外源TGF—β1基因在小鼠ES细胞中的表达及其对细胞分化的影响

本文利用逆转录病毒载体Ｄｏｌ,在其ＢａｍＨＩ酶切位点插入猪ＴＧＦ－β１ １．７ｋｂｃＫＮＡ,构建成表达质粒,并用磷酸钙沉淀法将该质粒ＤＮＡ转染到小鼠ＥＳ－５细胞,经Ｇ４１８筛选获得抗Ｇ４１８的ＥＳ－５细胞克隆（ＥＳ－Ｔ）,经ＲＮＡ点杂交,Ｎｏｒｔｈｅｒｎ印迹杂交证明有６个细胞克隆能表达外源猪ＴＧＦ－β１的ｍＲＮＡ,其中两个杂交信号较强的克隆进一步用Ｓｏｕｔｈｅｒｎ印迹杂交,也证明猪ＴＧＦ－μ     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ２总ＤＮＡ经ＰｓｔＩ部分酶切后分离２～１０ｋｂ的片段,插入质粒ｐＵＣ１９的ＰｓｔＩ位点,转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）,利用几丁质平板从约８０００个重组子中筛选到一个几丁酶基因阳性克隆（命名为ｐＣＨＴ１）。用１２种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长３０ｋｂ,其中各有一个ＫｐｎＩ,ＳａｃＩ和ＳｓｐＩ位点。把该克隆片段反向插入ｐＵＣ１９的ＰｓｔＩ位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Ｓｏｕｔｈｅｒｎ杂交证实了该片段来自于Ｂ．ｃｉｒｃｕｌａｎｓＣ２基因组,且以单拷贝形式存在,它不能与来自于其它７株几丁酶产生菌的总ＤＮＡ杂交。     

HBV—C基因探针的制备及应用

应用ＰＣＲ技术扩增出ＨＢＶ ＤＮＡ Ｃ基因片段并与ｐＡＴ１５３质粒重组,转化到Ｅ．ｃｏｌｉ ＲＲＩ中,经体内扩增,提纯,用光生物系标记,制备了Ｃ基因的重组质粒探针。该探检测灵敏度在Ｓｏｕｔｈｅｒｎ印迹中达１ｐｇ,在点印迹中为５ｐｇ。用此探针以Ｓｏｕｔｈｅｒｎ印迹方式配合ＰＣＲ技术检测乙肝病人血清中的ＨＢＶ ＤＮＡ,在５３例ＰＣＲ产物电泳检测阴性的样品中,Ｓｏｕｔｈｅｒｎ杂交又检出１８例阳性。     

含吸氢酶基因（hup）嵌合质粒的构建和吸氢酶活性在阴沟肠杆菌中的表达

以质粒ｐＲＫ４０４为载体亚克隆含大豆根瘤菌吸氢酶结构基因（ｈｕｐ）的片段,构建成嵌合质粒ｐＨＲ１１、ｐＨＲ４和ｐＨＲ１０。通过三亲本杂交将这些嵌合质粒导入无吸氢活性的Ｒｈｏｄｏｂａｃｔｅｒｓｐｈａｅｒｏｉｄｅｓ２４１菌株（Ｈｕｐ－）,均获得Ｈｕｐ＋的接合子。利用启动子检测质粒ｐＭＰ２２０证明,在ｈｕｐ对结构基因上游１．２ｋｂ内存在ｈｕｐ启动基因片段。以ｐＲＫ２０１３为助质粒可将ｐＨＲ１１导入Ｅｎｔｅｒｏｂａｃｔｅｒｃｌｏａｃａｅ和Ｋｌｅｂｓｉｅｌｌａｏｘｙｔｏｃａ等土壤固氮菌株。本文以接合子Ｅ．ｃｌｏａｃａｅＥＨ１１１３为例,通过对基因组ＤＮＡＳｏｕｔｈｅｒｎ杂交分析证明,嵌合质粒ｐＨＲ１１在ＥＨ１１１３中稳定贮存和复制。Ｈ２诱导接合子ＥＨ１１１３吸氢酶活性高表达,吸氢活性与放氢活性比值约为对照的两倍。当以延胡索酸为电子受体时,吸氢酶的吸氢作用支持菌株固氮酶活性的提高。     

应用cDNA文库快速构建法克隆高粱肌动蛋白基因

取生长一周的高粱嫩叶为材料,按照ｃＤＮＡ库快速构建法,用λｇｔ１０为载体成功地获得了含有２×１０＾０６人重组噬菌体的ｃＤＮＡ库。以水稻ＲＡｃｌ ｃＤＮＡ为探针,经噬菌体原位杂交筛选出两个阳性克隆片段,并对其中一个进行了Ｓｏｕｔｈｅｒｎ杂交鉴定。然后将重组体中含有的ｃＤＮＡ片段亚克隆至ｐＢｌｕｅｓｃｒｉｐｔ ＳＫ－载体上测序。     

大赖草及近缘物种原位杂交和Southern杂交的分析

用Ｔｈｉｎｏｐｙｒｕｍｂｅｓａｒａｂｉｃｕｍ（Ｓａｖｕｌ．＆Ｒａｙｓ）Ａ．Ｌｖｅ的基因组ＤＮＡ作探针,分别与大赖草Ｌｅｙｍｕｓｒａｃｅｍｏｓｕｓ（Ｌａｍ．）Ｔｚｖｅｌ．和脆轴偃麦草Ｔｈ．ｊｕｎｃｅｕｍ（Ｓａｖｕｌ．＆Ｒａｙｓ）Ａ．Ｌｖｅ的体细胞杂交,大赖草的１４对染色体均出现杂交信号,脆轴偃麦草只有７对染色体有杂交信号。在用重复ＤＮＡ序列ＰＨｖ６２作探针的原位杂交中,Ｔｈ．ｂｅｓａｒａｂｉｃｕｍ有４对染色体有杂交信号,大赖草有１３对染色体显示杂交信号,新麦草Ｐｓａｔｈｙｒｏｓｔａｃｈｙｓｊｕｎｃｅａ（Ｆｉｓｃｈ．）Ｎｅｖｓｋｉ和脆轴偃麦草无杂交信号。用ＰＨｖ６２作探针的Ｓｏｕｔｈｅｒｎ杂交结果与原位杂交相似。在被检测的１２个普通小麦大赖草异源染色体系中,除二体附加系中５Ｌｒ＃１和双二体附加系１Ｌｒ＃１＋５Ｌｒ＃１没有杂交信号外,其余的异染色体系与ＰＨｖ６２都有特异杂交信号。据此推测Ｔｈ．ｂｅｓａｒａｂｉｃｕｍ有可能参予了赖草属物种的形成过程。但是,大赖草的染色体组在进化过程中显然已发生过变异。     

HBV─C基因探针的制备及应用

应用ＰＣＲ技术扩增出ＨＢＶＤＮＡＣ基因片段并与ｐＡＴ１５３质粒重组,转化到Ｅ．ｃｏｌｉＲＲＩ中,经体内扩增,提纯,用光生物素标记,制备了Ｃ基因的重组质粒探针。该探针检测灵敏度在Ｓｏｕｔｈｅｒｎ印迹中达１ｐｇ,在点印迹中为５ｐｇ。用此探针以Ｓｏｕｔｈｅｒｎ印迹方式配合ＰＣＲ技术检测乙肝病人血清中的ＨＢＶＤＮＡ,在５３例ＰＣＲ产物电泳检测阴性的样品中,Ｓｏｕｔｈｅｒｎ杂交又检出１８例阳性。     

光照和温度对豌豆Lhcb2基因表达的影响

采用ＲＴ－ＰＣＲ技术,从豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）幼叶中克隆了１个约８００ｂｐ的Ｌｈｃｂ２ ｃＤＮＡ。以特异探针进行的Ｓｏｕｔｈｅｒｎ杂交结果表明,Ｌｈｃｂ２基因以单拷贝形成存在于豌豆基因组中。不同光照时间和温度对豌豆幼苗进行处理的ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ ｂｌｏｔｔｉｎｇ分析表明,Ｌｈｃｂ２基因转录水平上的表达受光照的控制,且明显表现出对光照时间的依赖性。光照０￣１．５ｈＬｈ     

酶母K.cicerisporus基因组中有启动子功能的DNA片段的克隆

利用以３′－氨基糖苷磷酸转移酶基因（ＡＰＨＩ）为报道基因的一套启动子探针质粒ｐＳＫ－ｋａｎ４０１、ｐＳＫ－ｋａｎ１１０５、ｐＳＫ－ｋａｎ１２３８,从酵母Ｋｌｙｖｅｒｏｍｙｃｅｓ ｃｉｃｅｒｉｓｐｏｒｕｓ基因组中克隆到８个有较强启动功能的ＤＮＡ片段,分析出３′端ＤＮＡ序列,并在酵母Ｋｌｕｙｖｅｒｏ８ｍｙｃｅｓｅ ｌａｃｔｉｓ中通过检测报告基因与３－磷酸甘油醛脱氢酶基因（ＡＰＤＨ）的ｍＲＮＡ表达量的比     

酵母K.cicerisporus基因组中有启动子功能的DNA片段的克隆

利用以３′－氨基糖苷磷酸转移酶基因（ＡＰＨＩ）为报告基因的一套启动子探针质粒ｐＳＫ－ｋａｎ４０１、ｐＳＫ－ｋａｎ１１０５、ｐＳＫ－ｋａｎ１２３８,从酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓｃｉｃｅｒｉｓｐｏｒｕｓ基因组中克隆到８个有较强启动功能的ＤＮＡ片段,分析出３′端ＤＮＡ序列,并在酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓｌａｃｔｉｓ中通过检测报告基因与３－磷酸甘油醛脱氢酶基因（ＧＡＰＤＨ）的ｍＲＮＡ表达量的比值,确定它们的功能强度,其中ｐＳＫ－ｋａｎ４０１－４１和ｐＳＫ－ｋａｎ１１０５－５１两个克隆的插入片段具有较强的启动子功能,并证明这两个片段在酵母Ｋ．ｃｉｃｅｒｉｓｐｏｒｕｓ中也有启动子功能。     

Localization of Nitrogen Fixation Gene in an Associated Nitrogen Fixing Bacteria Klebsiella planticola 19-1

A large plasmid in Klebsiella planticola 19-1 was identified from lysis of bacteria in an agarose gel and using a technique of electrical field overturn. The presence of nil HDK-like gene and localization of nif HDK-like gene in the large plasmid were confirmed by Southern hybridization with 32P-labelled nif HDK probe.     

nif HDK基因在W80-2上的初步定位

对几种质粒检测方法进行了比较 ,发现原位裂解法能比较满意地检测到巴西固氮螺菌 (Azospirillumbrasilense)的巨大质粒。利用改进后的原位裂解法能比较稳定地检测到W 80 2菌株中的巨大质粒。通过Southern blotting的方法将W 80 2菌的染色体及巨大质粒转到尼龙膜上 ,与用地高辛标记的含nifHDK基因的 pSA30质粒杂交 ,发现W80 2菌株的nifHDK基因定位在染色体上。     

nifV is contiguous to nifHDK in Frankia strain FaC1

The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain FaC1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pFAH 1. isolated from a cosmid library made from Frankia strain FaCl, resulted in the identification of a 7.2-kb Pst I fragment to which Klebsiella nif H, nif D and nif K probes hybridized. This nif -hybridizing fragment was subcloned and analyzed by restriction fragment mapping. Further subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Four of these ORFs have the potential to code for nif V-, nif H-, nif D- and nif K-like gene products and the two others are unidentified ORFs. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the nif V-like gene relative to the nif HDK cluster differs, A consensus nif -promoter-like sequence, found 5'to nif H. was not detected upstream of the nif V-like gene. Nine copies of a 7-bp direct repeat were found 5'to ORFA.     

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.     

Nucleotide sequence and expression of nifHD from Frankia strain EuIKl, a symbiont of Elaeagnus umbellata

Nucleotide sequences of nif HD from Frankia strain EuIKl were determined and analysed. The 3.2-kb and 5.5-kb Bam HI fragments of a genomic clone were previously shown to contain nif HD-like sequences and to be contiguous, based on hybridization experiments and partial sequencing data. Sequence analysis of about 3.0 kb from these fragments revealed two open reading frames and beginning of a third in the same orientation, each of which showed high degree of sequence homology with nif H, nif D and nif K, respectively. The deduced amino acid sequence of the nif H ORF, consisting of 861 bp, showed sequence similarity of about 91% with those of other Frankia strains, whereas that of nif D ORF, consisting of 1458 bp, showed about 85% similarity. Intergenic sequence between nif H and nif D was 45 bp and between nif D and nif K was 61 bp. The 5'of nif H revealed putative Shine-Dalgarno sequences; however, a sequence resembling a typical nif -promoter or NifA binding site was not found. Northern hybridization of RNA from the nodules showed that nif HDK were transcribed into a polycistronic mRNA in the symbiont of Elaeagnus umbellata .     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

华葵根瘤菌nifA基因的克隆和功能分析

华葵根瘤菌(Mesorhizobium huakuii即R.astragali)159的nifA基因的序列分析表明,该基因全长1227bp,编码分子量为44734D的Nif A蛋白。与其它NifA蛋白的序列比较发现,华葵根瘤菌NifA蛋白也存在保守的中间结构域和C末端DNA结合结构域,但其氨基端缺失。Tn5定点突变得到的突变体是Nif-表型。构建了nifA基因组成型表达的质粒,此质粒在大肠杆菌中对华葵根瘤菌nifHlacZ有激活作用。     

An open reading frame upstream from the nifH gene of Klebsiella pneumoniae

An open reading frame upstream from nifHDK operon of Klebsiella pneumoniae had been described. The orientation of this open reading frame is opposite to that of nifHDK and sequence homology was found between the open reading frame promoter and the promoter of nifHDK operon. A recombinant plasmid carrying the promoter region of the open reading frame fused to the beta-galactosidase gene was constructed. Strains of E.coli were transformed with the plasmid containing this open reading frame promoter-lacZ fusion or co-transformed with it and a plasmid carrying the nifA gene. An appreciable activity of beta-galactosidase was found in strains which received both plasmids, indicating that the promoter of the open reading frame can be activated by the product of nifA gene. Thus, the open reading frame found between nifHDK operon and nifJ behaves just like other nif genes of K.pneumoniae in requiring the product of nifA as the positive effector for expression.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.