A genetic strategy for differential screening of meiotic germ-cell cDNA libraries

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences that failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis. © 1996 Wiley-Liss, Inc.     

A novel serine/threonine kinase gene,Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects. © 1996 Wiley-Liss, Inc.     

Bimodal karyotype in Cynomorium coccineum L. and its systematic implications

The presence of a bimodal karyotype in Cynomorium coccineum (2n = 28) is used to support its separation from Balanophoraceae and the maintenance of Cynomoriaceae as a separate family.     

同翅类昆虫的雄性生殖系统及精子发生(昆虫纲:半翅目)

本文比较了同翅类昆虫雄性生殖系统的结构、减数分裂期间染色体的行为和精子尾部的超微结构。研究表明蜡蝉总科和异翅类的精巢具有被膜,而蝉总科、叶蝉总科、沫蝉总科、角蝉总科、木虱总科、蚜总科、粉虱总科和蚧总科的精巢均不具有被膜。也可以根据精巢小叶的形状将精巢分为三类,蝉总科、叶蝉总科、沫蝉总科、角蝉总科、蚜总科和粉虱总科的精巢小叶为球形,蜡蝉总科、木虱总科和蚧总科的精巢小叶为管状,而异翅类的精巢小叶为片层状。减数分裂可以被分为5类:①蝉型(Cicadoidtype);②蜡蝉型(Fulgoroidtype);③木虱型(Psyloidtype);④蚜型(Aphidoidtype);⑤粉虱型(Aleyrodoidtype)和⑥蚧型(Coccoidtype),至少具有四个类群的减数分裂前期I具有弥散期,它们是:木虱总科、蜡蝉总科、蚧总科和异翅类。除粉虱总科和蚧总科的精子尾部退化以外,其余种类的精子鞭毛均具有典型的9 9 2轴丝结构。     

Mutability of microsatellites developed for the ant Camponotus consobrinus

Five highly polymorphic (GA)n microsatellite loci are reported for the formicine ant Camponotus consobrinus. The occurrence of many nests with a simple family structure enabled a search for new mutations, 11 of which were found from 3055 informative typings. These mutations were not randomly distributed across loci, 10 of them occurring at the locus Ccon70. The spectrum of mutations across alleles at Ccon70 was also nonrandom, with all of them occurring in alleles in the upper half of the allele size distribution. Six of the Ccon70 mutations decreased allele size. The mutations observed fit the stepwise mutation model well, i.e. mutations could always be assigned to an allele which differed in size from them by one repeat unit. The parental origins of the Ccon70 mutations were established and appear more female biased than vertebrate mutations, significantly so compared with human haemophilia A and primate intron mutations. This result may indicate that the lack of meiosis in males (which are haploid in ants) reduces the mutation rate in that sex relative to species in which both sexes are diploid.     

A cytogenetic study of development in mechanically disrupted pairs of Tetrahymena thermophila

We examined the nuclear behavior of mating Tetrahymena cells that had been mechanically disrupted at various times throughout conjugation. Disruption was achieved by agitating conjugating Tetrahymena in the presence of 0.1-3 mm glass beads. Two minutes of agitation with 1 mm beads yielded optimal pair disruption (70%) with high viability (92%). Disrupting pairs between 0-4.7 h after the initiation of mating produced mostly disrupted conjugants in which development was aborted. However, as many as 20% of these early disrupted conjugants completed development even without their mating partners. After 5 h the percentage of disrupted conjugants completing development increased dramatically, reaching 80% by 6.7 h. These results support a model suggesting that events associated with nuclear exchange and fusion 5 h into conjugation trigger a commitment to completion of the postzygotic developmental program. The early conjugants that completed development following disruption suggest that development can be sustained even in the absence of a mating partner. This represents a novel method of bringing the micronuclear genome into macronuclear expression with minimal cytoplasmic exchange between partners. We discuss these results in light of a model relating cortical and nuclear signaling events that reciprocally drive conjugal development.     

Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes

Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.