Charlton S, Boulianne R, Chow Y C, Lu B C
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
Gene. 1992 Dec 1;122(1):163-9. doi: 10.1016/0378-1119(92)90044-p.
The naturally synchronous meiosis of the fungus, Coprinus cinereus, provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. We have cloned a cDNA from the fruiting body of C. cinereus encoding the 12-kDa subunit of a meiotic endonuclease (mENase). The identification of the 12-kDa subunit cDNA clone was achieved by the mENase antiserum against a lambda gt11 cDNA expression library. It was confirmed by a direct match of the amino acid (aa) sequence obtained from purified 12-kDa polypeptide with the nucleotide sequence. Northern blot analysis using the cDNA clone as a probe showed that the mENase-encoding gene (MenA) for the 12-kDa subunit was expressed mainly in fruiting bodies and at a very low level in the asexual vegetative mycelium. In addition, it was differentially expressed in the early meiotic stages. The MenA transcript was most abundant in fruiting body primordia prior to the premeiotic S-phase; it remained high from karyogamy to early pachytene, declined drastically by late pachytene and diplotene, and was undetectable by sterigma stage. Western blot analysis showed that the mENase protein was produced at a very low level in mycelium; it was produced in great quantity during the early meiotic stages and decreased to a low level at the end of meiosis.
真菌灰盖鬼伞自然同步的减数分裂为研究与减数分裂和子实体发育相关的差异基因表达提供了一个理想的系统。我们从灰盖鬼伞的子实体中克隆了一个cDNA，它编码一种减数分裂内切核酸酶（mENase）的12 kDa亚基。通过针对λgt11 cDNA表达文库的mENase抗血清实现了12 kDa亚基cDNA克隆的鉴定。从纯化的12 kDa多肽获得的氨基酸（aa）序列与核苷酸序列的直接匹配证实了这一点。使用该cDNA克隆作为探针的Northern印迹分析表明，编码12 kDa亚基的mENase基因（MenA）主要在子实体中表达，而在无性营养菌丝体中表达水平非常低。此外，它在减数分裂早期阶段差异表达。MenA转录本在减数分裂前S期之前的子实体原基中最为丰富；从核配到早粗线期一直保持高水平，在晚粗线期和双线期急剧下降，到担孢子梗期无法检测到。Western印迹分析表明，mENase蛋白在菌丝体中产生水平非常低；在减数分裂早期大量产生，在减数分裂结束时降至低水平。
