酿酒酵母5号染色体精细物理图谱绘制

利用脉冲电泳（ＰｕｌｓｅｄＦｉｅｌｄＧｅｌＥｌｅｃｔｒｏｐｈｏｒｅｓｉｓ,ＰＦＧＥ）分析了酵母菌Ａ３６４ａ的电泳核型,以５号染色体专一探针确定了该染色体在电泳核型中的位置,以内切酶ＢａｍＨＩ对该染色体ＤＮＡ进行部分酶切后,与整合型载体ＹＩｐ５连接获得了一个染色体专一的基因文库,其转化子数目超过了理论要求值。从文库中筛选与已知探针有同源性的片段并用内切酶ＢａｍＨＩ,ＥｃｏＲＩ,ＨｉｎｄＩＩ,ＰｓｔＩ和ＳａｌＩ分析这些插入片段,获得了一个覆盖Ａ３６４ａ５号染色体（其长度估计为６２０ｋｂ）９．４％的精细物理图谱。利用边界克隆和菌落杂交将使我们能够对整条染色体进行进一步的“步查”     

水稻抗稻瘟病基因Pi-2(t)物理图谱的构建

应用ＢＡＣ文库,采用基于分子标记的染色体着陆（ｍａｒｋｅｒ－ｂａｓｅｄ ｃｈｒｏｍｏｓｏｍｅ ｌａｎｄｉｎｇ）和染色体步查（ｃｈｒｏｍｏｓｏｍｅ ｗａｌｋｉｎｇ）等手段,建立了包含有裟抗稻瘟病基因Ｐｉ－２（ｔ）的物理图谱,该物理图谱由２２个ＢＡＣ克隆组成,遗传跨度８ｃＭ,而物理距离为９２５ｋｂ,该物理图谱的构建不仅为进一步分离和克隆该基因打下了基础,同时也可为分子标记辅助选择育种选择抗稻瘟病新材料     

人淋巴毒素基因的分子克隆及其物理图谱

本文报道用一种简便、快速并省时的方法——体内同源重组⑴,以小鼠淋巴毒素 (MuLT)cDNA为探针,从以粘粒pcos2EMBL为载体构建成的人基因组文库中分离出人淋巴毒素(HuLT)基因。然后,以同位素³²P标记重组粘粒的cos单链末端⑵,再将经限制酶部份酶切的这种重组粘粒DNA片段电泳分部后,制作出HuLT基因的EcoR I、BamH I、PstⅠ和PvuⅡ四种限制性内切酶的物理图谱。     

高粱叶绿体psbD基因在重组质粒pSD_2Ⅱ中的定位及psbD基因的亚克隆

利用pUC19我们构建了高粱叶绿体基因文库,从库中我们筛选到4个含psbD基因的阳性克隆。Southern分析证明重组质粒由pUC19和高梁叶绿体DNA的Pst10片段组成。通过8种限制性内切酶酶切分析和Southern分析测出了重组质粒pSD_2Ⅱ的物理图谱,并确定了高梁psbD基因在重组质粒中的精确位置。通过构建pSAC_2质粒,可以将Pst10片段中非psbD部分去除,使psbD可以直接用EcoRⅠ从pSAC_2上切下,为研究高粱psbD基因的表达和序列分析奠定了基础。     

水稻组蛋白H_3基因的分离和组织结构分析

用噬菌斑原位杂交法,从一个水稻新品系的EMBL,基因文库中筛选到了两个组蛋白H,基因克隆,并制作了它们的限制性内切酶图谱,分析了它们的组织结构。其中λRH_3-1克隆包含2个组蛋白H_3基因区(分别为0.5kb和0.8kb的BamHI-EcoRI片段,两基因区距离5.8kb)。而λRH_3-2克隆只带有1个结构特异的H_3基因区(5.2kb的EcoRI-HindⅢ片段)。本文还就水稻组蛋白H_3基因在整个基因组中的组织结构特点进行了讨论。     

黄地老虎颗粒体病毒DNA的基因文库和物理图谱

利用EMBL 4——携带多切点连接序列的λ取代载体,建成了黄地老虎颗粒体病毒(AsGV)DNA基因文库,并绘制出AsGV DNA基因组的物理图谱。当体外包装效率达3 x 104pFU／μg DNA时,Sal和BamHI烈酶完全酶切的EMBL 4和BamHI部分酶切的AsGV DNA,按2：1的接头克分子比,用T4连接酶连接后,在BHB2688与2690体系中进行体外包装,再经在L95和ED 8654中的转染和转导,获得1．5×103噬菌斑。根据Clarke和Carbon公式,筛选概率达到复盖99％的AsGV基因组只需35个重组噬菌斑。我们随机挑取了35个重组噬菌斑,经快速琼脂糖凝胶电泳分析得到13个不同类型的重组噬菌斑,以32P标记的AsGV DNA为探针进行分子杂交,确定了BamHI在AsGV DNA基因组上位点的相邻位置。     

图位克隆技术在分离植物基因中的应用

本文综述图位克隆技术的原理及基本技术环节,并与其它基因克隆技术相比较说明图位克隆技术的优缺点。本文还对近年来图位克隆技术在分离不同的植物发育基因上的广泛应用进行了简要概述,并对其应用前景和近期的预期进展作出展望。 Abstract:In this review,the principle and basis steps of map-based gene cloning on plant were introduced in detail.At the same time,the advantages and disadvantage of the method were evaluated as compared with the other methods.The extensive application of the map-based gene cloning method on cloning genes of different plants in the past was also summarized in brief.The prospect and the expected progress in several years were proposed in this paper.     

浑球红细菌谷氨酸合酶基因(glt)的克隆和图谱分析

利用转座子Tn5随机插入诱变筛选得到12株浑球红细菌(Rhodobacter sphaeroides)氨同化缺陷突变株(Asm~-)。这些突变株胞内均无GOGAT活性,同时它们均无固氮酶活性(Nif~-),并且具有氮代谢多效性缺失表型(Ntr~-)。将含有Azorhizobium sesbaniae ORS571的完整glt基因的质粒pHB10转入突变株中能互补上述表型。通过筛选携带Tn5的R-prime质粒克隆了glt::Tn5片段。Southern杂交证明所克隆glt::Tn5片段与E. coli的gltBD基因有同源性。用此片段与以pLAFR3为载体所构建的R. sphaeroides 601基因文库进行菌落原位杂交筛选到了携带glt基因的cosmid pLT27。pLT27能互补所有12株R.sphaeroides氨同化缺陷突变株。酶切分析表明在该cosmid中插人的染色体DNA片段大小约为26.5kb。以pRK415为载体亚克隆了4.0kb与10.5kh的pLT27的Hindlll酶切片段,分别命名为pLTRK271与pLTRK272。pLTRK272能互补变种GT6、GT10、GT11,pLTRK…     

人基因组表达图谱分析和疾病相关基因搜寻──现状与展望(续前)

人基因组表达图谱分析和疾病相关基因搜寻──现状与展望（续前）康毅滨,柴建华（复旦大学遗传学研究所上海２００４３３）二、ｃＤＮＡ随机部分ｆ三、测序及ＥＳＴ的建立以上所讨论的方法均是从基因组ＤＮＡ出发寻找表达序列,与之恰好相反的另一种构建表达图的思路是随机挑选ｃＤＮＡ文库中的克隆进行测序并在染色体上定位,即ｃＤＮＡ策略。人类基因组计划的一个重要目标是获得人基因组３０亿个碱基对的顺序,而以目前的测序技术要在近期内完成这一目标还略显力不从心。     

Construction of a high-density,high-resolution genetic map and its integration with BAC-based physical map in channel catfish

Construction of genetic linkage map is essential for genetic and genomic studies. Recent advances in sequencing and genotyping technologies made it possible to generate high-density and high-resolution genetic linkage maps, especially for the organisms lacking extensive genomic resources. In the present work, we constructed a high-density and high-resolution genetic map for channel catfish with three large resource families genotyped using the catfish 250K single-nucleotide polymorphism (SNP) array. A total of 54,342 SNPs were placed on the linkage map, which to our knowledge had the highest marker density among aquaculture species. The estimated genetic size was 3,505.4 cM with a resolution of 0.22 cM for sex-averaged genetic map. The sex-specific linkage maps spanned a total of 4,495.1 cM in females and 2,593.7 cM in males, presenting a ratio of 1.7 : 1 between female and male in recombination fraction. After integration with the previously established physical map, over 87% of physical map contigs were anchored to the linkage groups that covered a physical length of 867 Mb, accounting for ∼90% of the catfish genome. The integrated map provides a valuable tool for validating and improving the catfish whole-genome assembly and facilitates fine-scale QTL mapping and positional cloning of genes responsible for economically important traits.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994     

A comparative map of macrochromosomes between chicken and Japanese quail based on orthologous genes

In order to develop a comparative map between chicken and quail, we identified orthologous gene markers based on chicken genomic sequences and localized them on the Japanese quail Kobe-NIBS linkage map, which had previously been constructed with amplified fragment length polymorphisms. After sequencing the intronic regions of 168 genes located on chicken chromosomes 1-8, polymorphisms among Kobe-NIBS quail family parents were detected in 51 genes. These orthologous markers were mapped on eight Japanese quail linkage groups (JQG), and they allowed the comparison of JQG to chicken macrochromosomes. The locations of the genes and their orders were quite similar between the two species except within a previously reported inversion on quail chromosome 2. Therefore, we propose that the respective quail linkage groups are macrochromosomes and designated as quail chromosomes CJA 1-8.     

Effects of detraining on motor unit potential area,muscle function and physical performance based on CNTF gene polymorphism

[Purpose]

The purpose of this study was to identify the effect of detraining on motor unit potential area (SMUP), muscular function and physical performance, according to CNTF gene polymorphism.

[Methods]

For this study, GG (normal homozygote, n = 8) group and GA + AA (mutation heterozygote and homozygote, n = 10) group were divided by CNTF gene polymorphism and both groups were performed detraining for 4 weeks. The data was analyzed by two-way repeated measures ANOVA for verifying the differences between two groups and interaction using SPSS (ver. 20.0) statistical program.

[Results]

The results were as follows. First, changes in body composition were measured but there was no significant interaction effect between time and group. Seconds, changes in SMUP were measured by SEMG. Interaction effect between time and group was found lateral vastus during isokinetic exercise of 180°/sec (p < .05). Third, changes in isokinetic muscle strength of 60°/sec and 180°/sec were measured but there was no significant interaction effect. Fourth, significant statistical differences were not showed changes of sports performance after detraining.

[Conclusion]

In conclusion, there were no significantly differences between GG and GA + AA group after detraining, therefore, further study will be considered a matter in various its interventions such as serum levels of CNTF and changes in receptors and muscle fiber types.     

A genetic map of Cottus gobio (Pisces, Teleostei) based on microsatellites can be linked to the physical map of Tetraodon nigroviridis

To initiate QTL studies in the nonmodel fish Cottus gobio we constructed a genetic map based on 171 microsatellite markers. The mapping panel consisted of F1 intercrosses between two divergent Cottus lineages from the River Rhine System. Basic local alignment search tool (BLAST) searches with the flanking sequences of the microsatellite markers yielded a significant (e < 10(-5)) hit with the Tetraodon nigroviridis genomic sequence for 45% of the Cottus loci. Remarkably, most of these hits were due to short highly conserved noncoding stretches. These have an average length of 40 bp and are on average 92% conserved. Comparison of the map locations between the two genomes revealed extensive conserved synteny, suggesting that the Tetraodon genomic sequence will serve as an excellent genomic reference for at least the Acanthopterygii, which include evolutionarily interesting fish groups such as guppies (Poecilia), cichlids (Tilapia) or Xiphophorus (Platy). The apparent high density of short conserved noncoding stretches in these fish genomes will highly facilitate the identification of genes that have been identified in QTL mapping strategies of evolutionary relevant traits.     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

A genetic linkage map of Picea abies Karst., based on RAPD markers,as a tool in population genetics

Norway spruce (Picea abies Karst.) is a most important species among European forest trees for both economical and ecological reasons. However, this species has suffered from a lack of information on the genetic side due to the scarcity of linkage data. In this study we have used a population of 72 megagametophytes from a single tree in a natural Italian stand to produce a genetic linkage map by means of RAPD markers. Ninety-six random decamers used as primers yielded 185 polymorphic loci showing Mendelian inheritance. Analysis of the segregation by multipoint analysis allowed us to define 17 major linkage groups covering a total distance of 3584 cM, with an average spacing between markers of 22 cM. Possible uses of a genetic linkage map with respect to population ecology and genetics are discussed.