棒杆菌启动子片段的酶谱分析及定位

用大肠杆菌启动子探测质粒pSDS I (Ap^r,Tc^s)从钝齿棒杆菌(Corynebacterium crenatum)6282染色体的HindⅢ酶切片段中,克隆到两个具有启动功能的DNA片段,分别将两个重组质粒命名为pSDB5和pSDB21。含有这两个质粒的菌株均可以在含300μg/ml Tc的平板上生长。通过酶切分析,pSDB5的插入片段为1.6kb,pSDB21的插入片段为3.4kb,并分别作出了它们的限制性酶切图谱。对pSDB21利用其EcoR Ⅰ和BglⅡ位点,通过亚克隆删除了与启动功能无关片段,构建成pSDB210和pSDB211,从而使启动子定位于约0.1kb的BglⅡ/HindⅢ外源片段上。分子杂交实验证明所得到的这两个具有启动功能的DNA片段确实来源于钝齿棒杆菌6282的染色体DNA。     

噬菌体T7溶菌酶基因的克隆

以Pbr322及含有噬菌体T7 RNA聚合酶强启动子φ10的Pbr322衍生物作为克隆载体,经限制内切酶AvaⅡ+HaeⅢ切割的一段噬菌体T7 DNA片段分别克隆到Pbr322及其衍生质粒的BamHⅠ位点上。插入的DNA片段为632碱基对。该片段包括噬菌体T7基因3．5和T7 RNA聚合酶弱启动子φ3．8的全部编码序列。已知噬菌体T7基因3．5的功能为产生噬菌体T7溶菌酶,利用氯仿处理检测带有重组质粒的转化子胞内溶菌酶活力。证明两种克隆株整体细胞中,均有溶菌酶存在。用10一20％SDS-聚丙烯酰胺凝肢电泳检查噬菌体T7基因3．5蛋白带,结果表明T7基因3．5在Pbr322衍生质粒中的表达优于Pbr322。     

地衣芽孢杆菌温和噬菌体的具有启动子功能片段的克隆

载体pTG402与地衣芽孢杆菌噬菌体B1p7经限制酶酶切并连接后转化大肠杆菌。从全部转化子中抽提质粒DNA,转化枯草杆菌后经苗落原位显色选到22个有启动子功能片段的克隆子。利用邻苯二酚双加氧酶对底物的显色反应,测定了15个克隆子的启动子活性,并绘制了启动子功能最强的重组质粒的酶切图谱。此外,还测定了两个克隆子在枯草杆菌各个生长期的表达情况,发现在对数生长后期表达量太增,认为识别这两个启动子的σ因子可能是σ³⁷。     

乳酸乳球菌启动子—信号肽活性片段在大肠杆菌中的克隆和表达

利用pGPB14为启动子-信号肽序列探测载体,在大肠杆菌中克隆乳酸乳球菌总DNA中具有启动子-信号肽功能的DNA片段。共得到42个具有红霉索、氨苄青霉素双重抗性的转化子。转化子的氨苄青霉素抗性水平在100～800μg/ml之间,β-内酰胺酶活力大多积累于周质空间,说明重组质粒中的外源插入片段确实具有发动转录和促进分泌的功能。Southern杂交结果表明,插入片段的确来源于乳酸乳球菌总DNA,其大小在80～400bp之间。DNA序列测定发现pSEQ8和pSEQ12中插入片段分属于pSEQ4和pSEQ17插入片段的一部分。在测序的4个DNA序列中都找到了启动子和起始密码子。其中2个含有典型的S.D.序列和非典型的信号肽序列,其他2个则没有发现典型的S.D.序列和信号肽序列。另外发现启动子上游序列对启动子转录起始的效率具有促进作用。     

血管内皮细胞生长因子启动子突变体萤光素酶报告基因载体的构建

目的：构建含有不同片段血管内皮细胞生长因子（VEGF）基因的启动子的萤光素酶报告基因载体,并定位缺氧诱导因子1a（HIF-1d）结合VEGF启动子的区域。方法：以VEGF全长启动子为模板,PCR扩增VEGF启动子的不同片段,插入萤光素酶报告基因载体pGL4-basic,确定所扩增的DNA序列后,将其与HIF-1a表达载体共转染293T细胞,定位HIF-1a结合VEGF启动子的区域。结果：测序结果表明扩增的不同片段的VEGF启动子序列正确;萤光素酶活性实验表明,-1128～-728bp片段是HIF-1a与VEGF相互作用激活VEGF启动子转录活性的区域。结论：克隆了VEGF启动子5’端缺失突变体报告基因表达载体,为调控VEGF表达的转录因子的筛选及功能研究打下了良好基础。     

谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析

摘要：【目的】获得谷氨酸棒杆菌10147基因组中具有启动子活性片段的结构序列,为构建表达载体做准备。【方法】利用启动子探测载体pAKC6,采用鸟枪法克隆经过限制性内切酶Sau3A I完全酶切的谷氨酸棒杆菌10147染色体DNA片段,并测定pAKC6上报告基因编码的氯霉素乙酰转移酶（CAT）的比活力,以筛选有启动子功能的片段。【结果】共克隆到30个具有启动子功能的片段。其中有三个插入片段起动的氯霉素乙酰转移酶比活力大于24 U/mg,插入片段F57起动的CAT比活力为32.50 U/mg;而插入有启动子Ptrc的阳性对照的CAT比活力为26.33 U/mg。【结论】获得三个DNA插入片段具有与已知启动子Ptrc相当的启动活性,这些片段可以用于构建谷氨酸棒杆菌表达载体。     

具有原核基因启动子活性的蓖麻蚕染色体DNA片段

利用大肠杆菌启动子克隆载体pHE5,研究了蓖麻蚕染色体的Hind Ⅲ酶解片段在大肠杆菌中作为四环素抗性基因启动子的功能作用。蓖麻蚕染色体的Hind Ⅲ片段与pHE5重组,在大约10~6个重组子中获得953株启动子重组体。测定了这些含有重组质粒的菌株抗四环素能力,其中有4株在平板上抗四环素水平超过225微克/毫升。对其中pARP201的插入片段进行了限制性图谱分析和启动子活性区域的缺失定位,证明了pARP-DB(由pARP201衍生而来)中的约0.5kb的外源插入片段具有完整的原核启动子功能。我们分析了这段DNA的部分核苷酸顺序,发现它与原核基因启动子极其相似。     

菠菜叶绿体DNA 4.1 kb的SalⅠ片段的克隆

利用pBR 322衍生质粒DNA为载体,将菠菜叶绿体DNA的SalⅠ限制性片段插入质粒的Sal Ⅰ位点,获得52个含重组质粒的菌落(Ap~rTc~5)。并对每个克隆的质粒进行限制性内切酶分析,通过Southern吸印与探针杂交,证明了重组质粒pSS104含有的插入DNA是菠菜叶绿体DNA 4.1kb的SalⅠ片段。迄今在该片段尚未定位任何已知的叶绿体基因,用大肠杆菌的活体系统也未能发现这段DNA的转录产物,本文对此进行了讨论。     

编码酵母丙氨酸tRNA两个半分子的DNA的克隆

用DNA合成仪合成寡聚脱氧核苷酸。用T4-DNA连接酶把这些寡聚脱氧核苷酸重组成双链DNA。这两个双链DNA的上游是T7-启动子,下游分别编码酵母丙氨酸tRNA的5′半分子(1-35位核苷酸)和3′半分子(35-76位核苷酸)。再把这两个双链DNA克隆到PUC 12质粒中。经点杂交筛选和DNA顺序测定证明克隆是成功的。     

扣囊拟内孢霉α-淀粉酶基因的克隆和在酿酒酵母中表达

以穿梭质粒pCN60为载体,大肠杆菌C600为受体构建了扣囊拟内孢霉(Endomycopsis fibuligera)G45 Sau 3A基因文库。从基因文库中提取重组质粒DNA并转化酿酒酵母(Saccharomyces cerevisiae)BJ1991,选出四个具有o-淀粉酶活性的转化子,琼脂糖凝胶电泳结果证实插入的DNA片段为9.0kb。对插入DNA片段亚克隆,确定。-淀粉酶基因位于PstI-Sall 3.9kb片段上,启动子位于PstI-EcoRI 的1.3kb片段上。用亚克隆PGK11.9kb片段置换。-淀粉酶启动子区,其转化子的e-淀粉酶活性有明显提高。     

Large-scale preparation of a DNA fragment containing the strong promoter A1 of the phage T7

A procedure has been developed to isolate DNA fragments on a large scale. A DNA fragment of 130 base-pairs containing the strong promoter A1 of the phage T7 was purified to homogeneity in amounts of 10 mg. The procedure includes the rapid purification of gram amounts of plasmid DNA, a new, simple method to separate small DNA fragments from the vector by a phenol/water partitioning system, and a liquid-liquid PEG-dextran partition chromatography for the final purification of the fragment. The fragment was cloned in two vector systems: The vector pDS1, to1+ (1), containing an efficient terminator downstream from the promoter integration site, gives high yields, 3-4 mg plasmid DNA per liter medium. In the plasmid pWH802 (2), which is not specially designed for the amplification of a strong promoter, the integration of the promoter was possible but the yield decreased by a factor of about 50. The stability of the inserts was tested in both systems. Monomeric inserts were stable in both plasmids, multimeric inserts up to a tetramer were only stable in pWH802. Only one orientation of the fragment was found.     

Isolation of tobacco DNA segments with plant promoter activity.

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.     

Stability fluctuations of plasmid-bearing cells: immobilization effects

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE genet. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Structure and function of conjugative pili: monoclonal antibodies as probes for structural variants of F pili.

The lac-tra operon fusion plasmid pTG801 contains the known F plasmid DNA transfer (tra) genes required by Escherichia coli to elaborate functional F pili (T. Grossman and P. M. Silverman, J. Bacteriol. 171:650-656, 1989). Here, we show that these pili are actually structural variants of normal F pili and that the F plasmid must contain additional genes that affect pilus structure and function. We confirmed a previous report that two monoclonal antibodies that recognize epitopes at and near the amino terminus of F pilin do not decorate the sides of normal F pili, as determined by immunogold electron microscopy. However, both antibodies laterally decorated pTG801 pili. The epitope for one of the antibodies has been shown to include the amino-terminal acetyl group of F pilin, which must therefore also be present on pTG801 pilin. Normal antibody staining was restored to pTG801 pili when cells contained, in addition to pTG801, the compatible plasmid pRS31, which must therefore include at least one gene affecting F-pilus structure. One candidate, traD, was excluded as the sole such gene, since traD+ derivatives of a pTG801 strain still elaborated pili that could be laterally decorated with antibody. Moreover, although traD alone restored RNA bacteriophage R17 infectivity to pTG801 cells, as expected, it did not mimic pRS31 in restoring to pTG801 pili other characteristics of normal F pili. We conclude that pRS31 contains as yet uncharacterized genes required for elaboration of structurally normal F pili. Finally, we identified vesicular material, especially abundant in cultures of pTG801 transformants, that stained heavily with the anti-F-pilin monoclonal antibodies. This material may reflect the inner membrane pool of F pilin.     

Gene 26 of bacteriophage T4 basal plate. I. Two expression products of the cloned gene

We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter. The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression. We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed. Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26. The 8-9 kDa protein was shown to be expressed from the end region of gene 26. Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame.