Genetic Mapping of Four Mouse bHLH Genes Related toDrosophilaProneural Geneatonal

It has been shown that mammalian neurogenesis is partly controlled by multiple basic helix–loop–helix (bHLH) genes, as inDrosophila.Recently, mouse homologs ofDrosophila atonal,a proneural gene encoding a bHLH protein required for chordotonal organ and photoreceptor development, have been characterized to obtain further insights into the molecular nature of mammalian neurogenesis. Here, to assess their potential involvement in genetic neural disorders, we have determined genetic map positions for four mouseatonal-related genes,Atoh1, Atoh2, Atoh3,andNdrf,which encode MATH-1, MATH-2, MATH-3, and NDRF, respectively. Interspecific backcross analysis indicated thatAtoh1andAtoh2were located in separate positions of Chr 6 and thatAtoh3andNdrfwere mapped to Chr 10 and Chr 11, respectively. Thus, these structurally related genes are located separately on multiple chromosomes.     

八肋游仆虫Rab家族基因克隆和多样性分析

Rab蛋白是在真核细胞内膜泡运输过程中起重要调节作用的一类小分子Ras-like蛋白,为Ras超家族中最大的家族。Rab家族成员在不同的生物中表现出数量的多样性和功能上的分化。为进一步了解Rab蛋白的多样性及其在真核细胞内膜泡运输网络中的功能,本研究利用游仆虫大核染色体特异的端粒结构和基因大小的染色体结构特征,通过简并引物PCR方法从八肋游仆虫(Euplotes octocarinatus)中克隆到9种新的Rab基因,分别为EoRab1A、EoRab2b、EoRab2c、EoRab2d、EoRab6、EoRab7、EoRab2-like、EoRabL2和EoRan(GenBank登陆号为HM371131～HM371139)。序列分析表明,游仆虫中Rab基因家族成员既包括具有维持细胞结构核心功能保守基因,又包括为适应环境而进化出的特殊功能的新基因。     

Rab17 and Rab18, Small GTPases with Specificity for Polarized Epithelial Cells: Genetic Mapping in the Mouse

The Rab subfamily of small GTPases plays an important role in the regulation of membrane traffic in eukaryotic cells. While most Rab proteins are equally expressed in polarized and nonpolarized cells, Rab17 and Rab18 show epithelial cell specificity. Here we report the genetic mapping of Rab17 and Rab18 on mouse chromosomes 1 and 18, respectively. We also discuss some implications ofRab17andRab18mapping, including their candidacy for the mouse mutationsln(leaden),Tw(twirler), andax(ataxia).     

Genetic Mapping of the Mouse Genes Encoding the Voltage-Sensitive Calcium Channel Subunits

The genes encoding the α₁ and β subunits of voltage-sensitive calcium channel were mapped in the mouse by analysis of the progeny of two multilocus crosses. The α₁, β₂, and β₄ subunit genes, termed Cchna1, Cchb2, and Cchb4, are located at different sites on proximal Chr 2, while the β₃ subunit gene Cchb3 maps to Chr 15 near Wnt1. These results together with previous mapping data indicate that the calcium channel genes are dispersed in the mouse genome, unlike the sodium channel genes, which are clustered.     

拟南芥高迁移率族蛋白B族基因表达模式分析

为了解高迁移率族蛋白B族(HMGB)基因在拟南芥中的表达模式及作用方式,该研究克隆了拟南芥中5个编码HMGB的基因:AtHMGB1、AtHMGB2、AtHMGB3、AtHMGB4、AtHMGB5,并运用荧光实时定量PCR方法检测野生型拟南芥中以上5种基因在不同器官中的表达及在外源植物激素(ABA、2,4-D)处理前后的表达差异,选取AtHMGB2、AtHMGB4和AtHMGB5分别转化拟南芥并筛选出超表达株系,随即检测ABA诱导下超表达AtHMGB的转基因拟南芥的表型。研究证实:在野生型拟南芥中AtHMGB2在拟南芥各个器官中的表达量远高于其它家族成员,AtHMGB4和AtHMGB5在花、果荚和根中的表达略高于茎和叶;在ABA处理前后AtHMGB家族成员的表达水平有显著差异,其中AtHMGB2的表达被ABA显著负调控;ABA诱导下超表达AtHMGB2的转基因拟南芥与野生型相比出现萌发及生长迟缓现象,但超表达AtHMGB4与AtHMGB5的转基因拟南芥在ABA诱导下的种子萌发和幼苗生长与野生型相比差异不大。研究发现,AtHMGB家族成员在转录水平上响应ABA的方式各有不同,对理解AtHMGB家族成员的生物学功能提供了新的基础。     

异染色质相关蛋白TRIM28调控锌指蛋白和原钙黏蛋白家族基因的转录

目的 TRIM28是一种异染色质相关蛋白,通过和SETDB1、HP1相互作用参与H3K9me3修饰的建立,本文旨在更深入地研究TRIM28的相关功能。方法 本文利用CRISPR/Cas9技术、染色质免疫共沉淀技术、免疫印迹技术和实时荧光定量PCR技术,建立HEK293F Trim28基因敲除细胞系,分析一系列实验数据结果。结果 Trim28主要抑制内源表达水平较低的基因转录,进一步分析发现Trim28调控锌指蛋白家族基因和原钙黏蛋白β家族基因的转录。在Trim28敲除细胞系中,锌指蛋白家族基因H3K27ac修饰、H3K4me1修饰和H3K4me3修饰都显著上升,H3K9me3修饰下降。原钙黏蛋白β家族基因的H3K4me3修饰显著上升,H3K9me3修饰下降。结论 这些结果提示TRIM28通过改变染色质的开放程度调控锌指蛋白和原钙黏蛋白β家族基因的转录,为更深入研究TRIM28的功能提供了新的思路。     

鹅掌楸属GST家族基因的克隆与表达分析

该研究以鹅掌楸属植物为试验材料,在北美鹅掌楸和鹅掌楸转录组数据库中筛选出54条GST基因家族的EST序列,通过比对将其归为Tau、Zeta、Theta和Phi等4个亚类。在这4个亚类中各选取1条EST序列设计引物,通过反转录PCR(RT-PCR)与cDNA末端快速扩增(RACE)技术,从鹅掌楸属植物中克隆出4条全长分别为1 150、932、1 022和1 067bp的基因,并分别命名为LtGSTparCb、LtGSTZ-like、LtGSTF13a和LcGSTT1。对LtGSTparCb、LtGSTZ-like、LtGSTF13a和LcGSTT1这4个基因编码蛋白质的结构预测发现,其蛋白质的分子质量分别为25.17、25.34、24.49和28.09kD;理论等电点分别是5.60、6.53、6.66和9.20,即LtGSTparCb、LtGSTZlike、LtGSTF13a蛋白质属于酸性蛋白,LcGSTT1属于碱性蛋白;不稳定系数分别是33.01、36.97、43.19和47.96,即LtGSTparCb、LtGSTZ-like为稳定蛋白,LtGSTF13a、LcGSTT1为不稳定蛋白;α-螺旋(alpha helix)是这4个蛋白质二级结构的主要成分。利用在线软件TargetP并结合GST蛋白质在其他植物中亚细胞定位的结果,推测这4种蛋白质极有可能位于鹅掌楸属植物的细胞质内。实时荧光定量PCR分析结果表明,这4个基因在北美鹅掌楸各个组织中均有表达,其中LtGSTparCb和LtGSTF13a在叶中的表达量最高,在其他组织中的表达量均较低;LtGSTZ-like在花器官中的表达量最高,在叶中的表达量最低,在其他组织中的表达量居于两者之间;LcGSTT1在叶中表达量最高,花器官中表达量其次,在茎和叶芽中的表达量偏低。该研究结果可为鹅掌楸属GST家族基因的功能研究奠定基础。     

The Exon/Intron Organization and Chromosomal Mapping of the Mouse ADAMTS-1 Gene Encoding an ADAM Family Protein with TSP Motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescencein situhybridization revealed that the ADAMTS-1 gene is located in region C3–C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Genetic and Physical Mapping of a Gene Encoding a Methyl CpG Binding Protein, Mecp2, to the Mouse X Chromosome

The methyl CpG binding proteins (MeCP1 and MeCP2) are a class of proteins that bind to templates containing symmetrically methylated CpGs. Using an interspecific backcross segregating a number of X-linked markers, we have localized the Mecp2 gene in mouse to the X chromosome close to the microsatellite marker DXMit1. Detailed physical mapping utilizing an available YAC contig encompassing the DXMit1 locus has localized the Mecp2 gene to a 40-kb region between the L1cam and the Rsvp loci, indicating the probable position of a homologue on the human X chromosome.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.     

A New Family of Mouse Genes Homologous to the HumanMAGEGenes

The humanMAGEgenes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. TheMAGEgenes are located on the X chromosome, in three clusters denotedMAGE-A, B,andC,mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 humanMAGE-Agenes. Using aMAGE-Aprobe, we isolated 8 new murine genes that are homologous to theMAGEgenes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with theMAGE-AORFs than with theMAGE-BorMAGE-CORFs. Furthermore, likeMAGE-Agenes, they encode acidic proteins, whereas theMAGE-Bgenes encode basic proteins. Accordingly, these 8 murine genes were namedMage-a1to8(approved symbolsMagea1to8).Mage-agenes were mapped in two different loci on the mouse X chromosome.Mage-a4andMage-a7are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts,Mage-agenes were found to be transcribed in adult testis, but not in other tissues. Expression of someMage-agenes was also detected in tumor cell lines. TwoMage-agenes were found to be expressed in blastocysts.     

Genetic and Physical Mapping of the Mouse Ulnaless Locus

The mouse Ulnaless locus is a semidominant mutation which displays defects in patterning along the proximal-distal and anterior-posterior axes of all four limbs. The first Ulnaless homozygotes have been generated, and they display a similar, though slightly more severe, limb phenotype than the heterozygotes. To create a refined genetic map of the Ulnaless region using molecular markers, four backcrosses segregating Ulnaless were established. A 0.4-cM interval containing the Ulnaless locus has been defined on mouse chromosome 2, which has identified Ulnaless as a possible allele of a Hoxd cluster gene(s). With this genetic map as a framework, a physical map of the Ulnaless region has been completed. Yeast artificial chromosomes covering this region have been isolated and ordered into a 2 Mb contig. Therefore, the region that must contain the Ulnaless locus has been defined and cloned, which will be invaluable for the identification of the molecular nature of the Ulnaless mutation.     

Chromosomal Mapping of Genes in theRBCS Family in Arabidopsis thaliana

In Arabidopsis thaliana, four genes have been identified inthe RBCS gene family, one being assigned to subfamily RBCS-Aand the other three to subfamily RBCS-B (1B, 2B and 3B). Todetermine the chromosomal location ofthese genes, hybridizationanalysis with CIC YAC high-density filters was carried out forthe RBCS-A gene, and CAPS analysis for the three RBCS-B genes,based on the finding that restriction fragment length polymorphismis present in the upstream region of the gene RBCS-3B. The RBCS-Agene was mapped at 100.8 cM from the top of chromosome 1 andthe three RBCS-B genes at 62.70 cM from the top of chromosome5.     

三裂叶薯NBS-LRR类抗病基因的筛选鉴定与结构分析

为发掘甘薯近缘野生种三裂叶薯(Ipomoea triloba)的NBS-LRR类抗病基因,从基因数据库中对三裂叶薯基因组序列进行了筛选、鉴定和分析。结果表明,从三裂叶薯的98 025个基因中,筛选到282个编码NBS-LRR类蛋白的基因,其中N型80个,NL型83个,CN型28个,CNL型57个,TN型10个,TNL型23个,RN型1个。三裂叶薯的16条染色体上均含有NBS-LRR家族基因,数量最多的染色体含有65个,最少的只有1个。三裂叶薯基因组共有55个基因簇,包含了63.5%的NBS-LRR家族基因。在NBS-LRR抗病基因家族中,CNL和TNL亚家族分别对应到7和11个保守结构域。这为三裂叶薯抗性资源的利用提供了科学参考。