SNPkit: an efficient approach to systematic evaluation of candidate single nucleotide polymorphisms in public databases

There is widespread interest in devising genotyping methods for SNPs that are robust, inexpensive, and simple to perform. Although several high-throughput SNP genotyping technologies have been developed, including the oligonucleotide ligation assay, real-time PCR, and mass spectrometry, the issues of simplicity and cost-effectiveness have not been adequately addressed. Here we describe the application of a novel computer software package, SNPkit, which designs SNP genotyping assays based on a classical approach for discriminating alleles, restriction enzyme digestion. SNPkit can be used in genotyping assays for almost any SNPs including those that do not alter "natural" restriction sites. Using this method, 164 SNPs have been evaluated in DNA samples from 48 immortalized cell lines of randomly selected Chinese subjects. Sixty-two (37.8%) of the SNPs appeared to be common (frequencies of the minor alleles are > or = 5%) and were subsequently applied to a larger population-based sample. Overall, by using SNPkit, we have been able to validate and genotype accurately a large fraction of publicly available SNPs without sophisticated instrumentation.     

DNA transfer by highly asymmetric somatic hybridisation in Medicago truncatula (+) Medicago rugosaand Medicago truncatula(+) Medicago scutellata

A regenerable line of Medicago truncatula (Jemalong 2HA) as a recipient species, was fused with the sexually incompatible species Medicago scutellata or Medicago rugosa. The treatments described maintain the chromosome number of the recipient but enable the transfer of small amounts of DNA of the donor species, probably by intergenomic recombination. Without a chromosome number-change fusion products can readily regenerate to produce fertile plants; and potentially a library with a diverse array of new genetic material. The selection of fused cells is based on treatment of the recipient cells with iodoacetamide (IOA), a non-regenerable donor, γ-irradiation of the donor, and regeneration on a medium favouring the recipient. DNA transfer was demonstrated by amplified fragment length polymorphism (AFLP), Southern hybridisation and changed morphology. Received: 21 December 2000 / Accepted: 5 April 2001     

Semiochemicals from ovaries of gravid females attract ovipositing female houseflies,Musca domestica

Chemical signals originating from the ovaries of gravid females of Musca domestica (Diptera: Cyclorrhapha: Muscidae) attract ovipositing females to common egg-laying sites. Behavioral experiments indicated that females preferred to oviposit in fermented wheat bran containing ovaries from reproductively mature houseflies. Females preferred to oviposit in fermented wheat bran than wet wheat bran. This effect was additive with the attraction to housefly ovaries. Solvent extracts from housefly ovaries were attractive to gravid females. Extracts obtained with hexane were most attractive to gravid females for egg laying, and extracts obtained with ethyl acetate attracted more egg laying than extracts obtained by dichloromethane. Gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC-MS) analysis showed that tricosane and (Z)-9-tricosene were the main components of the hexane extracts. Both tricosane and (Z)-9-tricosene were shown to elicit dose-dependent aggregation of gravid females in oviposition bioassays, but high doses of either chemical were not attractive.     

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.     

A novel noncollagenous protein encoded by an alternative transcript of the chick type III collagen gene is expressed in cartilage,bone and muscle

RanBP1, a velocardiofacial syndrome/DiGeorge syndrome candidate gene, is expressed in the frontonasal processes, branchial arches, aortic arches, and limb buds. At these sites, RanBP1 apparently coincides with neural crest-derived mesenchymal cells. In addition, RanBP1 is expressed in the forebrain as well as in hindbrain regions previously associated with crest-derived mesenchymal cells.     

The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis

Formation of somatic embryos in plants is known to require high concentrations of auxin or 2,4-dichlorophenoxyacetic acid (2,4-D), which presumably acts to trigger a signalling cascade. However, very little is known about the molecular mechanism that mediates the vegetative-to-embryogenic transition. We have employed a genetic approach to dissect the signal transduction pathway during somatic embryogenesis. In a functional screen using a chemical-inducible activation-tagging system, we identified two alleles of Arabidopsis gene PGA6 whose induced overexpression caused high-frequency somatic embryo formation in all tissues and organs tested, without any external plant hormones. Upon inducer withdrawal, all these somatic embryos were able to germinate directly, without any further treatment, and to develop into fertile adult plants. PGA6 was found to be identical to WUSCHEL (WUS), a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and floral meristems. Transgenic plants carrying an estradiol-inducible XVE-WUS transgene can phenocopy pga6-1 and pga6-2. Our results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.     

Effects of different peptide fragments derived from proadrenomedullin on gene expression of adrenomedullin gene

Primary culture of vascular smooth muscle cells (VSMC) from rat aorta was used for the study of the effect of different peptides derived from proadrenomedullin on the expression of adrenomedullin (ADM) gene. ADM and preproADM(22-41) (PAMP) secreted by VSMC were measured by radioimmunoassay, and ADM mRNA in VSMC was determined by quantitative RT-PCR. After the incubation of VSMC in 10(-7)M ADM for 24h, PAMP in the medium and ADM mRNA in the VSMC were decreased by 34 and 41.3%, respectively, and cAMP concentration in the VSMC was increased by 385%. After the incubation of VSMC in 10(-7)M PAMP for 24h, ADM in the medium and ADM mRNA in the VSMC were decreased by 12.2 and 39.1%, respectively, and cAMP concentration in the VSMC was increased by 67%. The decreased ADM mRNA in VSMC induced by the ADM and PAMP treatment was completely reversed by the pre-treatment of the cells in 10(-7)M protein kinase inhibitor for 30 min. After the incubation of VSMC in 10(-7)M preproADM(153-185) (ADT) for 24h, however, ADM in the medium and ADM mRNA in the VSMC were increased by 21 and 35.2%. The increased ADM mRNA in VSMC induced by the ADT treatment was partially blocked by the co-incubation in ADM and ADT, and was totally blocked by the co-incubation in PAMP+ADM and ADT, but was not blocked by the co-incubation in PAMP and ADT. Our results suggest that the four peptides derived from proadrenomedullin may have different effects, possibly through a cAMP-dependent pathway, on the expression of ADM gene.     

蛋白酶活化受体-2的研究进展

蛋白酶活化受体(protease-activated receptors,PARs)属于G蛋白偶联受体家族成员,其N-末端被蛋白酶裂解后,可形成新的N-末端。新N-末端能够结合、激活自身受体。PAR-2是PARs的成员之一,其激活、灭活、脱敏、复敏、及其与信号转导途径的关系,尤其是与疾病(如呼吸道慢性炎症)的关系正倍受关注。