Human Molecular Genetics, 2000, Vol. 9, No. 14 2117-2124
© 2000 Oxford University Press
An evolutionarily conserved germ cell-specific hnRNP is encoded by a retrotransposed gene
1MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK and 2Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK
Received 17 January 2000; Revised and Accepted 28 June 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The gene encoding heterogeneous ribonucleoprotein (hnRNP) G recently has been mapped to the X chromosome. All mammals have a Y chromosome-encoded homologue of HNRNP G called RBMY, which is implicated with a role in male fertility and is a candidate for the azoospermia factor gene. We have identified a new member of this gene family, HNRNP G-T, and have mapped it as a single-copy gene on chromosome 11. This gene contains an uninterrupted open reading frame and no introns, consistent with derivation from a retroposon. However, unlike many retroposon-derived genes, HNRNP G-T is not a pseudogene. An antiserum raised to the conceptual reading frame of HNRNP G-T showed that it encodes a protein that is highly expressed in germ cells and in particular in the nuclei of meiotic spermatocytes. Surprisingly, although this antiserum was raised against human hnRNP G-T protein, it can also detect a similar protein in the testis of several mammals. This suggests that the protein is highly conserved and that the retrotransposition event generating the HNRNP G-T gene pre-dated at least the common ancestor of mouse and man. The existence of an additional testis-specific hnRNP G family member provides evidence for the importance of these proteins in normal germ cell development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of nuclear RNA-binding proteins (1). Recently, the gene encoding hnRNP G (RBMX) has been mapped to the X chromosome in both human and mouse (2,3). This gene has been proposed to be the bona fide HNRNP G gene since it both contains introns and after splicing has an open reading frame (ORF) corresponding to the cDNA. A previously mapped autosomal HNRNP G gene on human chromosome 6 contains no introns, suggesting that it is retroposon derived, and, as a result of a number of base pair substitutions and a frameshift interrupting the reading frame, is likely to be non-functional (2,4,5).
HNRNP G is homologous to the Y chromosome-encoded RBM gene family. These genes were identified originally as candidates for the azoospermia factor (AZF) gene, which is a Y chromosome gene hypothesized to have been deleted in some men, loss of which results in infertility. It is now known that microdeletions of at least three regions of the Y chromosome can cause infertility, and that removal of the AZFb region prevents expression of detectable RBM protein (6). Both RBMY and hnRNP G proteins interact with ubiquitously expressed proteins involved in splice site selection (SR proteins and Tra-2ß) and signal transduction (T-STAR and SAM68), suggesting a role in coupling signal transduction and cell-specific pre-mRNA splicing in germ cells (7–9).
Here we describe a new member of the HNRNP G gene family called HNRNP G-T. Unlike the other sex-linked members, we find that it is a single-copy autosomal gene which does not contain introns. Although it is likely to have been derived from a retroposon, HNRNP G-T encodes a germ cell-specific protein conserved in all mammals tested, suggesting a function for the cognate protein either in replacing hnRNP G protein function during meiotic prophase or as a new germ cell-specific splicing regulator. The existence of HNRNP G-T provides new evidence for a critical requirement for hnRNP G family proteins during germ cell development. We propose HNRNP G-T as a new candidate gene for autosomal mutations causing male infertility.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cloning and analysis of the HNRNP G-T gene
In a two-hybrid screen for proteins that interact with human RBMY protein (8), we identified a cDNA from a human testis library with a conceptual ORF encoding a protein 77.5% identical to the published hnRNP G protein (Fig. 1). Like hnRNP G and RBMY proteins, the predicted hnRNP G-T amino acid sequence includes an N-terminal RNA recognition motif and a C-terminus rich in tyrosine and basic amino acids. The extent of the RNA recognition motif is included within the solid black lines in the alignment in Figure 1, with the conserved RNP2 motif shaded and the RNP11 motif striped. The N-terminus including this motif is most conserved, but homology is spread over the entire protein sequence, consistent with these being members of the same protein family. In modular structure, hnRNP G-T is most similar to hnRNP G in that they do not have four repeats of 37 amino acids rich in serine/arginine/glycine/tyrosine (the so-called SRGY boxes: the actual SRGY tetrapeptides are shown in bold in Fig. 1) (10)—this absence is indicated as a gap. hnRNP G-T protein is 73% identical to human X-encoded hnRNP G protein, compared with 53% identical to human Y-encoded RBM. In particular, there are four gaps in the hnRNP G-T protein sequence when aligned with the other two, and three of these gaps are conserved with hnRNP G protein. The single non-conserved gap in hnRNP G-T is the loss of a single proline residue at the end of a run of prolines. None of the gaps in the hnRNP G-T protein sequence is conserved with RBMY protein. Overall, this comparison might be indicative that HNRNP G-T is derived by retrotransposition of an HNRNP G transcript, making these closer relatives to each other than to RBMY. However, the rates of sequence evolution might be different for each gene [in particular since they are encoded by different genetic compartments (see below)].
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HNRNP G-T maps to chromosome 11p15
This hnRNP G-T cDNA suggested the existence of a third transcribed member of the HNRNP G gene family. To test whether this is the case, we set out to identify and map such a gene. We first identified three phage P1-derived artificial chromosomes (PACs) containing the HNRNP G-T gene by hybridization on an arrayed library, and then used these PACs as probes to localize the chromosomal position of the gene by fluorescence in situ hybridization (FISH) on human chromosomes (Fig. 2A). A single hybridization signal (pseudocoloured red in Fig. 2A) was detected on 11p15 for each probe, closely adjacent to the insulin gene (pseudocoloured green). The signals for HNRNP G-T and insulin are separated more clearly in interphase chromosomes adjacent to the metaphase spread (Fig. 2A). We mapped the HNRNP G-T gene more precisely by designing a sequence-tagged site (STS) marker, and mapping this using the Genebridge radiation hybrid panel to 10.87 cRays from D11S922 on the Whitehead radiation map (see Materials and Methods).
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HNRNP G-T contains a single ORF uninterrupted by introns
Originally the gene encoding hnRNP G was mapped to chromosome 6p by in situ hybridization on human metaphase chromosomes (5). This 6p gene is likely to be a retroposon-derived pseudogene since it both lacks introns and has a number of frameshifts interrupting its reading frame (2). To test whether the HNRNP G-T gene contains introns, the cDNA and a genomic PAC were digested with BglII and SmaI, which would digest the cDNA into three distinct fragments (Fig. 2B). The prediction is that if the genomic copy does not contain introns, these fragments should be the same size in both the PAC clone and the cDNA clone. In contrast, if the HNRNP G-T gene contains introns, the genomic restriction fragments would be different. The digest was analysed by Southern blotting followed by hybridization with oligonucleotides specific for each of the fragments. As predicted for an intronless gene, identical fragments were detected in both cases (Fig. 2C).
These data suggested that the HNRNP G-T gene is intronless and so is likely to be derived from a processed retroposon. In order to confirm the proposed identity of the genomic clone and the cDNA, we directly sequenced the genomic HNRNP G-T gene from 20 nucleotides downstream of the initiation codon to within 20 nucleotides of the polyadenylation site in the 3'-untranslated region (UTR) (data not shown). As predicted from the restriction analysis described above, sequencing identified no introns in the genomic sequence. Moreover, there were no short deletions or insertions that would cause a frameshift in the reading frame. With the exception of a single base change, the cDNA and PAC HNRNP G-T nucleotide sequences were exactly co-linear. This single nucleotide difference results in a change from alanine to valine in the predicted primary amino acid sequence, hence making the hnRNP G-T protein encoded by this PAC identical to RBMY protein rather than hnRNP G at this position. This alanine is circled in Figure 1. This may be a polymorphism. A second cDNA had a further nucleotide difference from both the first cDNA and the PAC sequence (which were identical at this position), although this change was silent (i.e. it did not affect the encoded amino acid).
These differences could represent polymorphisms, since the cDNAs and the genomic PAC were not prepared from the same individuals. However, an alternative hypothesis is that the HNRNP G-T cDNA might be encoded by a second gene located elsewhere in the genome that contains introns. According to this scenario, the identified chromosome 11p15 HNRNP G-T gene might have been derived recently from this second gene by retrotransposition and so be very similar in sequence. If such a gene existed, we should be able to detect it by hybridization on human genomic DNA. To test this hypothesis, we digested human genomic DNA with two pairs of enzymes that flanked the HNRNP G-T gene, followed by Southern blotting and hybridization with an HNRNP G-T-specific probe. In both cases, we identified a single hybridizing fragment in two independent preparations of human genomic DNA (Fig. 2D), strongly consistent with the existence of a single gene. Moreover, the size of this fragment was identical to that obtained by an equivalent digestion of a preparation of PAC 297-C2 DNA (data not shown), again consistent with the existence of a single HNRNP G-T gene on chromosome 11p15.
HNRNP G-T encodes a meiosis-specific nuclear protein
Although many retrotransposons are inactive, a number are expressed at the RNA level. However, the lack of introns often leads to mRNAs being exported inefficiently from the nucleus to the cytoplasm and poorly translated (11). Natural intronless genes such as histones frequently have specific export sequences within their transcripts (12). In order to test whether HNRNP G-T does encode a protein, we raised an antiserum to a peptide corresponding to a unique stretch of amino acids (i.e. not found in either hnRNP G or RBMY). Consistent with HNRNP G-T encoding a protein, an affinity-purified antiserum raised against this peptide recognized a single protein of the predicted size of 
43 kDa on western blots containing human testis protein, but only weak background bands in human colon and prostate protein extracts (Fig. 3A). The intense signal corresponding to hnRNP G-T protein was pre-absorbed specifically by the immunizing peptide (Fig. 3A, lanes 1–3), but not by a non-specific peptide (lanes 4–6). In contrast, the weaker background bands were either not, or only marginally, reduced by pre-absorption.
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We directly tested the cellular expression pattern of hnRNP G-T protein using immunohistochemistry on sections of human testis with the anti-hnRNP G-T antiserum (Fig. 3B). We found that hnRNP G-T is a nuclear protein highly expressed in spermatocytes and to a much lesser extent in round spermatids (brown staining in Fig. 3B corresponds to a positive reaction). No hnRNP G-T was detected within somatic cells in the testis (Sertoli cells or myoid cells), or in transcriptionally quiescent elongating spermatids (blue counterstain detected only). No antibody reaction was detected after pre-absorption of the antiserum with the immunizing peptide (Fig. 3C; the blue counterstain only is visible). HnRNP G-T protein expression in spermatogonia was undetectable unless the colour reaction was overexposed (Fig. 3D). Under these conditions, Sertoli cells were still negative for hnRNP G-T expression, so this probably represents a real but very low relative level of expression. Hence, human hnRNP G-T protein is a nuclear protein predominantly expressed in spermatocytes.
HnRNP G-T protein is evolutionarily conserved
To try to address how long ago the retrotransposition event creating the HNRNP G-T gene took place, we performed a western zooblot to test whether hnRNP G-T protein is found in other mammals. A protein of almost identical molecular mass to human hnRNP G-T was recognized in testis extracts from mouse, rat and bull, but not in other tissues from these animals (Fig. 3E). Hence, the retrotransposition event must have pre-dated the last common ancestor of these mammals.
The western blotting data described above suggested that hnRNP G-T protein is testis specific in expression, and is conserved in different mammals. Because of the easy availability of tissues, we tested this hypothesis further in mouse, using multiple tissue western blots. We found that hnRNP G-T protein is expressed most strongly in mouse testis, but additional very weak expression of this protein was detected consistently in mouse brain (Fig. 3F, lanes 2–6). No hnRNP G-T protein expression was detected in other tissues tested. Hence, our antiserum cannot be cross-reacting with hnRNP G protein, which is expressed in all tissues (data not shown). We performed additional experiments to show that the antiserum does not cross-react with mouse RBMY protein. First, the anti-hnRNP G-T antiserum does not recognize RBMY ectopically expressed in COS cells (Fig. 3F, lane 1). Parallel experiments using an anti-mouse RBMY antiserum detected this ectopic expression (data not shown). Moreover, no reduction in mouse hnRNP G-T expression was observed in testis from mice with a YD1 chromosome deletion, although these mice show a much reduced level of RBMY gene expression (13). Hence, our anti-hnRNP G-T antiserum shows no detectable cross-reaction with endogenous RBMY protein in mouse testis.
We probed mouse testis sections with the anti-hnRNP G-T antiserum to compare directly expression of the mouse and human hnRNP G-T proteins. As in humans, mouse hnRNP G-T protein is highly expressed in pachytene spermatocytes, and much more weakly in spermatogonia and spermatids, although higher background staining was observed, probably since the antiserum was raised against the human protein and the epitope is not perfectly conserved (Fig. 3G). Consistent with it being specific, antiserum staining was pre-absorbed with the immunizing peptide (Fig. 3G, inset).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study, we describe a new member of the HNRNP G gene family. Unlike the other members, it is autosomal, and the lack of introns suggests that it was derived from these by retrotransposition. There has been considerable recent interest in the discovery of Y-linked genes implicated in male fertility, of which the HNRNP G-homologue RBMY is a candidate. Proof of RBMY as an essential spermatogenesis gene is difficult to obtain by point mutation screens in infertile men, since it is a multiple copy gene in human and mouse, and, moreover, the Y chromosome has proven to be refractory to gene inactivation by homologous recombination. However, the apparent conservation of HNRNP G-T genes in mammals does provide support for a crucial function for hnRNP G family proteins in germ cell development and, based on this, we propose HNRNP G-T as a new candidate autosomal male infertility gene.
Why might mammals need an HNRNP G-T gene? HnRNP G-T protein is expressed predominantly in the testis, although we do detect weak expression in the mouse brain [other ‘testis-specific’ mRNAs are also detected in the brain, e.g. T-STAR (7)]. One possibility is that HNRNP G-T might perform a specialized function geared to the specific requirements of germ cells which is either not required in somatic cells at all, or is somewhat different in this latter case. CstF-64, which encodes the cleavage-stimulating factor required for the polyadenylation of mRNA, is also X encoded. Interestingly, Cst-64 protein is not detected in meiosis, but in mouse spermatocytes is replaced by an antigenically related protein of different molecular mass, perhaps encoded by an autosomal gene (14). Since patterns of polyadenylation are slightly different in germ cells, this replacement protein might have a different function from the X homologue important for germ cell-specific processing. HnRNP G proteins have been implicated in pre-mRNA splice site selection (8,9). A number of mammalian genes are alternatively spliced in germ cells, for example DNA methyltransferase required for genomic imprinting, for which a sex-specific exon is included during meiosis (see ref. 15 for a review). Consistent with an important role for alternative splicing regulation during meiosis, the only known example of regulated alternative splicing throughout the entire life cycle of the single cell yeast Saccharomyces cerevisiae takes place during meiosis (16).
An alternative possibility is that HNRNP G-T may play a role in dosage compensation. The localization of genes on the sex chromosomes has important implications for expression, since the X and Y chromosomes are inactivated for transcription during meiosis in mammals by sequestration in a heterochromatic structure called the XY body (17). The meiotic pachytene stage which contains the XY body lasts 6 days in mice (18) and probably even longer in humans, and so any X-encoded genes essential for cell function during this time frame will need to be replaced.
The idea of autosomal homologues to replace sex chromosome gene expression in meiosis has been proposed before, but to the best of our knowledge the proteins encoded by these autosomal genes have not been identified directly in germ cells using specific antisera. Here we show that hnRNP G-T protein is most highly expressed in pachytene spermatocytes (and much more weakly in spermatids), consistent with a role in replacing HNRNP G genes on the X chromosome during male meiosis. This result would have interesting implications. The other autosomal homologues of X chromosome-encoded genes proposed to provide gene function during meiosis are clearly constitutively required for normal cellular metabolism. In contrast, none of the functions of any of the hnRNP proteins in mammalian development have been studied by reverse genetics, although hnRNP A1 is non-essential in cultured cells (19). A requirement for an HNRNP G-T gene to replace gene function during meiotic prophase would provide clear evidence for a crucial function for this family during male germ cell development.
There are several lines of evidence supporting the down-regulation of hnRNP G and RBMY during meiosis. The HNRNP G gene has been mapped to Xp26, and to the syntenic region on the mouse X chromosome (2,3). The flanking genes PGK1 (Xq13.3), glycerol kinase (Xp21.3) and glucose 6-phosphate dehydrogenase (Xp28) are duplicated by testis-specific autosomal copies (20–23), strongly implying that this whole region of the X chromosome is inactivated during meiotic prophase. Direct evidence in favour of meiotic down-regulation of hnRNP G protein was found by immunohistochemical analysis of testis sections with a dog autoantiserum recognizing hnRNP G (24); the levels of staining were very much reduced in spermatocytes and spermatids. However, it is not known how specific this antiserum is to hnRNP G rather than to other family members; it may recognize epitopes present on several hnRNP G family members. Mouse RBMY is inactivated in the XY body (13).
The ‘zoo-western’ data suggest that despite its derivation from a processed retroposon and corresponding absence of introns, HNRNP G-T is a highly evolutionarily conserved gene, being present at least in the common ancestor of mouse and man. Most retrotransposed genes rapidly accumulate mutations and become inactive pseudogenes. In contrast, the conservation of hnRNP G-T protein across such distant evolutionary time strongly implies an important function. The specialized sex chromosomes in mammals are thought to have evolved from a pair of autosomes (25). It is thought that HNRNP G (RBMX) and RBMY are among the most ancient genes on the X and Y chromosomes (26), consistent with a correspondingly long evolutionary history for HNRNP G-T. Based on the evidence described above and by others (20–22), it is likely that the evolution of the sex chromosomes has not occurred independently, but has required both the duplication by retrotransposition and parallel maintenance of all essential genes on autosomes. Hence, in facilitating specialization of the sex chromosomes, retroposons are likely to have had an important impact on the evolution of the extant mammalian karyotype
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Isolation of genomic clones
To avoid cross-reaction with hnRNP G sequences, we generated a probe corresponding to the 3'-UTR of HNRNP G-T. This probe was specific for the cognate mRNA on northern blots (8). A PCR product was generated between primers 5'-GAGCAGATATAAGCAGGA-3' and 5'-CAGGAGCTTGTTGAAAGAAACGTT-3' using a human HNRNP G-T cDNA as a template, and used to probe a gridded PAC library containing human genomic DNA (27). Incubation at 65°C overnight in Church and Gilbert solution [0.5 M NaPO4/1% bovine serum albumin (BSA)/1 mM EDTA/7% SDS] was followed by three washes of 15 min each in 1x SSC/0.1% SDS at 65°C. Positive PACs were ordered from the Human Genome Mapping Project (HGMP, Hinxton, UK), DNA prepared and re-screened by PCR using primers internal to the HNRNP G-T cDNA. Three positive PACs from RPCI1 were used in subsequent analyses: 109-A15, 297-C2 and 292-I15.
Sequencing of the genomic HNRNP G-T gene
HNRNP G-T was PCR amplified into four overlapping regions for sequencing. Each region was amplified three separate times, once with RedTaq polymerase (Sigma, Poole, UK) and twice with Pfu polymerase (Stratagene, La Jolla, CA). Reactions were purified from oligonucleotides and free nucleotides using a Promega (Madison, WI) DNA Clean-Up System kit with a vacuum manifold, and resuspended in warm TE pH 8.0 (10 mM Tris–HCl, 1 mM EDTA) before being quantified on a gel. Each region was then sequenced with the same primers (from PNACL-Protein and Nucleic Acid Chemistry Laboratory, Leicester University, Leicester, UK) as used in the amplifications. Each region was sequenced twice in the sense direction (one Pfu amplification and the RedTaq amplification) and once in the antisense direction (one Pfu amplification). All sequencing was done with these PCR products by PNACL on an ABI Prism 377 automated sequencing machine (Applied Biosystems, Warrington, UK). Sequence chromatograms and raw sequences were analysed using Editview 1.0.1 and GeneJockey II software, respectively. Overlapping sequences of the four regions were then aligned to produce a contiguous sequence of the gene.
FISH mapping of HNRNP G-T
Each PAC DNA was labelled by random priming, and used to probe human metaphase chromosomes by FISH.
Radiation hybrid mapping of HNRNP G-T
We designed PCR primers specific for human HNRNP G-T (5'-AGCGTGATTCTTACAGCC-3' and 5'-CAACTCTTCCTTGTACTAGTCC-3') to generate an STS and mapped this on the Genebridge 4 radiation hybrid panel (Research Genetics, Huntsville, AL) using 35 cycles of PCR and an annealing temperature of 56°C. Analysis of the results positioned HNRNP G-T on the radiation hybrid map constructed at the Whitehead/MIT Center for Genome Research (Cambridge, MA).
Southern blotting analysis
For Figure 1C, 0.5 µg of PAC 297-C2 from RCPI1 and HNRNP G-T cDNA was digested with SmaI, or double digested with SmaI and BglII according to the manufacturer’s instructions (Roche, East Sussex, UK). Digested DNA was electrophoresed on a 2% agarose gel (Flowgen 3:1 Nuesieve agarose), and transferred to a Hybond N+ membrane by Southern blotting (Amersham, Little Chalfont, UK). Oligonucleotide probes 460S (5'-CGATGCCCATGAAGCGT-3'), T880 (5'-AGCGTGATTCTTACAGCC-3') and T882 (5'-TCGAAACCGACGAGAAAG-3') were labelled by polynucleotide kinase (Boehringer) and the probe purified on an NAP5 column. Southern blots were hybridized overnight at 49°C in Church and Gilbert solution, followed by two brief washes at 49°C in 6x SSC/0.1% SDS, and two further washes of 10 min at room temperature in 6x SSC/0.1% SDS. Radioactivity was detected on a PhosphorImager (Molecular Dynamics, Buckinghamshire, UK).
For Figure 1D, 20 µg of human genomic DNA isolated from HT1080 cells was digested with the indicated restriction enzymes, electrophoresed on a 1% agarose gel and Southern blotted as above. The filter was then probed as above for isolation of the genomic HNRNP G-T clones.
Generation of antiserum
To generate the anti-hnRNP G-T antiserum, the peptide CGGGGRYEEYRGYSPDAYSGG-OH was synthesized, coupled to keyhole limpet haemocyanin (Research Genetics), and injected into a sheep (Diagnostics Scotland, Carluke, UK). After four injections, IgG was purified by caprylic acid fractionation (28), and antibodies specific to the peptide were affinity purified on a Sulfolink column containing immobilized peptide (according to the manufacturer’s instructions).
Detection of proteins
Proteins from surgically removed human tissues were extracted in 2x Laemmli sample buffer, fractionated by 8% SDS–PAGE and transferred to Immobilon filters by semi-dry blotting (Millipore, Watford, UK). Western filters were blocked for 1 h in blocking buffer [5% non-fat dry milk/5% normal horse serum (Gibco BRL, Paisley, UK)/Tris-buffered saline (TBS)], and then probed for 1 h with affinity-purified sheep anti-hnRNP G-T antiserum (1:400). Filters were given three washes of 5 min each in TBS + 0.2% Tween 20 (TBST), then incubated for 1 h with horseradish peroxidase (HRP)-conjugated donkey anti-sheep antibody (1:2000 dilution; Diagnostics Scotland). Filters were then given three 5 min washes in TBST, then two washes in TBS, and were developed by enhanced chemiluminescence. For the pre-absorption experiment shown in Figure 2A, the primary antibody dilutions were pre-incubated for 1 h with 20 µl of sulfolink beads coated in either the immunizing hnRNP G-T peptide or a non-specific peptide (derived from the mouse RBMY protein sequence). The beads were removed by microcentrifugation prior to the primary incubation.
Immunohistochemistry was performed on microtome sections of surgically removed, paraffin-embedded human testis as described (6), using a 1:100 dilution of the sheep anti-hnRNP G-T antiserum. For the pre-absorbtion experiments, the antiserum was pre-incubated with 10 µg of the immunizing peptide.
The sequences have been submitted to GenBank (cDNA accession no. AF069682; genomic sequence accession no. AF279289).
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ACKNOWLEDGEMENTS |
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We thank Prof. Nick Hastie for comments on the manuscript, and Dr Paul Burgoyne for suggesting that hnRNP G-T might be an autosomal back-up for hnRNP G during meiosis. D.J.E. is a Royal Society of Edinburgh Caledonian Research Fellow. This work was supported by the Caledonian Research Fund and the Medical Research Council.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Institute for Human Genetics, School of Biochemistry and Genetics, University of Newcastle upon Tyne, Ridley Building, Claremont Place, Newcastle upon Tyne NE1 7RU, UK. Tel: +44 191 222 6827; Fax: +44 191 222 6662; Email: davide@hgu.mrc.ac.uk
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