The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal -map and is a new member of the AMP-binding protein family

The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms.     

The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase

The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6- amino acid substitution into the putative active site (amino acids 249-254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464-523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.     

The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

Background  

Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogeniCity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogeniCity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene.     

Identification of a gene family (kat) encoding kinesin-like proteins in Arabidopsis thaliana and the characterization of secondary structure of KatA

A gene family, designated kat, has been characterized in Arabidopsis thaliana by genomic Southern hybridization and nucleotide sequencing analysis. The kat gene family includes at least five members, named katA, katB, katC, katD and katE, whose products share appreciable sequence similarities in their presumptive ATP-binding and microtubule-binding motifs with known kinesin-like proteins. The carboxyl-terminal region of the KatA protein deduced from the nucleotide sequence of the cDNA clone has considerable homology with the mechanochemical motor domain of the kinesin heavy chain. The predicted secondary structure of the KatA protein indicates two globular domains separated by a long a helical coiled coil with heptad repeat structures, such as are commonly found in kinesin-like proteins.     

The gene fimU affects expression of Salmonella typhimurium type 1 fimbriae and is related to the Escherichia coli tRNA gene argU

The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU ⁺ strain fimbriation did not require the fimU gene.     

金霉素生物合成基因簇中调控基因ctcB的功能

【目的】研究金霉素产生菌中SARP家族转录调控基因ctc B的作用。【方法】利用大肠杆菌、链霉菌的属间接合转移和同源重组双交换的方法,构建ctc B基因缺失突变株。通过c DNA在相邻同转录方向的基因间隔进行PCR验证,确定金霉素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株金霉素生物合成基因簇的转录水平检测。随后,生物信息学预测分析了金霉素生物合成基因簇内Ctc B与DNA的结合位点。【结果】获得了ctc B基因缺失的双交换突变株。发酵结果显示,该突变株失去产生金霉素与四环素的能力。金霉素生物合成基因簇内有6个共转录单元,其中4个共转录单元在ctc B基因缺失突变株中转录水平明显下降。软件分析预测到一致性较高的Ctc B结合重复序列。【结论】ctc B正调控金霉素生物合成结构基因ctc G-D、ctc H-K、ctc N-P、ctc W-T 4个转录单元和ctc Q,为进一步研究ctc B调控机制奠定了基础。     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial β-oxidation pathways. Prior to channelling the fatty acids into β-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB). FaaB was found to be the major long-chain synthetase for fatty acid degradation. FaaB was shown to localise to the peroxisomes, and the corresponding gene was induced in the presence of short and long chain fatty acids. Deletion of the faaB gene leads to a reduced/abolished growth on a variety of fatty acids. However, at least one additional fatty acyl-CoA synthetase with a preference for short chain fatty acids and a potential mitochondrial candidate (AN4659.3) has been identified via genome analysis.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.     

Genetic analysis and mapping of gene fzp ( t ) controlling spikelet differentiation in rice

A mutant of spikelet differentiation in rice called frizzle panicle (fzp) was discovered in the progeny of a cross between Oryza sativa ssp. indica cv. V20B and cv. Hua1B. The mutant exhibits normal plant morphology but has apparently fewer tillers. The most striking change in fzp is that its spikelet differentiation is completely blocked, with unlimited subsequent rachis branches generated from the positions where spikelets normally develop in wild-type plants. Genetic analysis suggests that fzp is controlled by a single recessive gene, which is temporarily named fzp(t). Based on its mutant phenotype, fzp(t) represents a key gene controlling spikelet differentiation. Some F₂ mutant plants derived from various genetic background appeared as the “middle type”, suggesting that the action of fzp(t) is influenced by the presence of redundant, modifier or interactive genes. By using simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) method, fzp(t) gene was mapped in the terminal region of the long arm of chromosome 7, with RM172 and RM248 on one side, 3.2 cM and 6.4 cM from fzp(t), and RM18 and RM234 on the other side, 23.1 cM and 26.3 cM from fzp(t), respectively. These results will facilitate the positional cloning and function studies of the gene.     

hpc2研究进展

生物个体的胚胎发育以及细胞的增殖、分化,都同时受到多种基因的严格调控,PcG基因家族就是一类重要的发育相关基因.而hPc2基因是人PcG基因家族中的一个重要成员,其编码的HPC2蛋白,不仅可以和HPH、BMI-1以及RING1等其他人类PcG蛋白结合形成HPC/HPH PcG复合体,以蛋白复合体的形式参与对homeotic基因的表达抑制,以维持机体的正常发育以及细胞的增殖和定向分化,还发现它能与其他多种蛋白质相结合,提示HPC2可能具有多种功能.因此,对hPc2的深入研究不仅有助于进一步阐明PcG基因家族的作用机理,扩展人们对基因表达调控的认识,还有助于发现PcG基因家族与其他信号转导通路的联系,更好地理解细胞信号网络系统.     

Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.     

Escherichia coli parA is an allele of the gyrB gene

Summary A thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations. The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd. A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110. The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA. This mutation was linked with the Par phenotype as shown unambiguously by 4,6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background. Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene. This strongly suggests that parA is an allele of gyrB.     

苏云金芽胞杆菌BkdR和CcpA对bkd基因簇的转录调控

【目的】通过分析苏云金芽胞杆菌(Bacillus thuringiensis,Bt)转录调控因子BkdR和多效调控因子CcpA对亮氨酸、异亮氨酸、缬氨酸代谢基因簇bkd的转录调控,明确bkd基因簇的转录调控机制。【方法】通过β-半乳糖苷酶活性测定分析bkd基因簇启动子的诱导转录活性,采用同源重组技术敲除Bt HD73菌株的ccpA基因,通过融合His标签的方法在大肠杆菌中表达纯化BkdR和CcpA蛋白,通过凝胶阻滞实验明确BkdR和CcpA蛋白与bkd基因簇启动子的结合作用。【结果】亮氨酸、异亮氨酸、缬氨酸可诱导bkd基因簇启动子Pptb的转录活性。Pptb的诱导活性在bkdR突变体中明显降低,而在ccpA突变体中明显上升。BkdR和CcpA蛋白与Pptb均有结合作用。【结论】bkd基因簇的转录活性受BkdR正调控,而受CcpA负调控。     

CDC7 protein kinase activity is required for mitosis and meiosis in Saccharomyces cerevisiae

Summary The product of the CDC7 gene of Saccharomyces cerevisiae has multiple cellular functions, being needed for the initiation of DNA synthesis during mitosis as well as for synaptonemal complex formation and commitment to recombination during meiosis. The CDC7 protein has protein kinase activity and contains the conserved residues characteristic of the protein kinase catalytic domain. To determine which of the cellular functions of CDC7 require this protein kinase activity, we have mutated some of the conserved residues within the CDC7 catalytic domain and have examined the ability of the mutant proteins to support mitosis and meiosis. The results indicate that the protein kinase activity of the CDC7 gene product is essential for its function in both mitosis and meiosis and that this activity is potentially regulated by phosphorylation of the CDC7 protein.     

A multicopy suppressor ofnin1-1 of the yeastSaccharomyces cerevisiae is a counterpart of theDrosophila melanogaster diphenol oxidase A2 gene,Dox-A2

NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G₁/S progression and G₂/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum^– transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.