were used to classify known pathogenic and polymorphic variants. All seven known disease-associated variants occur at fully conserved or partially conserved (examples are shown in Table 1). However, amongst the known polymorphisms only 37% (7/19) are fully or partially conserved. Thus there is a clear difference in distribution of conserved and non-conserved residues between pathogenic and polymorphic variants.
|
We then examined the remaining 282 unassigned variants present in the BIC database. This indicated that 32% (89/282) of the variants are at fully conserved residues, suggesting that a significant number may be disease-associated. A further 30% (84/282) are at non-conserved residues, suggesting they are likely to be polymorphisms. Hence, in a significant number of cases, examination of the evolutionarily conservation of individual residues can inform an assessment of whether a particular variant is likely to be disease-causing (Table 1). For example the unassigned variant I2285V might be thought likely to be a neutral polymorphism due to the relatively conservative isoleucine to valine substitution. However the conservation of this residue as isoleucine in both mouse and chicken proteins suggests that the change to valine may indeed have pathogenic consequences. Conversely the V2728I variant is present as V in the mouse but I in chicken, suggesting that it might be a neutral polymorphism. Clearly this type of analysis can only give an indication of the likelihood that a variant residue may be neutral or deleterious; additional confirmatory functional studies will be required.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Here we describe the cloning and sequence analysis of the chicken BRCA2 gene, the first non-mammalian BRCA2 gene to be identified. Overall the chicken BRCA2 protein shows only limited amino acid identity with human BRCA2. However, several lines of evidence strongly suggest that we have isolated the orthologous chicken BRCA2 gene. First, the gene we have isolated maps to chicken chromosome 1q. There are several ongoing chicken genome mapping projects worldwide and over 500 gene loci have been mapped (see http://www.ri.bbsrc.ac.uk/chickmap/; http://www.zod.wau.nl/vf/chickensite/chicken.html; http://poultry.mph.msu.edu/chickmap.html). The avian karyotype is complex with 78 chromosomes, the majority of which are microchromosomes. Among many rearrangements between chicken and human chromosomes, 81 autosomal conserved segments have been noted (29). One of these conserved regions on chicken chromosome 1 contains the RB1 and LAMP1 loci, which are located on human chromosome 13q14.2 and 13q34, respectively (29). As the human BRCA2 gene maps to chromosome 13q12.3 it seems possible that BRCA2, LAMP1 and RB1 form a conserved linkage group on chicken chromosome 1. Secondly, phylogenetic analysis indicates that the rate of sequence divergence of the gene we have isolated is consistent with what is already known of the rapid rate of evolution of mammalian BRCA2 genes. Thirdly, the intron/exon organization of the gene we have identified is essentially identical to that of mammalian BRCA2 genes.
One of the problems in analysing proteins as large as BRCA2 is identifying regions critical for function. Comparison of proteins across large evolutionary distances can facilitate the identification of functionally important regions. This comparison should be particularly valuable with the rapidly evolving BRCA2 protein; the chicken BRCA2 shows only 37% identity to human BRCA2 but there are areas of much higher conservation. The high conservation of these regions in the context of overall poor sequence similarity suggests that they are of functional significance.
As is seen in comparisons of mammalian BRCA2 proteins (5), the large region encoded by exon 11 is very poorly conserved apart from the BRC repeats which have functions in binding RAD51. The most highly conserved repeats, as seen across other species, are BRC1, 7 and 8 (Fig. 5). Some repeats are of lower conservation, suggesting that they may be non-functional in binding RAD51, at least in some species; for example BRC5 in chicken or BRC6 in mouse or hamster appear to lack critical residues. In this respect it is notable that different BRC repeats have varying abilities to competitively block nucleoprotein filament formation by RAD51 (30). It has been postulated that the duplication of the BRC repeats occurred prior to the mammalian species radiation 60–85 million years ago (5). Since the BRC repeats within chicken BRCA2 are also more highly conserved than the rest of exon 11, it seems highly likely that duplication of these repeats occurred before the divergence of birds and mammals 230–300 million years ago. The presence of BRC repeats in even more evolutionarily divergent organisms (31 and see below) suggests that the origin of the motif itself might be much older.
No BRCA2-like genes have been found in the complete genome sequences of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the worm Caenorhabditis elegans and the fly Drosophila melanogaster. This suggested that BRCA2 genes might be restricted to vertebrates. However, sequence database searching using chicken BRCA2 has revealed the presence of several new non-vertebrate genes carrying two distinct regions of sequence similarity to vertebrate BRCA2 proteins. These genes are present in surprisingly evolutionarily divergent organisms such as intracellular parasites, plants and possibly insects. The two regions of sequence conservation are multiple copies of BRC-like potentially RAD51 binding repeats and a domain encoded by exons 17–20 of vertebrate BRCA2 genes which we have named the BLAT domain. The coincidence of these two domains in these novel BRCA2-related proteins suggests that they have some functional properties in common with human BRCA2. It will be interesting to determine, for example, whether the BRC-like repeats in these novel proteins are able to bind RAD51. Perhaps the most unexpected of the species having a gene with sequence similarity to BRCA2 is the obligate intracellular parasite E.cuniculi. This remarkable primitive eukaryote, a member of the Microspiridium family, lacks mitochondria and peroxisomes and has a highly compact genome (27). This compaction has been achieved, in part, by eliminating unnecessary genes and it is predicted that the genome of this organism has less than 2000 protein coding genes. Therefore it seems likely that the BLAT domain-containing ORF we have identified is critical for the life cycle of this organism. This ORF lacks BRC domains but intriguingly E.cuniculi has retained a RAD51 gene (27).
The BLAT domain lies within the most highly conserved region of BRCA2 proteins. This region (amino acids 2480–3170 in human BRCA2) contains the binding sites for several proteins such as DSS1, hBUBR1, hsFLNa and BCCIP
(10,12–14), but its exact function is unknown. Only one of these interactions, with DSS1, occurs within the central part of this conserved region (amino acids 2472–2957) in which the BLAT domain is located. It is of note that DSS1 is itself highly conserved in eukaryotes (10). Although DSS1 is deleted in the human hereditary syndome split hand-split foot (32), it is not clear that this is the cause of the disease. In the yeasts S.cerevisiae and S.pombe mutation of DSS1 causes growth arrest and cell cycle defects (10). Further work is required to investigate whether the BLAT domain binds other proteins.
The isolation of BRCA2 was originally confirmed by the discovery of potentially pathogenic mutations in the gene which segregated with cancer incidence in large families exhibiting predisposition to the disease (3). Most of these original mutations resulted in truncation of the protein encoded by BRCA2 (3,19,20). However, many sequence variants have now been identified which result in amino acid changes rather than chain termination and in most cases it is not known whether these predispose to breast cancer or are benign polymorphisms. These are known as unassigned variants and have been compiled in the BIC database (21,22). Classification of these is hindered by the absence of a functional assay for the BRCA2 protein. Although BRCA2 has been implicated in DNA double-strand break repair and transcriptional regulation (16), no method of defining the possible effects of the large number of missense variants is available. Analysis of the chicken BRCA2 sequence described here may provide assistance in making a preliminary assignment of these variants in human BRCA2. There are two reasons why analysis of the chicken sequence should be useful. First, the BRCA2 gene is evolving at a much higher than average rate. Secondly, the chicken BRCA2 is the most phylogenetically diverse BRCA2 gene sequence available. These two factors act to maximize sequence divergence and this is reflected in the very low overall conservation of BRCA2 sequence among vertebrates. Hence, conservation across large evolutionary distances tends to suggest that particular residues are critical for function. Conversely, variation in residues which are not identical or conserved in chemical character are less likely to be pathogenic.
We have used these criteria to examine variation in the BRCA2 protein. A difference was observed between known disease-associated variants, all of which are at conserved or partially conserved positions, and known polymorphisms, the majority of which are non-conserved residues. We have examined the conservation of residues altered in the unassigned variant class and this indicates that, while such an analysis could not in itself be used as a diagnostic test, it can in many cases provide valuable information about the likelihood of the variant being disease-associated. This can be factored in to any advice that might be offered in a genetic counselling context. This assessment will also provide useful indications about which variants might be productively tested for altered function in existing assays (for example see 18).
Finally, the availability of the chicken BRCA2 sequence will facilitate efforts to analyse BRCA2 function by targeted modification in the highly recombinogenic chicken cell line, DT40 (33). It seems possible that these cells transgenic for human BRCA2 could be a useful system for the analysis of variants in the gene.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cloning chicken BRCA2
A chicken genomic library in the vector
FIXII (Stratagene) and a chicken BAC library (HGMP) in the vector pECBAC1 were screened by standard methods. Hybridizing fragments were subcloned into pBluescript and sequenced using ABI Big DyeTM technology, on an ABI 377 Sequencer (Applied Biosystems). Oligonucleotides designed from exons of chicken BRCA2 genomic clones were used to amplify cDNA fragments from reverse transcribed chicken DT40 cell RNA, using the Expand High Fidelity PCR Kit (Roche Diagnostics). The 5' terminus was amplified by modification of the SMART 5'-RACE protocol (Clontech), using two gene-specific reverse primers: GSP1, 5'-GAG CTC TTT CAT CCT GTT GCT TTG C-3' and NGSP1, 5'-AGG ACA TTT CTG CAT CCA CTT CTG C-3'. The SMART adapter oligonucleotide and universal primer mix oligonucleotides were used as the forward primers for first strand cDNA synthesis and subsequent PCR.
DNA sequence analysis
Sequencing of cDNA fragments was performed as above. Sequence contigs were assembled using Sequencher 4.0 software package (Gene Codes Corporation). Sequence analysis was performed using multiple programmes from the GCG sequence analysis software package at the HGMP (www.hgmp.mrc.ac.uk). Phylogenetic analysis was performed using the PHYLIP package of tree constructing programmes (HGMP). BIC data derived from (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic) was analysed in Visual Basic Editor/Microsoft Excel using the Dayhoff amino acid substitution matrix (24,27).
Mapping chicken BRCA2 by FISH
Metaphase spreads of chicken DT40 cells were prepared using standard protocols (34). A BAC clone carrying the chicken BRCA2 gene was labelled with digoxigenin by nick translation. Dual colour FISH was performed using this probe and a biotin labelled chicken chromosome 1 probe prepared by DOP-PCR from flow sorted chicken chromosomes (23). Metaphases were viewed using a Zeiss Axioskop 25 epifluorescence microscope, and the images analysed using the Smartcapture2 software package (Vysis).
|
ACKNOWLEDGEMENTS |
|---|
We are grateful to the Cancer Research Campaign, Breakthrough Breast Cancer and the Mary-Jean Mitchell Green Foundation for financial support and Graham Goodwin for the gift of chicken red blood cell RNA. M.W. is the recipient of a Cancer Research Campaign Research Fellowship for a Clinician.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +44 207 970 6058; Fax: +44 207 878 3858; Email: alana@icr.ac.uk
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Rahman,N. and Stratton,M.R. (1998) The genetics of breast cancer susceptibility. Annu. Rev. Genet., 32, 95–121.[ISI][Medline]
2
Miki,Y., Swensen,J., Shattuck-Eidens,D., Futreal,P.A., Harshman,K., Tavtigian,S., Liu,Q., Cochran,C., Bennett,L.M., Ding,W. et al. (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science, 266, 66–71.
3 Wooster,R., Bignell,G., Lancaster,J., Swift,S., Seal,S., Mangion,J., Collins,N., Gregory,S., Gumbs,C. and Micklem,G. (1995) Identification of the breast cancer susceptibility gene BRCA2. Nature, 378, 789–792.[Medline]
4
Connor,F., Smith,A., Wooster,R., Stratton,M., Dixon,A., Campbell,E., Tait,T.M., Freeman,T. and Ashworth,A. (1997) Cloning, chromosomal mapping and expression pattern of the mouse Brca2 gene. Hum. Mol. Genet., 6, 291–300.
5
Bignell,G., Micklem,G., Stratton,M.R., Ashworth,A. and Wooster,R. (1997) The BRC repeats are conserved in mammalian BRCA2 proteins. Hum. Mol. Genet., 6, 53–58.
6
Chen,P.L., Chen,C.F., Chen,Y., Xiao,J., Sharp,Z.D. and Lee,W.H. (1998) The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. Proc. Natl Acad. Sci. USA, 95, 5287–5292.
7
Spain,B.H., Larson,C.J., Shihabuddin,L.S., Gage,F.H. and Verma,I.M. (1999) Truncated BRCA2 is cytoplasmic : implications for cancer-linked mutations. Proc. Natl Acad. Sci. USA, 96, 13920–13925.
8
Sarkisian,C.J., Master,S.R., Huber,L.J., Ha,S.I. and Chodosh,L.A. (2001) Analysis of murine Brca2 reveals conservation of protein–protein interactions but differences in nuclear localization signals. J. Biol. Chem., 276, 37640–37648.
9 Sharan,S.K., Morimatsu,M., Albrecht,U., Lim,D.S., Regel,E., Dinh,C., Sands,A., Eichele,G., Hasty,P. and Bradley,A. (1997) Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature, 386, 804–810.[Medline]
10
Marston,N.J., Richards,W.J., Hughes,D., Bertwistle,D., Marshall,C.J. and Ashworth,A. (1999) Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals. Mol. Cell. Biol., 19, 4633–4642.
11 Marmorstein,L.Y., Kinev,A.V., Chan,G.K., Bochar,D.A., Beniya,H., Epstein,J.A., Yen,T.J. and Shiekhattar,R. (2001) A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression. Cell, 104, 247–257.[ISI][Medline]
12
Futamura,M., Arakawa,H., Matsuda,K., Katagiri,T., Saji,S., Miki,Y. and Nakamura,Y. (2000) Potential role of BRCA2 in a mitotic checkpoint after phosphorylation by hBUBR1. Cancer Res., 60, 1531–1535.
13
Liu,J., Yuan,Y., Huan,J. and Shen,Z. (2001) Inhibition of breast and brain cancer cell growth by BCCIP, an evolutionarily conserved nuclear protein that interacts with BRCA2. Oncogene, 20, 336–345.[ISI][Medline]
14
Yuan,Y. and Shen,Z. (2001) Interaction with BRCA2 suggests a role for filamin-1 (hsFLNa) in DNA damage response. J. Biol. Chem., 276, 48318–48324.
15
Bertwistle,D., Swift,S., Marston,N.J., Jackson,L.E., Crossland,S., Crompton,M.R., Marshall,C.J. and Ashworth,A. (1997) Nuclear location and cell cycle regulation of the BRCA2 protein. Cancer Res., 57, 5485–5488.
16 Kerr,P. and Ashworth,A. (2001) New complexities for BRCA1 and BRCA2. Curr. Biol., 11, R668–R676.[ISI][Medline]
17 Moynahan,M.E., Pierce,A.J. and Jasin,M. (2001) BRCA2 is required for homology-directed repair of chromosomal breaks. Mol. Cell, 7, 263–272.[ISI][Medline]
18 Tutt,A., Bertwistle,D., Valentine,J., Gabriel,A., Swift,S., Ross,G., Griffin,C., Thacker,J. and Ashworth,A. (2001) Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences. EMBO J., 20, 4704–4716.[ISI][Medline]
19 Tavtigian,S.V., Simard,J., Rommens,J., Couch,F., Shattuck-Eidens,D., Neuhausen,S., Merajver,S., Thorlacius,S., Offit,K., Stoppa-Lyonnet,D. et al. (1996) The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat. Genet., 12, 333–337.[ISI][Medline]
20 Lancaster,J.M., Wooster,R., Mangion,J., Phelan,C.M., Cochran, C, Gumbs,C., Seal,S., Barfoot,R., Collins,N., Bignell,G. et al. (1996) BRCA2 mutations in primary breast and ovarian cancers. Nat. Genet., 13, 238–240.[ISI][Medline]
21 Shen,D. and Vadgama,J.V. (1999) BRCA1 and BRCA2 gene mutation analysis: visit to the Breast Cancer Information Core (BIC). Oncol. Res., 11, 63–69.[ISI][Medline]
22 Szabo,C., Masiello,A., Ryan,J.F. and Brody,L.C. (2000) The breast cancer information core: database design, structure, and scope. Hum. Mutat., 16, 123–131.[ISI][Medline]
23 Griffin,D.K., Haberman,F., Masabanda,J., O’Brien,P., Bagga,M., Sazanov,A., Smith,J., Burt,D.W., Ferguson-Smith,M. and Wienberg,J. (1999) Micro- and macrochromosome paints generated by flow cytometry and microdissection: tools for mapping the chicken genome. Cytogenet. Cell Genet., 87, 278–281.[ISI][Medline]
24 Li,W.-H. (1997) Molecular Evolution. Sinauer Associates, Sunderland, MA.
25 Mazoyer,S., Dunning,A.M., Serova,O., Dearden,J., Puget,N., Healey,C.S., Gayther,S.A., Mangion,J., Stratton,M.R., Lynch,H.T. et al. (1996) A polymorphic stop codon in BRCA2. Nat. Genet., 14, 253–254.[ISI][Medline]
26 Milner,J., Ponder,B., Hughes-Davies,L., Seltmann,M. and Kouzarides,T. (1997) Transcriptional activation functions in BRCA2. Nature, 386, 772–773.[Medline]
27 Katinka,M.D., Duprat,S., Cornillot,E., Metenier,G., Thommarat,F., Prensier,G., Barbe,V., Peyretaillade,E., Brottier,P., Wincker,P. et al. (2001) Genome sequence and gene compaction of the eukayrote parasite Encephalitozoon cuniculi. Nature, 414, 450–453.[Medline]
28 Dayhoff,M.O., Schwartz,R.M. and Orcutt,B.C. (1972) A model of evolutionary change in proteins. In Dayhoff,M.O. (ed.), Atlas of Protein Sequence and Structure. National Biomedical Research Foundation, Silver Spring, MD, pp. 345–352.
29 Burt,D.W., Bruley,C., Dunn,I.C., Jones,C.T., Ramage,A., Law,A.S., Morrice,D.R., Paton,I.R., Smith,J., Windsor,D. et al. (1999) The dynamics of chromosome evolution in birds and mammals. Nature, 402, 411–413.
30 Davies,A.A., Masson,J.Y., McIlwraith,M.J., Stasiak,A.Z., Stasiak,A., Venkitaraman,A.R. and West,S.C. (2001) Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol. Cell, 7, 273–282.[ISI][Medline]
31 Bork,P., Blomberg,N. and Nilges,M. (1996) Internal repeats in the BRCA2 protein sequence. Nat. Genet., 13, 22–23.[ISI][Medline]
32
Crackower,M.A., Scherer,S.W., Rommens,J.M., Hui,C.C., Poorkaj,P., Soder,S., Cobben,J.M., Hudgins,L., Evans,J.P. and Tsui,L.C. (1996) Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3–q22.1 and analysis of a candidate gene for its expression during limb development. Hum. Mol. Genet., 5, 571–579.
33 Buerstedde,J.M. and Takeda,S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell, 67, 179–188.[ISI][Medline]
34 Fantes,P. and Brooks,R. (1993) Mammalian cell cycle analysis. In Rickwood,B.D. (ed.), The Cell Cycle. A Practical Approach. The Practical Approach Series. Hames IRL Press, Oxford, UK, pp. 45–67.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Saeki, N. Siaud, N. Christ, W. W. Wiegant, P. P. W. van Buul, M. Han, M. Z. Zdzienicka, J. M. Stark, and M. Jasin Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions PNAS, June 6, 2006; 103(23): 8768 - 8773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ohashi, M. Z. Zdzienicka, J. Chen, and F. J. Couch Fanconi Anemia Complementation Group D2 (FANCD2) Functions Independently of BRCA2- and RAD51-associated Homologous Recombination in Response to DNA Damage J. Biol. Chem., April 15, 2005; 280(15): 14877 - 14883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Aspen Cancer Conference Fellows Toxicol Pathol, October 1, 2004; 32(6): 749 - 761. [PDF]
|
|
|
|
 |
|
 J. S. Masabanda, D. W. Burt, P. C. M. O'Brien, A. Vignal, V. Fillon, P. S. Walsh, H. Cox, H. G. Tempest, J. Smith, F. Habermann, et al. Molecular Cytogenetic Definition of the Chicken Genome: The First Complete Avian Karyotype Genetics, March 1, 2004; 166(3): 1367 - 1373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Warren, C. J. Lord, J. Masabanda, D. Griffin, and A. Ashworth Phenotypic effects of heterozygosity for a BRCA2 mutation Hum. Mol. Genet., October 16, 2003; 12(20): 2645 - 2656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hussain, E. Witt, P. A.J. Huber, A. L. Medhurst, A. Ashworth, and C. G. Mathew Direct interaction of the Fanconi anaemia protein FANCG with BRCA2/FANCD1 Hum. Mol. Genet., October 1, 2003; 12(19): 2503 - 2510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yang, P. D. Jeffrey, J. Miller, E. Kinnucan, Y. Sun, N. H. Thoma, N. Zheng, P.-L. Chen, W.-H. Lee, and N. P. Pavletich BRCA2 Function in DNA Binding and Recombination from a BRCA2-DSS1-ssDNA Structure Science, September 13, 2002; 297(5588): 1837 - 1848. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||