Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues
Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues
复制标题
hMSH4 的选择性剪接：睾丸中的两种亚型和体细胞组织中的异常转录物
作者：
S. Santucci;R. Paul;J. Michiels;A. Saunières;C. Desnuelle;V. Paquis
大肠杆菌MutHLS错配修复途径在整个进化过程中都是保守的。大量的工作导致了真核MSH和MLH基因在体细胞DNA修复中的各自作用的研究（Fishel和Wilson 1997）。如果现在清楚了这些基因中的一些也参与减数分裂，那么对它们的功能就知之甚少了。在酿酒酵母中，mut和MutL同源物可以任意区分。像MSH2、MLH1或PMS1这样的蛋白质属于第一组，它们是DNA修复和减数分裂重组所必需的（Hunter et al. 1996）。第二组包括两种蛋白，MSH4和MSH5，仅用于减数分裂重组（Ross-Macdonald and Roeder 1994; Hollingworth et al. 1995）。尽管这些减数分裂特异性基因的产物与大肠杆菌MutS蛋白同源，但它们似乎在错配修复中没有作用。在酿酒酵母中，MSH4基因仅在减数分裂细胞中表达，并且MSH4蛋白在粗粒期定位于染色质上。酵母msh4突变体在第一次减数分裂时表现出与相互重组减少和同源染色体异常分离相关的孢子活力降低。这些结果表明MSH4可能是晚期重组结节的一个组成部分。在哺乳动物中，MLH1或PMS2基因敲除突变的小鼠纯合子的产生证实了muthls样途径在减数分裂中的重要性。缺乏MLH1的小鼠不育（Edelmann et al. 1996）。pms2雄性不育，而雌性保持生育能力（Baker et al. 1995）。此外，有研究表明，在减数分裂中，MSH2可能被MSH家族的另一个成员所取代，因为MSH2缺陷的小鼠是可生育的（Reitmar et al. 1995; Edelmann et al. 1997）。我们最近发现了人类MSH4基因（Paquis-Flucklinger et al. 1997）。通过Northern blot分析，我们发现该基因仅在睾丸和卵巢中表达。这一结果表明，hMSH4基因的产物可能参与哺乳动物减数分裂重组，正如在酵母中看到的那样。这种哺乳动物减数分裂特异性MSH基因的鉴定将有助于研究减数分裂重组、分子机制以及MSH和MLH基因产物之间的关系。为了确定hMSH4基因的外显子-内含子连接，我们使用PCR方法在未克隆的基因组DNA中行走。对未克隆的接头连接的基因组DNA片段的5个人类“文库”（Genome Walker kit, Clontech (Palo Alto， CA)）进行pcr；图1 a)。PCR产物在两条链上克隆并测序。我们发现hMSH4基因包含19个外显子（图1B）。每个外显子边界的序列与剪接结的一致序列是相容的。每个外显子连接处的核苷酸序列和用于上述分析的hmsh4特异性引物可根据要求提供。通过Northern blot分析，我们最初在睾丸和卵巢中检测到一个约3.1 kb的转录本（PaquisFlucklinger et al. 1997）。接下来，我们用RT-PCR分析了hMSH4在人睾丸中的表达。逆转录反应之后是两步PCR程序，允许扩增总cDNA编码区。PCR产物直接克隆到pGEM-T载体（Promega, Madison， WI）。细菌转化后，对两条链上的个体克隆进行测序。除了含有预期的hMSH4 cDNA的克隆外，我们还获得了插入较短的质粒。序列分析表明，该短cDNA为hMSH4的选择性剪接产物，命名为DhMSH4。它包含一个帧内缺失（174 bp），并且缺少所有的外显子5（图1C）。为了研究hMSH4蛋白在成人睾丸中的分布，我们对一种与hMSH4最后c端138个氨基酸相对应的细菌蛋白制备了兔多克隆抗体（1pA血清）。为了验证其特异性，采用1pA血清对大肠杆菌中作为融合蛋白表达的hMSH4和DhMSH4进行Western blot分析。为此，构建了编码细菌谷胱甘肽s -转移酶（GST）融合蛋白的质粒。采用上述的RT-PCR方法生成hMSH4和DhMSH4睾丸cdna，巢式PCR引物含有末端延伸的smi位点，用于随后的亚克隆。扩增产物用SmaI酶切，并在正确的阅读框内连接到细菌表达载体pGEX 5X-2 （Pharmacia, Uppsala, Sweden）的SmaI位点。通过DNA测序证实插入的cdna无突变。细菌蛋白的表达、提取和纯化基本上与描述的一样（Smith and Johnson 1988），只是做了一些修改：在加入1mm IPTG后1小时30分钟收集细胞，以降低蛋白水解降解产物的水平。从编码GST- hmsh4或GST- dhmsh4蛋白的pGEX载体转化的诱导或非诱导细菌细胞中，从谷胱甘肽- sepharose微球和总蛋白样品中洗脱的GST融合蛋白，用多克隆1pA抗体进行Western blot实验分析。GST-hMSH4和GST-DhMSH4蛋白的预测分子量分别为130和122 kDa。如图2A所示，1pA血清识别出iptg诱导的细菌细胞的全蛋白提取物和谷胱甘肽多糖珠洗脱液中存在的预期大小的多肽。本实验表明，1pA抗体特异性识别hMSH4和DhMSH4蛋白。采用亲和素-生物素-过氧化物酶复合物法，用同一抗hmsh4血清对成人睾丸石蜡切片进行间接免疫染色。在精管内的一些生殖细胞中存在特定的核信号，最有可能在精母细胞中（图2B）。在减数分裂后的细胞中未观察到信号（图2C）。从人睾丸cDNA中，用hmsh4特异性引物扩增2 × 25个周期才能获得在溴化乙酯染色凝胶上可见的PCR产物（数据未显示）。这一结果和之前的Northern blot分析（Paquis-Flucklinger et Correspondence to: V. Paquis-Flucklinger哺乳动物基因组10,423 - 427(1999)）。
The Escherichia coli MutHLS mismatch repair pathway has been conserved throughout evolution. A large body of work has led to study of the respective roles of eukaryotic MSH and MLH genes in somatic DNA repair (Fishel and Wilson 1997). If it is now clear that some of these genes are also involved in meiosis, less is known about their functions. In Saccharomyces cerevisiae, two groups of MutS and MutL homologs can arbitrarily be distinguished. Proteins like MSH2, MLH1, or PMS1 belong to the first group and are required for both DNA repair and meiotic recombination (Hunter et al. 1996). The second group includes two proteins, MSH4 and MSH5, required only for meiotic recombination (Ross-Macdonald and Roeder 1994; Hollingworth et al. 1995). Although the products of these meiosis-specific genes are homologous to the E. coli MutS protein, they seem to have no role in mismatch repair. In Saccharomyces cerevisiae, the MSH4 gene is expressed only in meiotic cells, and the MSH4 protein localizes to chromatin during pachytene. Yeast msh4 mutants show a reduced spore viability associated with a decrease in reciprocal recombination and an abnormal segregation of homologous chromosomes at the first meiotic division. These results suggest that MSH4 could be a component of late recombination nodules. In mammals, the generation of mice homozygous for knockout mutations of the MLH1 or PMS2 genes has confirmed the importance of the MutHLS-like pathway in meiosis. Mice deficient in MLH1 are sterile (Edelmann et al. 1996). The pms2 males are sterile, while the females remain fertile (Baker et al. 1995). Moreover, it has been suggested that in meiosis MSH2 might be replaced by another member of the MSH family because MSH2-deficient mice are fertile (Reitmar et al. 1995; Edelmann et al. 1997). We have recently identified the human MSH4 gene (Paquis-Flucklinger et al. 1997). By Northern blot analysis, we have shown that this gene is expressed only in testis and ovary. This result suggests that the product of the hMSH4 gene could be involved in meiotic recombination in mammals as seen in yeast. The identification of this mammalian meiosis-specific MSH gene should facilitate the study of meiotic recombination, molecular mechanisms, and the relationships between the products of MSH and MLH genes. In order to determine the exon-intron junctions of the hMSH4 gene, we used a PCR method for walking in uncloned genomic DNA. PCRs were performed on five human “libraries” of uncloned, adaptor-ligated genomic DNA fragments (Genome Walker kit, Clontech (Palo Alto, CA); Fig. 1A). PCR products were cloned and sequenced on both strands. We have found that the hMSH4 gene contains 19 exons (Fig. 1B). The sequences of each exonintron boundary are compatible with the consensus sequences for the splicing junction. The nucleotide sequences of each exonintron junction and of hMSH4-specific primers used for the analysis described above are available upon request. By Northern blot analysis, we had initially detected a single transcript of approximately 3.1 kb in testis and ovary (PaquisFlucklinger et al. 1997). We next analyzed the expression of hMSH4 in human testis by RT-PCR. Reverse transcription reaction was followed by a two-step PCR procedure that allowed amplification of the total cDNA coding region. PCR products were directly cloned into the pGEM-T vector (Promega, Madison, WI). After bacterial transformation, individual clones were sequenced on both strands. In addition to the clones containing the expected hMSH4 cDNA, we obtained plasmids with a shorter insert. Sequence analysis revealed that this shorter cDNA, designated DhMSH4, was an alternatively spliced product of hMSH4. It contained an in-frame deletion (174 bp) and lacked all of exon 5 (Fig. 1C). To examine the distribution of hMSH4 proteins in the adult testis, rabbit polyclonal antibodies (1pA serum) were raised against a bacterial protein corresponding to the last C-terminal 138 amino acids of hMSH4. In order to verify its specificity, 1pA serum was used to perform Western blot analysis of hMSH4 and DhMSH4 expressed in E. coli as fusion proteins. For this purpose, plasmids encoding bacterial glutathione S-transferase (GST) fusion proteins were constructed. hMSH4 and DhMSH4 testis cDNAs were generated by RT-PCR as described above with nested PCR primers containing a terminal extension SmaI site for subsequent subcloning. Amplified products were digested with SmaI and ligated in the correct reading frame into the SmaI site of the bacterial expression vector pGEX 5X-2 (Pharmacia, Uppsala, Sweden). The cDNAs inserts were confirmed to be free of mutations by DNA sequencing. Expression, extraction, and purification of bacterial proteins were essentially as described (Smith and Johnson 1988), with a minor modification: cells were collected 1 h 30 min after the addition of 1 mM of IPTG to decrease the level of proteolytic degradation products. The GST fusion proteins eluted from glutathione-Sepharose beads and total protein samples, from either induced or non-induced bacteria cells transformed with pGEX vectors encoding GST-hMSH4 or GST-DhMSH4 proteins, were analyzed by Western blot experiments with polyclonal 1pA antibodies. The predicted molecular masses of GST-hMSH4 and GST-DhMSH4 proteins were respectively 130 and 122 kDa. As can be seen in Fig. 2A, 1pA serum recognized polypeptides of the expected size that were present in whole protein extracts from IPTG-induced bacterial cells and in eluates from glutathioneSepharose beads. This experiment showed that 1pA antibodies recognized specifically hMSH4 and DhMSH4 proteins. The same anti-hMSH4 serum was used for indirect immunostaining on paraffin sections of human adult testis, with the avidin biotin peroxidase complex method. A specific nuclear signal was present in some germ cells within the seminiferous tubules, most probably in spermatocytes (Fig. 2B). No signal was observed in post-meiotic cells (Fig. 2C). From human testis cDNA, 2 × 25 cycles of amplification with hMSH4-specific primers were necessary to obtain a PCR product visible on an ethidium bromide-stained gel (data not shown). This result and a previous Northern blot analysis (Paquis-Flucklinger et Correspondence to: V. Paquis-Flucklinger Mammalian Genome 10, 423–427 (1999).
影响因子：
4
作者：
Fishel, R;Wilson, T
通讯作者：
Wilson, T
影响因子：
10.5
作者：
HOLLINGSWORTH, NM;PONTE, L;HALSEY, C
通讯作者：
HALSEY, C