毛萼田菁茎瘤菌（Azorhizobium caulinodans）吸氢活性缺陷株的筛…

利用转座子Ｔｎ５随机插入突变法,从毛萼田菁茎瘤菌中筛选得到两株吸氢活性缺陷突变株Ｒ－４９和Ｒ－３０９,其固氮酶活性也大大降低,约为野生型固氮酶活性的６－８％。以带有Ｂｒａｄｙｒｈｉｚｏｂｉｕｍ ｊａｐｏｎｉｃｕｍ的５．９ｋｂ的ｈｕｐ基因的质粒ＰＨＲ１１为探针与Ａ．ｃｏｕｌｉｎｏｄａｍｓＯＲＳ５７１和Ｂ．ｊａｐｏｎｉｃｕｍ１２２ＤＥＳ（Ｈｕｐ）的总ＤＮＡ进行分子杂交,放射自显影表明,Ａ．ｃｏｕｌｉｎ     

利用Tn5定位诱变筛选紫云英根瘤菌Exo^—变种

利用Ｔｎ５定位诱变方法,对质粒ｐＪＢＢ５进行Ｔｎ５插入诱变,得到１０个Ｔｎ５在５９ｋｂＢ５外源片段上有不同插入位点的质粒ＴＮ１１,ＴＮ１１２,ＴＮ２２,ＴＮ２３,ＴＮ３１,ＴＮ４１,ＴＮ９１,ＴＮ１０１,ＴＮ１３１,ＴＮ１４１。将Ｔｎ１１等分别转移到已经含有不相容质粒ｐＰＨ１ＪＩ的紫云英根瘤菌１０７菌株中,使之发生同源变换。通过抗性选择及表型鉴别,筛选到３株菌落表型干燥（Ｍｕｃ－）的酸性胞外多糖（ＥＰＳ）合成缺陷菌株（Ｅｘｏ－）１０７（ＴＮ２２）,１０７（ＴＮ１０１）,１０７（ＴＮ１３１）。Ｓｏｕｔｈｅｒｎ杂交分析证明这３株变种的Ｔｎ５插入确实是同源交换而不是转座产生,表明经过适当改良的Ｔｎ５定位诱变法可以应用于紫云英根瘤菌Ｅｘｏ－变种的筛选。     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

p35Nck5a基因重复性打靶载体的构建和ES细胞基因打靶研究

为了在小鼠胚胎于细胞（ＥＳ）中引起神经细胞ｃｄｃ２类激酶调节亚基ｐ３５Ｎｃｋ５ａ基因的定点 重复,采用常规的分子克隆技术,构建得到长约１２．２ｋｂ的基因重复性打靶载体ｐＧＤＴＶ。用电 穿孔法将线性化的ｐＧＤＴＶ载体转入ＥＳ细胞,经过Ｇ４１８和ＧＡＮＣ分组药物选择,获得２４５个 双药物抗性的细胞克隆,细胞存活率为６．２２ × １０－５。经ＰＣＲ和基因组Ｓｏｕｔｈｅｒｎ杂交鉴定,２个 ＥＳ细胞克隆发生了ｐ３５Ｎｃｋ５ａ基因的重复,同源重组率为５．０８×１０－７、负向选择系统的应用使 同源重组事件的富集效率提高了７倍。为建立Ａｌｚｈｅｉｍｅｒ病的转基因小鼠模型打下了基础。     

紫云英根瘤菌107菌株exo基因簇非连锁突变位点的克隆

用鸟枪法从３株紫云英根瘤菌１０７菌株的胞外多糖合成缺陷变种ＮＡ－０５、ＮＡ－０７和ＮＡ－０８中克隆获得含有１０７菌株ｅｘｏ基因及Ｔｎ５的ｅｘｏ：Ｔｎ５片段。以ｐＲＫ４１５为载体构建１０７菌株ＥｃｏＲＩ酶切后ＤＮＡ片段的部分库,用ｅｘｏ：Ｔｎ５做探针原位杂交得到一个阳性克隆。该克隆的外源片段４．２ｋｂ能恢复３个变种的多糖表型及结瘤固氮能力。酶切分析和Ｓｏｕｔｈｅｒｎ杂交表明,３株变种中Ｔｎ５插入位点     

α－淀粉酶基因表达中启动子上游区EcoRI－BclI片段的生理功能

本文从ｐＡｍｙ４１和ｐＮＬ２０１出发,构建了一系列α－淀粉酶基因上游区ＥｃｏＲＩ－ＢｃｌＩ片段缺失或部分缺失的质粒,并构建了含有ｐＡｍｙ４１系列单质粒ｐＡｍｙ４１系列、ｐＮＬ２０１系列双质粒工程菌。通过对这些工程菌α－淀粉酶基因表达水平的分析显示,Ｂ．Ｌｉｃｈｅｎｉｆｏｒｍｉｓα－淀粉酶基因上游区ＥｃｏＲＩ－ＢｃｌＩ片段在α－淀粉酶基因表达中行使负的调控功能。     

鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探

将含有鸡传染性支气管炎病毒 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 的重组转移质粒ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３ Ｓ１ ． Ｈｏｌｔｅ 和ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１ ． Ｈｏｌｔｅ 分别与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ,ｇａｌ＋ ） 共转染草地夜蛾（ Ｓｆ９） 细胞,经空斑纯化得到重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 和 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。将重组毒株分别感染 Ｔｎ５ Ｂ１ 细胞,并进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗｅｓｔｅｒｎｂｌｏｔ 检测。结果表明, Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 蛋白, Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 凝胶薄层色谱分析结果显示,感染病毒后７２ ｈ Ｓ１ 蛋白的表达量占细胞内总蛋白量的３５ ．８ ％ ,而 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 感染的细胞内检测不出 Ｓ１ 蛋白。经分析认为这一差异主要来自 Ｓ１ 基因翻译起始位点及其附近的周围环境。     

α—淀粉酶基因表达中启动子上游区EcoRI—BclI片段的生理功能

本文从ｐＡｍｙ４１和ｐＮＬ２０１出发,构建一系列α－淀粉酶基因上游区ＥｃｏＲｉ－ＢｃｌＩＩ片段的人或部分缺失的质粒,并构建了ｐＡｍｙ４１系列质粒ｐＡｍｙ４１系列、ｐＮＬ２０１系列双质粒工程菌,通过对这些工和力α－淀粉酶基因表达水平的分析显示,Ｂ．ｌｉｃｈｅｎｉｆｏｒｍｉｓ α－淀粉酶基因上游区ＥｃｏＲＩ－Ｂｃｌｌ片段在α－淀粉酶基因表达中行使负的调控功能。     

携带p53基因的重组腺病毒的构建及对肿瘤细胞抑制的研究

１　材料与方法１１　质粒ｐＣＭＶｐ５３ＢＡＭ由美国约翰·霍普金斯肿瘤中心ＢｅｒｔＶｏｇｅｌｓｔｅｉｎ教授赠送［５］;ｐＲＳｅｔＡ购于Ｉｎｖｉｔｒｏｇｅｎ公司;ｐＣＡ１３和ｐＢＨＧ１１为ＭｉｃｒｏｂｉｘＢｉｏｓｙｓｔｅｍＩｎｃ．产品。ｐＢＨＧ１１包含腺病毒Ａｄ５基因组３６ｋｂ中的约３４ｋｂ序列,但Ａｄ５Ｅ１区１８８ｂｐ至１３３９ｂｐ缺失,代之以氨苄青霉素抗性基因和复制起始位点。ｐＢＨＧ１１缺少包装信号φ（２２ｂｐ至３４２ｂｐ）和Ａｄ５Ｅ３区（２７８６５ｂｐ至３０９９５ｂｐ序列）。ｐＣＡ１３…     

绿色木霉纤维素酶CBHⅡ基因的结构研究

实验经限制性酶切和双向Ｓｏｕｔｈｅｒｎ杂交将重组质粒ｐＣＢＨＩＩ－１４的１４ｋｂ外源片段上的绿色木霉纤维素酶ＣＢＨＩＩ基因定位于４．４ｋｂ的ＥｃｏＲＩ片段上,将该片段克隆于质粒ｐＵＣ１９,获得重组质粒ｐＣＢＨＩＩ。通过限制性分析构建了ｐＣＢＨＩＩ上４．４ｋｂＥｃｏＲＩ片段的物理图谱。在此基础上用质粒制作该片段的亚克隆,采用Ｓａｎｇｅｒ双脱氧链终止法测定了含绿色木霉纤维素酶ＣＢＨＩＩ基因的４０７４ｂｐ的ＤＮＡ序列,由ＧＴ－ＡＧ规则和ｃＤＮＡ序列推知该结构基因由４个外显子和３个内含子组成,总长１６１３ｂｐ,５′端存在与一般真核基因类似的两个特征性调控序列,但３′端不存在真核基因通常具有的特征序列而存在功能未明的短重复序列和嘧啶富集区。并对纤维素酶基因间的同源性和可能的功能作了初步的探讨。     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens.

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutant was not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome. The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A. tumefaciens.     

荧光假单胞菌Tn5转座诱变及对青枯病生防特性

目的：利用Tn5转座诱变荧光假单胞菌PF20001,研究所获得的突变株对青枯病的生防效果。方法：利用三亲本杂交方式,将带有转座子Tn5的Tn5-102（含luxAB）的质粒pTR102成功地转入PF20001,利用平板相互拮抗法分析突变株对青枯病致病菌的拮抗作用。结果：通过诱导Tn5转座,得到荧光假单胞菌PF20001的Tn5插入突变库。经平板相互拮抗实验发现,菌株PF20001-lux-48拮抗圈明显大于野生型（半径达0.35cm）。用Tn5-lux特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到300bp的扩增产物,证实该菌株基因组中有Tn5插入。结论：Tn5的插入使菌株PF20001对青枯病生物防治能力增强。     

Unusual genetic phenomena associated with Tn5 mutagenesis in Alcaligenes eutrophus strain H1

Tn5 was introduced into Alcaligenes eutrophus strain H1 by a suicide vector pSUP1011. Physical characterization of mutants obtained after Tn5 mutagenesis revealed a relatively high frequency of plasmid curing, or deletion of a 50 kb plasmid DNA segment. Results of Southern hybridization and chromosomal walking indicate that the same continuous stretch of plasmid DNA (designated as D region of plasmid) is deleted in four independent isolates. Moreover, the same deletion of plasmid DNA is also observed in a mitomycin C-generated mutant strain H1-4.Journal Paper No. J-12095 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2607, supported in part by a grant from the Iowa High Technology Council     

质粒RSF1010寄主范围特性的遗传学分析

采用Tn5插入诱变、限制性核酸内切酶作图以及DNA转化等方法,对广泛寄主范围型质粒SF 1010的衍生体－pKT 2 40进行研究。证实质粒的寄主围决定于它在遗传背景不同的寄主中复制并保存自身的能力,而repA,rcpB和repC基因为该质拉复制所必需。     

Cloning and expression of the rfe–rff gene cluster of Escherichia coli

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.     

Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

Molecular cloning of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum.

A pLAFR1 cosmid genomic library of wild-type Bradyrhizobium japonicum J1063 was constructed. A cosmid clone designated pBjJ4, containing a 26-kilobase (kb) DNA insert, was identified as being able to confer the ability to convert alpha-naphthaleneacetamide acid on B. japonicum J1B7 Rifr, which cannot perform this conversion. The gene coding for the enzyme that converts alpha-naphthaleneacetamide to alpha-naphthaleneacetic acid was localized in the 3.5-kb region of pBjJ4 by recloning in plasmid pSUP202. The gene coding for the enzyme was also mapped by Tn5 insertion mutagenesis to a region of ca. 2.3 kb. When the gene was placed behind the lacZ promoter and used to transform Escherichia coli, a high level of expression of indole-3-acetamide hydrolase activity was found. Since there have been no reports of this activity in E. coli, we have thus confirmed that the gene cloned here is a structural gene for indole-3-acetamide hydrolase and have designated it as the bam (Bradyrhizobium amidehydrolase) gene. Southern hybridization with the central region of the bam gene indicated that a high degree of similarity exists among the bam gene, the iaaH gene from Pseudomonas savastonoi, and the tms-2 gene from Agrobacterium tumefaciens. The result suggests that there is a common origin for the gene that encodes the enzyme that catalyzes the biosynthesis of indoleacetic acid.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.