kasT gene of Streptomyces kasugaensis M338-M1 encodes a DNA-binding protein which binds to intergenic region of kasU-kasJ in the kasugamycin biosynthesis gene cluster

We previously reported that a 4.2 kb SacI-EcoRI DNA region from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM transporter genes (kasKLM). As an extension of that study, a 3.7 kb Psti-SacI DNA region, located at 1.5 approximately 5.2 kb upstream of kasK, was cloned and sequenced, revealing three complete open reading frames, designated kasT, kasU and kasJ. The kasJ gene encodes a protein (KasJ) with a conserved dinucleotide (FAD)-binding motif Homology search for KasJ showed its similarity to NADH: N-amidino-scyllo-inosamine oxidoreductase (StsB) which is involved in biosynthesis of the streptidine moiety of streptomycin (SM) in S. griseus. The kasT gene encodes a DNA-binding protein (KasT), including a helix-turn-helix motif near the center of the sequence. This protein is similar in structure to a pathway-specific activator protein (StrR) that plays a role in regulating the SM biosynthesis gene cluster of S. griseus. A fusion protein (Trx-KasT) clearly showed DNA binding activity with the intergenic region of kasU-kasJ, suggesting that KasT is a pathway-specific regulator of the KSM biosynthesis gene cluster.     

稀疏链霉菌基因文库柯斯质粒对异源宿主致死现象的研究

目的对稀疏链霉菌基因文库中编号为13F2:pJTU2463的柯斯质粒对异源宿主的致死现象进行探索性研究。方法通过构建若干克隆及相应的接合转移实验,限制性内切酶酶切图谱分析,质粒测序及序列分析,从若干角度探讨这一质粒发挥致死作用的可能性及途径。结果编号为13F2:pJTU2463的柯斯质粒中包含致死基因的核酸片段被缩小至亚克隆lth24;将lth24测序并分析其可能编码的蛋白,检索相关文献并进行生物信息学分析,推测三组基因可能与致死现象具有关联。这三组基因可能分别负责编码蛋白酶,双组分激酶系统中的双组分感应激酶和双组分调节因子,ABC转运系统中的ABC转运蛋白和ATP结合蛋白。结论虽然没有完全阐明致死现象的具体机制,但目前的研究已经可以推断该致死现象是由一种新颖且仍未充分阐明的机制造成,并为后续研究奠定了信息学和遗传学操作的基础。     

南京地区TTV部分基因的克隆及序列测定

目的：了解中国南京地区有无输血传播病毒（TTV）感染。方法：用套式聚合酶链反应（PCR）检测病因未明肝炎患者血清中TTVDNA，并对PCR扩增产物进行序列测定。结果：14例非甲-非戊型肝炎患者血清中共检出8例TTVDNA阳性者，对其中一株进行序列测定，该序列与日本TTV部分基因（CLON_22）和TTV中国株1相对应位置的核苷酸同源性很高，分别达97.65％和98.99％。结论：中国南京地区肝炎患者存在TTV感染。     

遗传工程在产抗生素链霉菌方面的研究进展

对链霉菌遗传工程中的载体——受体系统、抗生素抗性基因的克隆、抗生素生物合成结构基因的克隆、调节和分化基因的克隆、杂合抗生素的产生以及链霉菌作为外源基因克隆的受体等几个方面的研究报道作了综述。     

Diversity of polyketide synthases found in the Aspergillus and Streptomyces genomes

Natural products produced by biological organisms have played an important role in human health. The genomes of several Aspergillus species and several Streptomyces species have recently been sequenced. Interestingly the genomes revealed a large range of secondary metabolite genes, for many of which the products are currently unknown, suggesting that there is a wealth of secondary metabolites yet to be discovered. This brief review will discuss the current knowledge in polyketides produced by the various different classes of polyketide synthases (PKSs) present in the Aspergillus and Streptomyces genomes.     

MRP class of human ATP binding cassette (ABC) transporters: historical background and new research directions

1. The adenosine triphosphate (ATP) binding cassette (ABC) transporters form one of the largest protein families encoded in the human genome, and more than 48 genes encoding human ABC transporters have been identified and sequenced. It has been reported that mutations of ABC protein genes are causative in several genetic disorders in humans. 2. Many human ABC transporters are involved in membrane transport of drugs, xenobiotics, endogenous substances or ions, thereby exhibiting a wide spectrum of biological functions. According to the new nomenclature of human ABC transporter genes, the 'ABCC' gene sub-family comprises three classes involving multidrug resistance-associated proteins (MRPs), sulfonylurea receptors (SURs), and a cystic fibrosis transmembrane conductance regulator (CFTR). 3. Molecular cloning studies have identified a total of ten members of the human MRP class including ABCC11, ABCC12, and ABCC13 (pseudo-gene) that have recently been characterized. 4. This review addresses the historical background and discovery of the ATP-driven xenobiotic export pumps (GS-X pumps) encoded by MRP genes, biological functions of ABC transporters belonging to the MRP class, and regulation of gene expression of MRPs by oxidative stress.     

Biology of Membrane Transport Proteins

Membrane transporter proteins are encoded by numerous genes that can be classified into several superfamilies, on the basis of sequence identity and biological function. Prominent examples include facilitative transporters, the secondary active symporters and antiporters driven by ion gradients, and active ABC (ATP binding cassette) transporters involved in multiple-drug resistance and targeting of antigenic peptides to MHC Class I molecules. Transported substrates range from nutrients and ions to a broad variety of drugs, peptides and proteins. Deleterious mutations of transporter genes may lead to genetic diseases or loss of cell viability. Transporter structure, function and regulation, genetic factors, and pharmaceutical implications are summarized in this review.     

Cloning from Streptomyces cellulosae of the gene encoding beta-lactamase, a blue-dextran binding protein.

A beta-lactamase gene was cloned from Streptomyces cellulosae as a 2.3-kb DNA fragment using Streptomyces lividans 1326 and PIJ385 as a host-vector system. During the course of cloning, a part of the chromosomal DNA fragment cloned together with a part of the vector plasmid were deleted, indicating instability of this contiguous DNA region. The enzyme from the clone showed similar properties with respect to binding of blue dextran and isoelectric point to the enzyme from S. cellulosae. The cloned gene hybridized not only to DNA of S. cellulosae, the source of DNA, but also to DNAs of several Streptomyces species, irrespective of their formation of beta-lactamase. These results suggest that this gene may have homology to genes other than the one for beta-lactamase.     

几种调控基因对除虫链霉菌工业生产株的抗生素效价的影响

在模式链霉菌（如天蓝色链霉菌和变铅青链霉菌）中导入许多抗生索生物合成的调控基因可以大幅度提高抗生索的含量。本文报道利用链霉菌的整合质粒克隆几种已知的调控基因。并通过接合转移从大肠埃希菌中导入产生avermectin和多拉菌素的除虫链霉菌工业生产菌株中。发现3种调控基因afiR、aveR和orfX对菌株MMR630中avermectin的含量均可以提高约1倍。但是，以上的3种，加上另外3种调控基因分别导入菌株G11后，发现除aft8提高约13％外，其余调控基因使菌株产生多拉菌素的含量反而有不同程度的降低。将调控基因币B置于链霉菌强启动子PerrnE^＊下表达降低了菌株G11中多拉菌素的含量。上述结果表明，调控基因对不同链霉菌的抗生素生物合成具有不同的影响，反映了抗生素生物合成确实受到了复杂网络的调控。     

Studies on biosynthetic genes and enzymes of isoprenoids produced by actinomycetes

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMG-CoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.     

宁夏枸杞根际土壤链霉菌V-1-3次级代谢产物分析

摘要：目的 获得链霉菌V-1-3的基因组序列信息,分析其次级代谢产物生物合成基因簇并预测其代谢产物,为发现潜在新抗生素奠定基础。方法 基于16S rRNA基因序列进行菌株属水平鉴定,利用Illumina HiSeq+PacBio测序技术对菌株V-1-3进行基因组测序,采用antiSMASH(v6.0.1)在线工具分析次级代谢产物生物合成基因簇,液-质联用技术检测产生的次级代谢产物。结果 链霉菌V-1-3基因组序列全长8 243 417 bp,平均(G+C)含量为72.14 %,共编码7578个基因,预测到33个生物合成基因簇,利用液-质联用检测到4个代谢物：oxalomycin B、geosmin、coelichelin和ishigamide。结论 来自盐碱地的链霉菌V-1-3具有丰富的次级代谢产物生物合成基因簇,能产生多种次级代谢产物,具有进一步发掘新抗生素的价值。     

MRP class of human ATP binding cassette (ABC) transporters: historical background and new research directions

1.?The adenosine triphosphate (ATP) binding cassette (ABC) transporters form one of the largest protein families encoded in the human genome, and more than 48 genes encoding human ABC transporters have been identified and sequenced. It has been reported that mutations of ABC protein genes are causative in several genetic disorders in humans.

2.?Many human ABC transporters are involved in membrane transport of drugs, xenobiotics, endogenous substances or ions, thereby exhibiting a wide spectrum of biological functions. According to the new nomenclature of human ABC transporter genes, the ‘ABCC’ gene sub-family comprises three classes involving multidrug resistance-associated proteins (MRPs), sulfonylurea receptors (SURs), and a cystic fibrosis transmembrane conductance regulator (CFTR).

3.?Molecular cloning studies have identified a total of ten members of the human MRP class including ABCC11, ABCC12, and ABCC13 (pseudo-gene) that have recently been characterized.

4.?This review addresses the historical background and discovery of the ATP-driven xenobiotic export pumps (GS-X pumps) encoded by MRP genes, biological functions of ABC transporters belonging to the MRP class, and regulation of gene expression of MRPs by oxidative stress.     

委内瑞拉链霉菌中氯霉素生物合成基因的克隆

目的克隆委内瑞拉链霉菌(Streptomyces venezuelae)ISP5230中的氯霉素生物合成基因。方法以pabAB基因片段为探针,通过菌落杂交技术从该菌的基因文库中钓取相关基因。结果得到一7.5kbBamHIBamHI DNA片段,突变株的互补实验表明,该7.5kb DNA片段使氯霉素生物合成阻断变株CML-4恢复了氯霉素的生物合成。从野生菌株的染色体上删除该7.5 kb DNA片段中的一段3.5 kbNcoINcoI DNA片段后,野生菌株丧失了氯霉素的生物合成能力。结论该7.5 kb DNA片段含有氯霉素生物合成所需的基因。     

一种新的链霉菌表达载体启动子

eryA基因直接控制着红霉素母环6-脱氧-红霉内酯B的合成,在红霉素生物合成过程中具有重要作用。本文克隆了eryA基因的启动子PeryA,以绿色荧光蛋白基因为报告基因,构建了大肠埃希菌-糖多孢红霉菌穿梭型质粒。PEG介导原生质体转化法将穿梭型质粒分别转入糖多孢红霉菌A226与变铅青链霉菌JT46,荧光显微镜检测发现,此启动子在两菌株中都具有功能。随后,以变铅青链霉菌JT46为宿主,对PeryA启动子区域进行了深入研究,结果发现该启动子的-35区并不是必需的,仅有-10区、长度为41bp的该启动子在链霉菌中仍具有功能。定点突变证明-10区对于该启动子是必不可少的。因此,41bp的该启动子片段可作为链霉菌的有效启动子,这是迄今为止所发现的最短的启动子之一,可用于构建新的链霉菌表达载体。     

Cloning and Characterization of the Murine and Rat mrp1 Promoter Regions

The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents. The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression. As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene. All three promoters are GC-rich, TATA-less, and CAAT-less. Conservation of sequence between rodent and human promoters is limited to a proximal region of 100 nucleotides containing binding sites for members of the Sp1 family and a putative activator protein-1 element. The 5'-untranslated region (UTR) of human MRP1 contains an insertion of approximately 160 nucleotides comprising a GCC-triplet repeat and a GC-rich tandem repeat that is absent from the rodent sequences. Transient transfection analyses demonstrated that the conserved GC-boxes of all three genes are the major determinants of basal activity. Based on electrophoretic mobility shift assays, each GC-box can be bound by Sp1 or Sp3. Unlike the rodent genes, the human MRP1 5'UTR also binds Sp1 but not Sp3, and the human promoter retains substantial activity even in the absence of the conserved GC-boxes. Finally, we show that the tumor suppressor protein p53 can repress the human and rodent promoters by a mechanism that is independent of the Sp1 elements.     

中国南京TTV部分基因的克隆及对其扩增物序列的测定

目的 了解中国南京地区有无TT病毒(TTV)感染。方法采用套式聚合酶链反应(PCR)检测病因未明的肝炎患者血清中的TTV-DNA,并对PCR扩增产物进行序列测定。结果 在14例非甲～戊型肝炎患者的血清中,共检出8例TTVDNA阳性者,对其中一株进行序列测定,该序列与日本TTV部分基因(CLON22)和TTV中国株1相对应位置的核苷酸同源性很高,分别达97.65％和98.99％。结论 在中国南京地区的肝炎患者中存在着TTV感染。