Kerr S M, Vambrie S, McKay S J, Cooke H J
Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, UK.
Mamm Genome. 1994 Sep;5(9):557-65. doi: 10.1007/BF00354930.
Few mammalian proteins involved in chromosome structure and function during meiosis have been characterized. As an approach to identify such proteins, cDNA clones expressed in mouse testis were analyzed by sequencing and Northern blotting. Various cDNA library screening methods were used to obtain the clones. First, hybridization with cDNA from testis or brain allowed selection of either negative or differentially expressed plaques. Second, positive plaques were identified by screening with polyclonal antisera to prepubertal testis nuclear proteins. Most clones were selected by negative hybridization to correspond to a low abundance class of mRNAs. A PCR-based solid-phase DNA sequencing protocol was used to rapidly obtain 306 single-pass cDNA sequences totaling more than 104 kb. Comparison with nucleic acid and protein databases showed that 56% of the clones have no significant match to any previously identified sequence. Northern blots indicate that many of these novel clones are testis-enriched in their expression. Further evidence that the screening strategies were appropriate is that a high proportion of the clones which do have a match encode testis-enriched or meiosis-specific genes, including the mouse homolog of a rat gene that encodes a synaptonemal complex protein.
在减数分裂过程中,很少有参与染色体结构和功能的哺乳动物蛋白质得到表征。作为鉴定此类蛋白质的一种方法,通过测序和Northern印迹分析了在小鼠睾丸中表达的cDNA克隆。使用了各种cDNA文库筛选方法来获得这些克隆。首先,与来自睾丸或大脑的cDNA杂交可以选择阴性或差异表达的噬菌斑。其次,通过用针对青春期前睾丸核蛋白的多克隆抗血清进行筛选来鉴定阳性噬菌斑。大多数克隆是通过阴性杂交选择的,对应于低丰度类别的mRNA。基于PCR的固相DNA测序方案被用于快速获得306个单通道cDNA序列,总计超过104 kb。与核酸和蛋白质数据库的比较表明,56%的克隆与任何先前鉴定的序列没有显著匹配。Northern印迹表明,这些新克隆中的许多在表达上富含睾丸。筛选策略合适的进一步证据是,确实有匹配的克隆中有很大一部分编码富含睾丸或减数分裂特异性的基因,包括一个编码联会复合体蛋白的大鼠基因的小鼠同源物。