Molecular and functional analysis of the ribosomal L11 and S12 protein genes ( rplK and rpsL ) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants. Received: 12 May 1997 / Accepted: 22 July 1997     

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m²) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m²) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.     

结核分枝杆菌三种耐药基因的检测方法

建立3种结核分枝杆菌耐药基因的检测方法,探讨耐药基因突变与耐药性的关系。将58株临床分离株均做聚合酶链反应-单链构象多态性分析(PCR—SSCP)和传统药物敏感试验。结果表明,结核分枝杆菌耐药基因突变与耐药水平有密切联系,绝大多数结核分枝杆菌耐药基因突变发生在高耐药株,少部分在低耐药株发生基因突变。     

检测纳米材料毒性的若干实验方法

纳米材料进入生命体和环境以后可能带来的生物安全问题需要定量的测定。现有的检测纳米材料生物安全性的方法大致可分为体内和体外实验两种,但是还没有证据表明既有的某个方法单独能作为一种检测所有纳米材料毒性的通用方法,也没有证据表明这些方法合在一起就能全面评估纳米材料的生物安全性。本文在总结既有的若干方法的同时,报道了一种基于rpsL基因的复制保真性来定量检测纳米材料毒性的方法。纳米材料对rpsL基因的体内体外复制过程保真性的影响均可方便地定量测定。     

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

A 5′-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches ～330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment.     

多耐药临床分离株结核分支杆菌耐药基因rpsL 的克隆表达及功能的初步研究

制备多耐药临床分离株结核分支杆菌基因组DNA,PCR扩增其耐药基因rpsL基因和rrS基因,同时进行测序分析,结果显示该株多耐药临床分离株结核分支杆菌的rpsL基因的93、94bp处的碱基发生了突变,碱基C、C突变为T、T,使得相对应的编码氨基酸由脯氨酸(Pro)突变为亮氨酸(Leu);而rrS基因未发生核苷酸的插入、缺失或取代;进一步构建该株多耐药临床分离株结核分支杆菌耐药基因rpsL高效原核表达重组我体pGEX-λ T／rpsL,所构建的重组质粒pGEX-λ T／rpsL在大肠杆菌中高效表达了38kD的融合蛋白．结果提示,该株多耐药临床分离株结核分支杆菌对链霉素耐药仅仅是由于rpsL基因的个别碱基突变,是单基因型的,在国内外属首次研究发现．