Expression of Radiation-Induced Mutations at the Arginine Permease (CAN1) Locus in SACCHAROMYCES CEREVISIAE

In the yeast Saccharomyces cerevisiae, the expression of resistance to the L-arginine analog, L-canavanine, after mutagenesis, is strongly dependent on the metabolic state of the cell. The frequency of mutations recovered after exposure to ultraviolet light or X rays was measured under a variety of culture conditions. The results indicate that the frequency of mutants recovered is determined by the following three factors: (1) The potential mutants still possess enough permease activity to take up some of the cell poison, and some are therefore killed before they can express the mutant genotype. The sensitivity is strongly influenced by the endogenous free arginine, which is in turn influenced by the growth medium. (2) The rapid decay of the permease molecules and the inability of the potential mutants to resynthesize this protein results in a rapidly increasing change of expression when selection is delayed. (3) During the time when the permease activity is decaying, repair of the mutagen-induced damage appears to occur.     

Molecular Analysis of 36 Mutations at the Mouse Pink-Eyed Dilution (P) Locus

Thirty-six radiation- or chemically induced homozygous-lethal mutations at the p locus in mouse chromosome 7 have been analyzed at 17 loci defined by molecular probes to determine the types of lesions, numbers of p-region markers deleted or rearranged, regions of overlap of deletion mutations, and genetic distances between loci. A linear deletion map of the [Myod1, Ldh3]-[Snrpn, Znf127] region has been constructed from the molecular analyses of the p-locus deletions. The utility of these deletions as tools for the isolation and characterization of the genes specifying the neurological, reproductive, and developmental phenotypes genetically mapped to this region will grow as more detailed molecular analyses continue.     

Molecular Analysis of Two Novel Missense Mutations in the GDF5 Proregion That Reduce Protein Activity and Are Associated with Brachydactyly Type C

Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5^T201P and proGDF5^L263P revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.     

Molecular Analysis of Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Deficiencies: Novel Mutations and the Spectrum of Japanese Mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

Molecular Analysis of Diepoxybutane-Induced Mutations at the rosy Locus of Drosophila melanogaster

We have analyzed at the molecular level diepoxybutane-induced mutants determined to have lesions affecting expression of the ry locus. Of the 21 mutants analyzed here, genetic analysis suggested that five were putative deficiencies involving ry and adjacent lethal loci. However, molecular analysis confirmed that only two of these five putative deficiencies were in fact deletions detectable by the methods used in the analysis. The remaining 16 mutants were viable as homozygotes, suggesting that their lesions were confined to the ry locus. Seven of these 16 intragenic mutants were determined to be deletions of genetic material as evidenced by altered restriction patterns relative to the wild type patterns. Thus, nine of 21 (43%) diepoxybutane-induced mutants are due to deletions ranging in size from approximately 50 base pairs to more than 8 kilobase pairs. Most of the deletions (seven of nine or 78%) are intragenic and less than 250 base pairs in size; it seems that most, if not all, affect coding rather than regulatory sequences.     

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology^1,2, behavior³, and memory and learning^4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. II. Complementation Analyses of Lethal Brown Deletions

Numerous new mutations at the brown (b) locus in mouse chromosome 4 have been recovered over the years in germ-cell mutagenesis experiments performed at the Oak Ridge National Laboratory. A large series of radiation- and chemical-induced b mutations known to be chromosomal deletions, and also known to be prenatally lethal when homozygous, were analyzed by pairwise complementation crosses as well as by pseudodominance tests involving flanking loci defined by externally visible phenotypes. These crosses were designed to determine the extent of each deletion on the genetic and phenotype map of the chromosomal region surrounding the b locus; the crosses also provided basic data that assigned deletions to complementation groups and defined four new loci associated with aberrancies in normal development. Specifically, the pseudodominance tests identified deletions that include the proximally mapping whirler (wi) and the distally mapping depilated (dep) genes, thereby bracketing these loci defined by visible developmental abnormalities with landmarks (deletion breakpoints) that are easily identified on the physical map. Furthermore, the complementation crosses, which were supplemented with additional crosses that allowed determination of the gross time of lethality of selected deletions, defined four new loci required for normal development. Homozygous deletion of one of these loci (b-associated fitness, baf) results in a runting syndrome evident during postnatal development; deletion of one locus [l(4)2Rn] causes death in the late gestation/neonatal period; and deletion of either of two loci [l(4)1Rn or l(4)3Rn] results in embryonic death, most likely in pre-, peri- or postimplantation stages. The placement of these new functionally defined loci on the evolving molecular map of the b region should be useful for continuing the analysis of the roles played in development by genes in this segment of chromosome 4.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. I. Origin and Molecular Mapping of Radiation- and Chemical-Induced Lethal Brown Deletions

Over a period of many years, germ-cell mutagenesis experiments using the mouse specific-locus test have generated numerous radiation- and chemical-induced alleles of the brown (b; Tyrp1) locus in mouse chromosome 4. We describe here the origin, maintenance and initial molecular characterization of 28 b mutations that are prenatally lethal when homozygous. Each of these mutations is deleted for Tyrp1 sequences, and each of 25 mutations tested further is deleted for at least one other locus defined by molecular clones previously found to be closely linked to b by interspecific backcross analysis. A panel of DNAs from mice carrying a lethal b mutation and a Mus spretus chromosome 4 was used in the fine structure mapping of these molecularly defined loci. The deletional nature of each of these prenatally lethal mutations is consistent with the hypothesis that the null phenotype at b has an effect only on the quality (color) of eumelanin produced in melanocytes. The resulting deletion map provides a framework on which to build future molecular-genetic and biological analyses of this region of mouse chromosome 4.     

中国田鼠卵巢细胞gpt基因自发和亚砷酸钠诱发突变的分子分析

本文研究了亚砷酸钠对CHO-AS52细胞gpt基因的致突变作用。实验结果表明,亚砷酸钠能诱发该基因发生突变,且其突变频率随砷浓度的增加而增高。PCR分析指出,绝大多数亚砷酸钠诱发的CHO-AS52突变体的gpt基因完全缺失。在CHO-AS52细胞自发的、50μmol/L和100μmol/L亚砷酸钠诱发的突变体中,gpt基因完全缺失者所占比率分别为36.00％、54.72％及66.67％。对亚砷酸钠诱发的非缺失型gpt基因突变的PCR产物直接进行DNA序列分析表明,在9个突变细胞克隆中,有2个发生移码突变,其余7个突变细胞克隆的gpt基因结构未发现改变,碱基的改变可能发生在基因启动子区。     

Development of Rhesus Macaque Monkeys (Macaca mulatta) at Early Stages of Ontogenesis at the Artificial Nurturing and Rearing by Human

The experience of the artificial nurturing and rearing of rhesus macaque kids from the moment of their birth to the age of half a year is described. Data are presented about development of sensomotor functions, peculiarities of manifestation of complete forms of the species-specific behavior and communicative response of socially isolated monkeys for the first six months of life at their rearing by human. The obtained results promote enlargement of the biological knowledge of ontogenesis of the lower monkeys.     

靶点药物Cetuximab(C225)研究新进展

在许多肿瘤组织中均有表皮生长因子受体(epidermal growthfactor receptor,EGFR)的过表达,它的失调与肿瘤对化疗和放疗的耐受以及不良预后相关,为肿瘤的治疗提供了一个理想的分子靶点.Cetuximab(C225)是特异性EGFR单克隆抗体,与化疗或放疗联合应用时具有协同作用,具有毒副作用少、靶向性好等优点.Cetuximab(C225)已被批准用于对伊利替康抵抗的结直肠癌和头颈部鳞癌的治疗,对非小细胞肺癌、乳腺癌、胰腺癌等具有EGFR高表达肿瘤治疗的临床试验正在进行之中,为肿瘤治疗开辟了一个全新的领域.     

杜仲雄花芽2个发育时期转录组分析

杜仲（Eucommia ulmoides）雄花富含多种活性成分和营养成分,具有重要的药用和营养价值。为了揭示杜仲雄蕊原基发育相关基因的表达情况,为杜仲雄花芽发育分子调控机制研究提供理论参考。本文以杜仲良种"华仲11号"（ "Huazhong No.11" ）为材料,采用lllumina高通量测序技术,分别对苞叶原基分化期和雄蕊原基分化期的花芽进行转录组测序,通过生物信息学对2个发育时期的转录组进行比较分析,筛选出与雄花芽形态发育相关的差异基因。结果显示,转录组测序共获得40.48 Gb过滤数据,各样品的clean reads与杜仲基因组进行序列比对,比对效率为90.56%~93.01%。在2个发育时期筛选出583个差异表达基因,其中在雄蕊原基发育期上调基因315个,下调基因267个。差异基因GO和KEGG功能分析显示,差异基因富集在与生长发育、光周期途径、激素合成和信号传导、碳代谢等相关的生物过程和代谢通路。结果显示光周期途径是杜仲成花诱导的重要途径,同时雄花芽在形态分化过程中受碳水化合物、植物激素和其他代谢物质调控。此外,MADS-box家族成员FLC、SOC1、AGL3和AGL8参与杜仲雄蕊器官发育。本研究为杜仲花发育基因调控提供了基础数据,也为雄花用杜仲的分子育种提供了参考。     

处于性腺不同发育阶段斜带石斑鱼垂体的EST表达差异分析

斜带石斑鱼是重要的海产经济鱼类,在其个体发育过程中存在先雌后雄的天然性反转现象。垂体是调节生长和生殖等生理过程的重要内分泌器官。构建了斜带石斑鱼分别处于卵巢发育起始和性反转后期的垂体SMARTcDNA的质粒文库,并通过测序分别筛选到232个和258个表达序列标签(expressed sequence tags,EST)。将所得EST与GenBank数据库中的序列进行比对,结果表明,处于卵巢发育起始和性反转后期斜带石斑鱼垂体EST中,激素所占比例均为最高,分别为40.5%和34.9%。进一步比较分析了这两个性腺发育时期斜带石斑鱼垂体EST中各种激素相对表达丰度,表明生长/催乳激素家族(GH、PRL和SL)和阿黑皮素原(POMC)表达水平下降;促性腺激素α亚基(GTHα)表达水平急剧上升,促滤泡激素β亚基(FSHβ)、促黄体激素β亚基(LHβ)表达水平上升。