用原位分子杂交技术定位生长激素基因于5号染色体

本工作利用放射性标记的ｂＧＨ基因为探针，通过原位杂交定位牛生长激素基因于染色体５ｑ２２－２６内，该结果与以前的ｂＧＨ基因定位的结果不同，讨论了基因探针、基因定位方法等方面与定位准确性的关系。     

Localization of An Exogenous GH Gene on Chromosomes of Transgenic Pig by in situ Hybridizaiton

近些年来随着原位杂交技术的不断改进,该技术已广泛用于染色体的基因定位。非放射性标记探针的应用使基因定位变得更加简单易行,从而有可能对动物的转基因进行定位研究。本文首次采用胶体金标记药盒（Ａｎｔｉｄｉｇｏｘｉｇｅｎｉｎｇｏｌｄ）和银加强试剂（Ｓｉｌｖｅｒｅｎｈａｎｃｅｍｅｎｔｒｅａｇｅｎｔｓ）的非同位素原位杂交技术对转基因猪外源基因进行了定位研究。如Ｆｉｇ．１所示：表达质粒ｐＳＭＴＰＧＨ含有载体ｐＵＣ１９,羊启动子ＭＴ０１１和猪生长激素ＰＧＨ基因。选５头带有ｐＳＭＴＰＧＨ的转基因猪,分别制备含有染色体ＤＮＡ的杂交膜。用ＢｇｌＩＩ和ＳｍａＩ对ｐＳＭＴＰＧＨ进行完全酶切,收集０．９Ｋｂ片段作为探针,以ｄｉｇ１１ｄＵＴＰ进行标记。探针与ＤＮＡ杂交后,用Ａｎｔｉｄｉｇｏｘｉｇｅｎｉｎｇｏｌｄ和Ｓｉｌｖｅｒｅｎｈａｎｃｅｍｅｎｔｒｅａｇｅｎｔｓ进行显色反应。胰酶法Ｇ—显带后,用光学显微镜检查。选择分散相良好、显影银颗粒清楚的玻片进行摄影记录（Ｆｉｇ．２）。对染色体上的显影银颗粒进行统计分析,参照家猪的染色体标准带型,确定外源ＰＧＨ基因整合位点。Ｆｉｇ．３为４１０４号转基因猪染色体上的银颗粒分布情况。对５头     

牛生长激素基因的人工合成,重组克隆及高效表达

本文通过设计选择大肠杆菌高频利用密码子代替天然密码子,人工全合成３２个寡聚核苷酸片段;连接并克隆获得Ａ（２７８ｂｐ）、Ｂ（８８ｂｐ）、Ｃ（２２４ｂｐ）３个较大ＤＮＡ片段;经重组克隆、定位突变等获得阳性克隆;双向ＤＮＡ序列测定分析表明获得了两种人工全合成牛生长激素（ｂＧＨ）编码基因,即编码Ｎ－Ｍｅｔ－Ａｌａ－ｂＧＨ和Ｎ－Ｍｅｔ－Ｐｈｅ－ｂＧＨ的两个基因,而至今尚未见有人工全合成ｂＧＨ基因的报道。构建了在ＰＬｐｒｏｍｏｔｅｒ控制下含上述合成基因的表达质位ｐＢＬｂＧＨＥ７Ａ１和ｐＢＬｂＧＨＥ８,并在大肠杆菌中进行了温敏诱导表达。经ＳＤＳ－ＰＡＧＥ分析,表达产物占全菌蛋白的３７％以上。Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析和纯化产物的Ｎ端氨基酸序列测定结果都表明上述两种人工全合成ｂＧＨ基因在大肠杆菌中得到了高效表达,表达量为现今国际文献报道的高限水平。     

Studies on Pattern of Producing Transgenic Fish I.Production and Detection of All fish TRansgenic Common Carp With cMT sGH Gene

转基因鱼工作的开展被用于水产科学基础与应用研究的各个领域。应用方面的一项重要研究就是利用转生长激素基因来提高鱼的增长速度。需要解决两个问题：一是转基因鱼所使用的基因元件;二是外源基因的高整和率与高表达。已有许多报道认为：不同种动物的生长激素不一定相互促进生长。我们通过显微注射,将鲤鱼金属硫蛋白启动子（ｃＭＴ）与大马哈鱼生长激素基因（ｓＧＨ）的融合基因（ｃＭＴｓＧＨ）导入鲤鱼单细胞后期的早期胚胎,构建了全鱼转基因鲤鱼。通过斑点杂交、Ｓｏｕｔｈｅｒｎｂｌｏｔ结合ＰＣＲＳｏｕｔｈｅｒｎｂｌｏｔ,对外源ｓＧＨ的整和进行了精确的检测和分析。实验选取性成熟鲤鱼,收集卵子和精子,湿法受精获得受精卵。经过基因注射,孵化后的鱼苗放入水族箱。待鱼苗平游,提取总ＤＮＡ进行检测。以ＰｓｔＩ酶切质粒ｐｃＭＴｃＧＨ（Ｆｉｇ．４）分离３．４ＫｂｓＧＨ片段,用随机引物标记,作为杂交探针。同时,设计并合成ｓＧＨ基因的ＰＣＲ特异引物。Ｆｉｇ．１的斑点杂交结果与Ｆｉｇ．２的ＰＣＲＳｏｕｔｈｅｒｎ结果相比较（Ｔａｂｌｅ１）,说明斑点杂交存在着高的假阳性。而ＰＣＲＳｏｕｔｈｅｒｎ将ＰＣＲ的快速、方便与Ｓｏｕｔｈｅｒｎ的准确性相结合,排除了Ｐ     

人胎儿胸腺内bc1－2基因表达与分布

采用免疫酶组织化学与地高辛标记的单链ｃＤＮＡ探针原位杂交技术,研究了抗凋亡基因ｂｃｌ－２在人胎儿胸腺组织中的表达与分布。结果ｂｃｌ－２ｍＲＮＡ及其蛋白均优势定位于髓质区。提示胸腺中ｂｃｌ－２基因表达调控发生在转录水平;ｂｃｌ－２基因介导的细胞凋亡状态可能参与Ｔ淋巴细胞的成熟过程。     

气道上皮细胞的抗氧化损伤保护及微环境调控

本工作建立了臭氧对原代培养的兔支气管上皮细胞（ＢＥＣ）损伤模型,观察到血管活性肠肽（ＶＩＰ）、表皮生长因子（ＥＧＦ）、热应激等微环境理化因子可减轻细胞损伤,具有细胞保护作用,其保护机制与提高还原谷胱甘肽（ＧＳＨ）含量有关,并依赖于蛋白激酶的磷酸化调节及基因转录。ＢＥＣ细胞在基础情况下有ｂｃｌ－２基因的低水平表达。ＶＩＰ和ＥＧＦ可促进ｂｃｌ－２基因转录,增强ＢＥＣ的抗氧化损伤能力。ＥＧＦ或热应激促进     

小麦HMW-GS1D×5基因时间特异性表达的研究

利用小麦高分子量谷蛋白１Ｄ×５亚基探针,研究ＨＭＷ－ＧＳ１Ｄ×５基因表达的规律。结果表明,ＨＭＷ－ＧＳ１Ｄ×５基因从开花初期开始表达。这为从生理、营养、施肥等诸多方面人为地对ＨＭＷ谷蛋白基因的表达加以调控,使之在特定时期能够充分表达该基因从而改善小麦品质打下基础。     

香菇的电泳核型分析

采用钳位均匀电场（ＣＨＥＦ）凝胶电泳进行香菇的电泳核型分析,香菇染色体ＤＮＡ呈现８个谱带,结果与香菇联会丝复合物电镜研究一致。以酿酒酵母、裂殖酵母染色体作参照物,香菇染色体大致在７～２．２Ｍｂ之间,染色体组总计约３３Ｍｂ。电泳核型分析也可研究ｒＲＮＡ基因定位。     

中国棉铃虫核型多角体病毒几丁质酶基因的定位与克隆

以α３２ＰｄＡＴＰ标记含ＣｆＭＮＰＶ几丁质酶基因的重组质粒为探针,在６８℃条件下对棉铃虫单粒包埋核型多角体病毒（ＨａＳＮＰＶ）进行Ｓｏｕｔｈｅｒｎ杂交,将ＨａＳＮＰＶ的几丁质酶基因分别定位在ＢａｍＨＩＥ、ＢｇｌⅡＥ、ＥｃｏＲＩＧ、ＨｉｎｄⅢＦ、ＸｂａＩＨ、ＢａｍＨＩ＋ＨｉｎｄＩＩＭ和ＢａｍＨＩ＋ＸｂａＩＨ,并以ｐＴＺ１９Ｒ为载体获得了ＸｂａＩＨ片段克隆。     

肌糖原磷酸化酶a,b亚基的杂交

本文用标记在肌糖原磷酸化酶ａ（ＧＰＡ）亚基上的荧光探针ＩＡＥＤＡＮＳ与标记在肌糖原磷酸化酶ｂ（ＧＰＢ）亚基上的ＩＡＦ之间无辐射能量转移;高压力下亚基交换动力学和外源荧光探针荧光偏振等三种方法证实了肌糖原磷酸化酶ａ、ｂ的亚基可相互交换,形成磷酸化酶ａ、ｂ的杂交形式。     

Chromosomal localization of the ovine beta-casein gene by non-isotopic in situ hybridization and R-banding.

The ovine beta-casein gene (CNS2) has been mapped to a specific chromosome band using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The probe pTZ-E4 was a fragment of the ovine beta-casein gene inserted in the plasmid pTZ18R and labeled with biotin-11-dUTP. It hybridized to band q32 of ovine chromosome 4. The discrepancy between this result and the previous localization of this gene on cattle chromosome 6 may be explained by the very great similarity of the banding patterns of ovine and bovine chromosomes 4 and 6.     

Localization of the ß-subunit of follicle stimulating hormone in cattle and sheep by in situ hybridization

The locus for the beta-subunit of the follicle stimulating hormone gene (FSHB) has been determined in both cattle and sheep by in situ hybridization of a bovine and an ovine cDNA probe, respectively, to metaphase chromosomes. Our results show that the FSHB locus is on cattle chromosome 15 in the region of bands q24-qter and in sheep on the cytogenetically homologous chromosome 15, also in the region q24-qter. The mapping of the FSHB gene in cattle together with the location of other genes (CAT, HBB and PTH) previously found to be syntenic in cattle and on human chromosome 11p, defines an evolutionarily conserved synteny. The localization of the FSHB gene to a cytogenetically homologous region in cattle and sheep is consistent with the hypothesis of extensively conserved chromosome structure in these two species.     

用原位杂交法定位猪乳铁蛋白基因于染色体2q^12

本研究以非放射性标记的猪乳铁蛋白（Ｐｏｒｃｉｎｅ Ｌｉａｃｔｏｆｅｒｒｉｎ简称ＰＬＦ）ｃＤＮＡ为探针,通过染色体原位杂交法,对ＰＬＦ基因了染色体进行了染色体定位。实验中采用金胶抗体技术并结合使用银增强系统,提高了方法的灵敏度。利用染色体的组型分析,对杂交点的分布进行了统计学分析。５２％（２６／５０）的分裂相在第２号染色体具银粒分布,实验结果表明：ＰＬＦ基因定位于猪２号染色体２ｑ＾１２区域。     

Assignment of the growth hormone gene locus to 19q26-qter in cattle and to 11q25-qter in sheep by in situ hybridization

The growth hormone gene locus (GH) of cattle and sheep was mapped to a chromosomal region in each species by using in situ hybridization. The probe employed was an 830-bp cDNA sequence from the ovine growth hormone gene. Based on QFQ chromosome preparations, our results show that the GH locus is on cattle chromosome 19 in the region of bands q26-qter and in sheep on chromosome region 11q25-qter. The GH assignments together with previous localizations of type I cytokeratin genes (KRTA) and one homeobox (HOX2) gene in cattle and one type I cytokeratin gene (KRTA) in sheep identify a strongly conserved chromosomal segment on human chromosome 17, bovine chromosome 19, and sheep chromosome 11.     

Assignment of the HOX2 and HOX3 gene clusters to the bovine chromosome regions 19q17-qter and 5q14-23

The homeobox 2 (HOX2) and homeobox 3 (HOX3) clusters have been chromosomally assigned in cattle by in situ hybridization. The probes employed were a murine probe for the mapping of HOX2 to 19q17-qter and human probes for the mapping of HOX3 to 5q14-q23. These assignments confirm the chromosomal assignment of two syntenic groups, consisting of loci located on human chromosome 12 (bovine chromosome 5) and the long arm of human chromosome 17 (bovine chromosome 19).     

Gene mapping on maize pachytene chromosomes by in situ hybridization

A sensitive in situ hybridization technique which was effective for mapping genes of low copy number on human metaphase chromosomes was used for gene mapping on maize pachytene chromosomes. A cloned genomic EcoR1 fragment of 10.8 kb, containing most or all of the sequence encoding the Waxy locus mRNA, was used as the probe. Southern DNA blotting analyses performed by Shure et al. (1983) indicated that the Waxy locus was a single copy sequence. In our in situ hybridization experiment, the probe hybridized to a specific site on chromosome 9. Labeling at this site was detected in 48.6% of 154 randomly selected copies of chromosome 9. To test the sensitivity of the method, subclones of the fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and 0.8 kb were used for in situ hybridizations. Labeling efficiency for each probe was determined. The data showed that a single copy probe of 1.9 kb could be detected at the correct position in 18% of 183 randomly selected number 9 chromosomes.     

肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中克隆的研究

在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.     

Purification and characterization of recombinant bovine growth hormone produced in Escherichia coli

Using the pUBJ10 plasmid containing the modified bovine growth hormone (bGH) cDNA, large production has been attempted in E. coli BL21 strain. The bGH was highly expressed upto the level of 35% of total cell proteins by IPTG induction and temperature shift to 40°C. The recombinant bGH (rbGH) was isolated from inclusion bodies by solubilization in 10 M urea and followed by DEAE-TOYOPEARL 650C ion exchange and Sephadex G-100 column chromatography. The pUBJ10-derived bGH was eluted at 25.28 min similar to the standard bGH (at 25.18 min) by reverse-phase HPLC. The analysis of N-terminal amino acid showed that the mature bGH has glutamic acid as a first amino acid in agreement with DNA sequencing data. The biological activity was indirectly measured by radioreceptor assay and compared with a pituitary-derived bGH.     

Comparative FISH-mapping of the survival of motor neuron gene (SMN) in domestic bovids

A comparative fluorescence in situ mapping of the SMN gene was performed on R-banded chromosome preparations of cattle (Bos taurus, BTA, 2n = 60), river buffalo (Bubalus bubalis, BBU, 2n = 50), sheep (Ovis aries, OAR, 2n = 54) and goat (Capra hircus, CHI, 2n = 60), as well as on those of a calf from Piedmont breed affected by arthrogryposis. SMN was located on BTA20q13.1, OAR16q13.1, CHI20q13.1 and BBU19q13. These chromosomes and chromosome bands are believed to be homeologous, confirming the high degree of chromosome homeologies among bovids. The position of SMN was refined in cattle, compared to the two previous localizations, while it is a new gene assignment in the other three bovids. A comparative fiber-FISH performed on extended chromatin of both normal cattle and calf affected by arthrogryposis revealed more extended FITC signals in the calf, compared to the normal cattle (control), suggesting a possible duplication of the SMN gene in the calf affected by arthrogryposis. .     

Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.