The transfer RNA genes in Oryza sativa L. ssp. indica

The availability of the draft genome sequence of Oryza sativa L. ssp. indica has made it possible to study the rice tRNA genes. A total of 596 tRNA genes, including 3 selenocysteine tRNA genes and one suppressor tRNA gene are identified in 127551 rice contigs. There are 45 species of tRNA genes and the revised wobble hypothesis proposed by Guthrie and Abelson is perfectly obeyed. The relationship between codon usage and the number of corresponding tRNA genes is discussed. Redundancy may exist in the present list of tRNA genes and novel ones may be found in the future. A set of 33 tRNA genes is discovered in the complete chloroplast genome of Oryza sativa L. ssp. indica. These tRNA genes are identical to those in ssp. japonica identified by us independently from the origional annotation.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.     

The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.     

Rearrangements of mitochondrial transfer RNA genes in marsupials

Summary The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNA^Tyr gene at the same position as in placental mammals andXenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and, in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNA^Tyr gene has been evolving under more constraints than the other tRNA genes.Deceased July 21, 1991     

Mutation and cloning of clustered Streptomyces genes essential for sulphate metabolism

Summary A range of mutants auxotrophic for cysteine (cys) and resistant to selenate (sel) were isolated from many Streptomyces strains but chiefly from S. coelicolor A3(2) and S. lividans 66. Two of the classes of sel/cys mutants probably contained simple biochemical lesions of sulphate permease (selC) and ATP sulphurylase (selA) activities, while a further two classes (selD and selE) were pleiotropic and possibly regulatory. Most classes of sel mutations were clustered around the cysD locus of S. coelicolor. Segments of chromosomal DNA cloned from S. coelicolor, S. cattleya and S. clavuligerus and able to complement various sel/cys mutations allowed the relative positions of these mutations and the cysC and cysD mutations of S. coelicolor to be determined. The sel/cys DNA can be used for two-way selection: Cys⁺Sel^sCys^-Sel^r.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Cloning and expression of the SalI restriction-modification genes of Streptomyces albus G

Summary The Streptomyces albus G genes (salR and salM) for the class II restriction enzyme SalI (SalGI) and its cognate modification enzyme were cloned in Streptomyces lividans 66. Selection was initially for the salR gene. From a library of S. albus G DNA in the high copy number plasmid pIJ486 several clones of S. lividans were obtained that were resistant to phage C31 unmodified at the many SalI sites in its DNA, but were sensitive to modified phages last propagated on a restriction-deficient, modification-proficient mutant of S. albus G. SalI activity was detected in cell-free extracts of the clones, though only at levels comparable with that in S. albus G. Five different recombinant plasmids were isolated, with inserts of 5.6, 5.7, 8.9, 10 and 18.9 kb that contained a common region of 4.5 kb. These plasmids could not be digested by SalI, although the vector has four recognition sites for this enzyme, indicating that the salM gene was also cloned and expressed. Subcloning experiments in S. lividans indicated the approximate location of salR and salM, and in Escherichia coli led to detectable expression of salM but not of salR. A variety of previously isolated S. albus G mutants affected in aspects of SalI-specific restriction and modification were complemented by the cloned DNA; they included a mutant temperature-sensitive for growth apparently because of a mutation in salM. Southern blotting showed that DNA homologous to the cloned sal genes was present in Xanthomonas and Rhodococcus strains, but not detectably in Herpetosiphon strains, all of which produce SalI isoschizomers.     

Mutant of the glutamine transfer RNA gene as UGA suppressor in Escherichia coli

Summary A UGA suppressor derived from a glutamine tRNA gene of Escherichia coli K 12 was isolated and characterized. Phages carrying the suppressor su⁺2_UGA could be obtained only from a hybrid transducing phage, h ⁸⁰ cI ₈₅₇psu ⁺2_oc, but not from the original transducing phage cI ₈₅₇psu ⁺2_oc. By DNA sequence analysis, it was found that the su ⁺2 UGA suppressor obtained has two mutations; one is in the anticodon (TTATCA), as expected, and the other (CT) is at the 7th position from the 3 end of tRNA ₂ ^Gln . The significance of these mutations and the lethal effect on phage of the increased amounts of UGA suppressor tRNAs are discussed.     

Nucleotide sequence of genes hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2) having similarity to rpoD genes

Summary The complete nucleotide sequences were determined of hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2). They indicate the presence of a single open reading frame in each gene coding for polypeptides of 396 (43747 daltons), 339 (38173 daltons), and 332 amino acid residues (37190 daltons), respectively. These amino acid sequences revealed extensive similarities with the principal sigma factors of Bacillus subtilis, Escherichia coli, Mxyococcus xanthus, Pseudomonas aeruginosa, and also the katF gene product of E. coli. Besides the highly conserved amino acid residues in the rpoD box region, alignment of hrd gene products and the known principal sigma factors and sigma-related factors allowed us to postulate a common basic structure for the principal sigma type factors as distinct from the alternative sigma factors.     

海洋链霉菌酮合成酶结构域基因的克隆及分析

海洋链霉菌通过聚酮合酶(PKS)合成许多结构和功能多样且具有药用价值的聚酮化合物(PKs),酮合成酶结构域(KS)作为PKS的核心结构域,可催化底物与伸长的聚酮之间的脱羧缩合,在聚酮化合物生物合成中起着重要作用。本文通过对从海洋链霉菌Streptomyces sp. X66基因组DNA克隆获得的ks基因的生物信息学分析表明,该ks基因序列长945 bp, BLAST序列比对显示其具有典型的酮合酶结构域的功能区域。理化分析显示其拟编码309个氨基酸,理论等电点为6.60,原子组成为C₁₄₀₁H₂₂₃₉N₄₂₅O₄₁₉S₈,不稳定指数为42.11,平均亲水系数为0.112,编码产物为酸性疏水不稳定蛋白,且不含信号肽和跨膜结构,二级结构以无规则卷曲和α-螺旋为主,SDS-PAGE显示其分子量约为55 kDa。通过对ks基因的研究,为进一步解析聚酮化合物合成代谢中的调控机制及组合生物学和体外酶系合成聚酮化合物提供参考。     

浅紫灰链霉菌CQ01819及其次级代谢产物的定向发掘

【目的】采用特征次级代谢产物生物合成的保守功能基因探针,定向分离土壤中产生特征次级代谢产物的菌种资源,借助基因转录分析为导向的培养基优化方法,获得目标次生代谢产物。【方法】首先,根据5种特征次级代谢产物保守的合成功能基因设计简并引物,定向从土壤样品中筛选、分离并纯化菌株。然后,以RT-qPCR为指导开展目标产物的发酵培养基优化;最后,对菌株进行发酵,利用多种色谱技术分离纯化目标天然产物,并结合高分辨质谱与核磁共振等技术对所获得的化合物进行结构鉴定。【结果】从土壤中筛选得到了一株AHBA合酶基因和环氧化酶基因均为阳性的链霉菌菌株(编号为CQ01819),根据转录分析优化发酵培养基,最终从该菌株分离纯化得到了含有AHBA结构单元的丝裂霉素C、聚醚类抗生素莫能霉素A和缬吲霉素。【结论】本研究通过菌株的定向分离纯化,筛选得到了产生预期抗生素的浅紫灰链霉菌菌株CQ01819;基于RT-qPCR指导的发酵培养基优化,确定了菌株的发酵条件;获得发酵粗提物后,采用多种色谱整合技术和光谱分析策略,快速分离并鉴定了目标产物。该研究为目标菌种资源的定向筛选、菌株的发酵条件的快速优化和化合物的定向分离提供了较...     

Mapping of the sor genes for l-sorbose degradation in the chromosome of Klebsiella pneumoniae

Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.     

欧洲疮痂链霉菌F5的鉴定及其抗菌活性

【背景】木豆(Cajanus cajan)是一种具有多种药理活性的药用植物,目前对其根际功能放线菌的认识和研究有限,有必要对其应用开发潜力进行研究。【目的】从木豆根际土中筛选一株对植物病原菌和常见病原菌具有广谱拮抗活性的放线菌菌株,鉴定菌株的分类地位、相关代谢产物及可能的生物合成途径,为该菌株的开发应用提供数据支撑。【方法】以7种常见植物病原真菌及8种常见病原菌为指示菌,采用平板对峙法和滤纸片扩散法筛选具有广谱抗菌活性的放线菌菌株,基于形态观察与系统发育分析对该菌株进行分类鉴定,并通过高分辨质谱和超高效液相色谱-串联质谱(UPLC-MS/MS)对活性菌株的次生代谢产物进行鉴定与验证。采用PCR扩增菌株聚酮合成酶Ⅰ(polyketide synthase,PKS-Ⅰ)和非核糖体多肽合成酶(non-ribosomal peptide synthetase,NRPS)基因,明确其活性代谢产物可能的生物合成途径。【结果】通过抑菌试验筛选得到拮抗放线菌F5,确定其为欧洲疮痂链霉菌(Streptomyces europaeiscabiei),F5菌株基因组中含有编码PKS-Ⅰ和NRPS合成的相关基...     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Hox genes in the echiuroid Urechis unicinctus

An echiuroid species, Urechis unicinctus, was surveyed for Hox genes using polymerase chain reaction with homeobox-specific degenerate primers. We identified nine distinct homeodomain-containing gene fragments. These nine fragments were classified by comparative analysis. This analysis revealed that this echiuroid possessed at least three Hox genes from the anterior group, five from the central group, and one from the posterior group.Sung-Jin and Dae-Hee Lee contributed equally to this work.An erratum to this article can be found at