Molecular mechanisms of gynogenetic clonal reproduction in polyploid crucian carp

这项为期三年的项目旨在研究日本鲫鱼种群或亚种(即鲫鱼)雌核发育的分子机制。这种极其罕见和有趣的脊椎动物(鱼类)已知产生未减少的卵子，其基因组组成与体细胞完全相同，方法如下：1.跳过第一次减数分裂；2.受精后，保持传入精子核膜不受损害，从而阻止雄性基因组参与胚胎发育。这种类型的克隆繁殖在基本的发育生物学和人工大规模生产克隆后代的方法方面都是值得注意的。在本研究中，我们研究了相当数量的C.a.langsdorfi(双性二倍体和全雌性三倍体或四倍体)和其他几个C.A.包括金鱼在内的日本许多地方的亚种的线粒体D-loop序列，我们发现的特异/多态的DNA标记，以及精心挑选的引物的RAPD-PCR图谱，我们肯定地阐明了雌核发育群体基因组的母系起源。在本次调查过程中，三倍体基因组与两个二倍体C.A.为了寻找与雌核发育机制相关的基因，我们选择了两种方法：仔细研究重要的细胞周期调控基因Wee1基因，以及从二倍体和三倍体鱼类的覆盖物中相互减去cDNA。对于前者，我们对Wee1基因及其产物进行了全面的鉴定。对于后者，我们分离了4个与雌核发育现象相关的基因，并对它们的碱基序列和表达模式进行了分析。

This three-year project was to study the molecular mechanisms underlying gynogenesis practiced by a population, or subspecies, of Japanese crucian carp, namely, Carassius auratus langsdorfi. This extremely rare and interesting vertebtare(fish) has been known to produce unreduced eggs with its genomic constitution exactly identical to that of somatic cells by means of 1. skipping the first meiotic division and 2. upon insemination, keeping the membrane of incoming sperm nucleus undamaged, thus prohibiting the male genome from participating in embryo development. Such a type of clonal reprodction as this one is remarkable in terms of both basic developmental biology and developing an artificial way of mass production of clonal offspring.In this study, we examined a sizable collection of C.a.langsdorfi (both the bisexsual diploid and all-female tri- or tetraploid) and several other C.a. subspecies, including the goldfish, from many parts of Japan for their mitochondrial D-loop sequences, DNA markers which we found specific/polymorphic, electrophoretograms of RAPD-PCR with carefully selected primers, and we almsot surely clarified the maternal origin of the genomes of the gynogenetic populations. In the course of this investigation, a strong link between the triploid genome and genomes of both diploid C.a. and goldfish was also found.In search for genes that are related to gynogenetic mechanisms, we chose two approaches : close examination of wee1 gene, an important cell-cycle regulating gene, and mutual subtraction of cDNAs from overies of both diploid and triploid fish. For the former, we conducted a thorough characterization of wee1 gene and its product. For the latter, four genes were isolated which were reasonably expected to be related to the gynogenetic phenomena and they were analyzed for the base sequences and patterns of expression.