VAP1, with cystatin C motif, an oocyte protein encoded by a novel ovarian-specific gene during oogenesis in the common brushtail possum (Trichosurus vulpecula)

In the brushtail possum oocyte, vesicles accumulate in a polarized fashion at the vegetal pole and cytoplasm rich in mitochondria and containing the germinal vesicle comprise the animal pole. During cleavage to early blastocyst stages, animal pole cytoplasm locates to the cells of the embryonic hemisphere (pluriblast) and vegetal pole vesicular cytoplasm to cells of the abembryonic hemisphere (trophoblast). Previously identified 16 amino acid residues, associated with the vesicle-rich cytoplasm were used for molecular cloning and characterization of a vesicle associated protein, VAP1. The degenerate primer was used in a 3'RACE for vap1 gene cloning. The cDNA encoding VAP1 was 516 bp in length with no significant homologies and coded for 172 amino acid residues for the mature protein. The N-terminal domain of VAP1 showed a structural homology to the cysteine protease inhibitor, Cystatin. Gene expression studies during oogenesis revealed that vap1 had an ovary-specific, possibly oocyte-specific expression, which occurs during follicle formation and growth and in adult ovaries. Recombinant VAP1 fusion protein generated polyclonal antibodies in the mouse and in the brushtail possum.     

A simple method for discriminating between cell membrane and cytosolic proteins

* Transgenic plants expressing either green fluorescent protein (GFP)-genomic DNA or GFP-cDNA fusions have been used as powerful tools to define the subcellular localization of many proteins. Because most plant cells are highly vacuolated, the cytosol is confined to a thin layer at the periphery of the cells, making it very difficult to distinguish among cell wall, cell membrane and cytosolic GFP-fusion proteins. * Plasmolysis tests inform about cell-wall localization of GFP-tagged proteins, but they do not discriminate between its cell membrane and/or cytoplasmic localization. By observing the GFP signal in transgenic protoplasts placed at a hypotonic solution, it was possible to distinguish between cell membrane and cytosolic GFP-tagged proteins. * The osmotic disruption of the protoplast vacuole in the hypotonic solution allows the diffusion of the GFP signal from the cell periphery to the central part of the cell volume when the GFP is fused to a soluble protein. By contrast, such diffusion does not occur when the protein under study is attached to the cell membrane. * The present method is easier, faster and cheaper than subcellular fractionating studies and/or immunoelectron microscopy, which have been traditionally used to discern between cell membrane and cytosolic proteins.     

Mating system and reproductive skew in the black rhinoceros

Only approximately 2600 black rhinoceros survive today, mainly in small, isolated populations of < 100 animals. The management of remaining black rhinoceros populations aims at preserving natural levels of genetic relatedness and optimizing breeding success, which requires an accurate knowledge of the mating system, reproductive skew and effective population size. DNA was extracted from faecal samples from a community of 35 wild black rhinoceros, and microsatellites were used to characterize patterns of paternity of 19 offspring born from eight females in this community. Paternity could be ascribed unequivocally for each offspring. Although our conclusions must be considered tentative, we present the first genetic evidence that black rhinoceros males are polygynous, with a high variance in reproductive success. We also describe a noninvasive management tool that can be used for the genetic management of this critically endangered species, both in the wild and in captivity.     

Induction of androgenetic development of the brook charr (Salvelinus fontinalis) × Arctic charr (Salvelinus alpinus) hybrids in eggs derived from the parental species

Failure of interspecific androgenesis between brook charr (Salvelinus fontinalis, Mitchill 1814) and Arctic charr (Salvelinus alpinus, L.) has been attributed to the conflict between the egg cytoplasm of one species and the sperm nucleus of the other species. To overcome this incompatibility, sperm derived from the brook charr × Arctic charr hybrid male was used to induce androgenetic development in eggs originating from the parental species as well as their hybrids. The eggs were subjected to 420 Gy of X-radiation to damage the maternal nuclear DNA and inseminated with untreated sperm. Haploid zygotes were exposed to high hydrostatic pressure shock (7000 psi for 4 min), which was applied 420 min after insemination to inhibit the first cell cleavage and recover the diploid state of the zygote. The androgenetic diploid offspring that hatched from the brook charr, the Arctic charr and the hybrids eggs had survival rates of 4.7 ± 0.6%, 1.2 ± 0.4% and 16.8 ± 0.5%, respectively. Drastic mortality among the hatched androgenetic individuals was observed within the first five months of rearing. Cytogenetic analysis of the androgenetic progenies exhibited residues of the irradiated maternal nuclear genome in the form of radiation-induced chromosome fragments in 47% of the specimens that were examined. Interactions between the egg cytoplasm and the sperm nucleus, the low quality of the gametes, the expression of homozygous paternal lethal alleles and the incomplete inactivation of the maternal chromosomes were identified as factors responsible for the large mortality among androgenetic embryos and hatchlings.     

Stem diversity, cauline domatia, and the evolution of ant-plant associations in Piper sect. Macrostachys (Piperaceae)

Plants possess a variety of structures that harbor ant nests, and the morphology of these domatia determines the nature of ant-plant mutualisms in a given plant species. In this study, we report on the differences in anatomy between myrmecophytes of Piper, which are regularly excavated by an obligate ant mutualist (Pheidole bicornis) and nonmyrmecophytes of Piper, which consistently have solid stems. Stems of excavated plant species lacked outward evidence of modification; however, striking anatomical differences were apparent between hollow-stemmed species before excavation and the remainder of the solid-stemmed species studied. Prior to excavation by ants, stems of myrmecophytes were characterized by strongly heterogeneous piths in which a large, central area had relatively large cells lacking intracellular crystals with a periphery of smaller cells containing numerous crystals. The domatium excavated by the ants was restricted to the large-celled region. This is the first report of the absence of crystals in ant-excavated portions of stems of myrmecophytes. Cauline domatia became lined with 3-8 cell layers of suberized wound tissue, which may have an impact on nutrient absorption by Piper myrmecophytes.     

A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity

Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNA^Leu. LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain–domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS.     

Comparison on the genotoxic effects of nuclear vs cytoplas-mic irradiation from the alteration of CD59 gene locus

Using microbeam to irradiate human-hamster hybrid AL cells with defined number of a particles in a highly localized spatial region, this paper showed that cytoplasmic irradiation induced very little toxicity. For example, the cell killing by 4α particle traversal through the cytoplasm was about 10%, and about 70% cells survived after their cytoplasm was irradiated with 32 a particles. In contrast, the survival fractions for nuclear irradiation at the same doses were 35% and less than 1% respectively. Mutation induction showed that while nuclear irradiation induced 3-4-fold more CD59- mutants than cytoplasmic irradiation at equivalent particle traversal, at an equitoxic dose level of 90% survival, the latter exposure mode induced 3.3-fold more mutants than nuclear irradiation. Moreover, using multiplex PCR to analyze five marker genes on chromosome 11 (WT, CAT, PTH, APO-A1 and RAS), the results showed that the majority of mutants induced by cytoplasmic irradiation had retained all of the marker genes ana     

Comparison on the genotoxic effects of nuclear vs cytoplasmic irradiation from the alteration ofCD59 gene locus

Using microbeam to irradiate human-hamster hybrid A_L cells with defined number of α particles in a highly localized spatial region, this paper showed that cytoplasmic irradiation induced very little toxicity. For example, the cell killing by 4 α particle traversal through the cytoplasm was about 10%, and about 70% cells survived after their cytoplasm was irradiated with 32 a particles. In contrast, the survival fractions for nuclear irradiation at the same doses were 35% and less than 1% respectively. Mutation induction showed that while nuclear irradiation induced 3—4-fold more CD59^- mutants than cytoplasmic irradiation at equivalent particle traversal, at an equitoxic dose level of 90% survival, the latter exposure mode induced 3.3-fold more mutants than nuclear irradiation. Moreover, using multiplex PCR to analyze five marker genes on chromosome 11 (WT, CAT, PTH, APO-A1 and RAS), the results showed that the majority of mutants induced by cytoplasmic irradiation had retained all of the marker genes analyzed. By comparison, the proportion of mutants suffering loss of additional chromosomal markers increased with increasing number of particle traversal through nuclei.     

作物雄性不育性在育种中的应用概评

概述总结了作物雄性不育性的类别与遗传特点。雄性不育性的遗传机理涉及细胞质遗传的现象,目前已初步探明玉米C群不育系的胞质基因可能是atp6-c,芝麻不育胞质基因拟为atpA。雄性不育化杂交种在实践中主要应用于玉米、水稻和蔬菜中。尽管现有近交理论、DNA甲基化效用、水稻胞质与核不育系遗传等理论提出,雄性不育化育种的基本理论尚需进一步探讨。在雄性不育化育种技术上,要逐步解决难点作物,如小麦、荞麦、菜豆等的不育化育种问题。