Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Derepression of Phosphomannose Isomerase by Regulator Gene Mutations Involved in Capsular Polysaccharide Synthesis in Escherichia coli K-12

A regulator gene mutation (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation (capS, which maps separately from capR) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR(+) to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR(+)man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.     

Identification of the Escherichia coli lytB Gene, Which Is Involved in Penicillin Tolerance and Control of the Stringent Response

The Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response, was identified as a previously described open reading frame designated orf316 located in the ileS-lsp operon (0.4 min on the linkage map).     

PSA基因启动子中一个与雄激素调节相关的序列

人前列腺特异抗原（PSA）基因的表达受雄激素的调节,其雄激素应答元件（ARE）位于－170附近.为了确定雄激素对该基因的诱导作用是否受ARE上游序列的影响,把PSA启动子区的不同长度的天然的和变异的DNA片段分别与报告基因CAT相连,构建了不同的pBLCAT3-PSA质粒.用它们转染人前列腺肿瘤细胞PC-3.结果表明15 bp的RF15序列（－340～－326）的缺失和变异可显著降低雄激素的诱导作用.区带转移测定表明人前列腺肿瘤细胞LNcap和PC-3中的某些核内调节蛋白可与RF15结合,而且其结合能力受Zn²⁺的影响.这些结果表明RF15可能是PSA启动子中的一个新的附属调节元件.与之结合的调节蛋白可能是通过与雄激素受体的相互作用促进雄激素对PSA基因的诱导作用.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

牛凝乳酶原基因在大肠杆菌中表达调控的研究

Shine-Dalgarno序列与起始密码子之问的距离与组成对凝乳酶原基因表达有明显的影响,可导致其表达水平有15倍之差。SD序列至ATG之间为15bp不利于表达,表达质粒中sD-ATG在7-11bp之间都有可能获得高效表达;但决定因素不是简单的长度,而是RBS附近可能的二级结构即△G的大小、SD序列及ATG中参与配对的碱基数目。将pTLC23中凝乳酶原cDNA3＇端端非翻译区插入终止密码子TGA与转录终止子rrnBT₁T₂之间适当位置可提高凝乳酶原基因的表达,这可能是因为这段序列能形成由53个碱基对和8个碱基组成的稳定的mRNA二级结构,起到转录终止子的作用,而一般认为串联终止子对终止转录更为有效。     

The RpoS-Mediated Regulation of Isocitrate Dehydrogenase Gene Expression in Escherichia coli

The Escherichia coli NADP⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42), encoded by an icd gene, is a tricarboxylic acid (TCA) cycle enzyme responsible for the oxidative decarboxylation of isocitrate to α-ketoglutarate. In order to examine how the icd gene expression is regulated, an icd-lacZ reporter fusion was constructed. While the icd gene was induced in exponential growth phase, it was repressed in stationary growth phase. Genetic inactivation of an rpoS gene, whose product is an alternative sigma factor, induced the icd gene expression approximately 4.8 times more in the stationary phase and the IDH enzyme activity in the rpoS mutant was 3.2 times higher than that in the wild type, indicating that the RpoS factor acts as a negative regulator of the icd gene expression in the stationary phase.     

Chromosomal Location of a Gene Involved in Potassium Ion Uptake in Escherichia coli B

A chromosomal lesion responsible for defective potassium ion uptake in Escherichia coli B has been mapped by use of standard interrupted-mating crosses. The mutation, kac-1, is in strain RD-2, which is deficient in K⁺ intake, exchanges cell K⁺ for extracellular isotope at a reduced rate, and has an abnormality of phosphorus metabolism associated with its potassium deficiency. This report places kac-1 at about 4 min clockwise from pro, close to gal. The locus of kac-1 is distinctly different from the potassium retention mutant in strain B-207, the only other potassium accumulation mutant mapped in E. coli so far. In this study, two other potassium accumulation mutations, kac-2 and kac-3, whose particular type of accumulation defects have not yet been determined, were mapped. These mutations are in the same region of the chromosome as kac-1.     

The Listeria monocytogenes Homolog of the Escherichia coli era Gene Is Involved in Adhesion to Inert Surfaces

Two transposon-insertional mutants of Listeria monocytogenes showing smaller viable surface-attached cell populations after disinfection with N,N-didecyl-N,N-dimethylammonium chloride were identified. In both mutants, transposon Tn917-lac was found to be inserted into the same gene, lmo1462, which is homologous to the essential Escherichia coli era gene. Both L. monocytogenes lmo1462-disrupted mutants displayed lower growth rates, as was also shown for several E. coli era mutants, and the lmo1462 gene was able to complement the growth defect of an E. coli era mutant. We showed that the disruption of lmo1462 decreased the ability of L. monocytogenes cells to adhere to stainless steel. Our results suggest that this era-like gene is involved in adhesion and contributes to the presence of L. monocytogenes on surfaces.     

胆固醇氧化酶基因的克隆及在E.coli中的表达

根据NCBI中报道的BrevibacteriumsterolicumATCC21387胆固醇氧化酶基因序列,采用PCR方法以Brevibacteriumsp.DGCDC-82的基因组为模板,扩增得到了编码胆固醇氧化酶的基因,该基因与来源于BrevibacteriumsterolicumATCC21387的胆固醇氧化酶基因(choB)同源性为98%。将得到的基因定向克隆到pET28a载体中,转化至含有编码argU和proL基因的大肠杆菌BL21-CodonPlus(DE3)-RP中表达。经过IPTG诱导后,经SDS-PAGE检测在约55kD处有一蛋白表达条带,目的蛋白表达量约占总蛋白的16%,经测定酶活为340U/L。     

ubiI,a New Gene in Escherichia coli Coenzyme Q Biosynthesis,Is Involved in Aerobic C5-hydroxylation

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.     

Site-specific mutagenesis of cysteine 148 to serine in the lac permease of Escherichia coli

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the lac Y gene of Escherichia coli such that Cys148 in the lac permease is converted to Ser. A mutagenesis protocol is used that significantly improves the efficiency of mutant recovery by in vitro methylation of closed-circular heteroduplex DNA containing the mutation, followed by nicking with HindIII in the presence of ethidium bromide and heat denaturation prior to transfection. In contrast to Gly148 permease (Trumble, W.R., Viitanen, P.V., Sarkar, H.K., Poonian, M.S., and Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860-867), permease containing Ser at position 148 catalyzes active lactose transport at a rate comparable to wild-type permease. Like Gly148 permease, however, transport activity is less sensitive to inactivation by N-ethylmaleimide, and galactosyl-1-thio-beta-D-galactopyranoside affords no protection against inactivation. The observations provide strong support for the contention that Cys148 is obligatory for substrate protection against inactivation by sulfhydryl reagents, but does not play an essential role in lactose:H+ symport.