Sugiyama Ryuji, Kazama Yusuke, Miyazawa Yutaka, Matsunaga Sachihiro, Kawano Shigeyuki
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
DNA Res. 2003 Oct 31;10(5):213-20. doi: 10.1093/dnares/10.5.213.
Dioecy in the model dioecious plant Silene latifolia is determined genetically by its heteromorphic sex chromosomes. A bacterial artificial chromosome (BAC) clone, #19B12, was isolated by screening a BAC library from S. latifolia using polymerase chain reaction (PCR) with a set of sequence tagged site (STS) primers, ScD05, which are specific to the Y chromosome. A portion of #19B12 was subcloned to construct plasmid #25-1, with an insert of 7.8 kb. This 7.8-kb fragment encodes ScD05 homolog and an anther-specific gene, CCLS96.1. Northern blot analysis of CCLS96.1 indicated a faint band of 1.8 kb in male and female flower buds. 5' and 3' rapid amplification of cDNA ends (RACE) indicated that transcripts of CCLS96.1 are very varied in size. Moreover, semi-quantitative reverse transcription-PCR (RT-PCR) showed that CCLS96.1 was also expressed in both male and female leaves. RACE produced at least ten species of transcripts, with 79-97% similarity among them. However, no significant ORFs could be predicted from their nucleotide sequences, since each has numerous stop codons throughout all three reading frames. Genomic Southern hybridization showed that the S. latifolia genome contains numerous CCLS96.1 homologs. These results suggest that the transcripts of CCLS96.1 play some role as multiple non-coding RNAs in S. latifolia.
模式雌雄异株植物宽叶蝇子草的雌雄异株由其异形性染色体遗传决定。通过使用一组对Y染色体特异的序列标签位点（STS）引物ScD05进行聚合酶链反应（PCR）筛选宽叶蝇子草的细菌人工染色体（BAC）文库，分离出一个BAC克隆#19B12。将#19B12的一部分亚克隆以构建质粒#25-1，其插入片段为7.8 kb。这个7.8 kb的片段编码ScD05同源物和一个花药特异性基因CCLS96.1。对CCLS96.1的Northern印迹分析表明，在雄花芽和雌花芽中出现一条1.8 kb的微弱条带。5'和3' cDNA末端快速扩增（RACE）表明CCLS96.1的转录本大小差异很大。此外，半定量逆转录PCR（RT-PCR）表明CCLS96.1在雄叶和雌叶中也有表达。RACE产生了至少十种转录本，它们之间的相似性为79-97%。然而，由于每个转录本在所有三个阅读框中都有大量终止密码子，因此无法从其核苷酸序列中预测出明显的开放阅读框（ORF）。基因组Southern杂交表明宽叶蝇子草基因组包含大量CCLS96.1同源物。这些结果表明CCLS96.1的转录本在宽叶蝇子草中作为多种非编码RNA发挥了某些作用。
