Cloning,expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain

The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.     

嗜热古菌S.solfataricus小热激蛋白基因的克隆及表达

目的:从嗜热古菌Sulfolobus solfataricus中克隆一种新的小热激蛋白SsHsp14.1的基因,并研究其表达和生物活性。方法:用PCR技术以S.solfataricus基因组为模板扩增得到目的基因序列片段,并将其克隆到pET-28a(+)中,转化到E coli BL21(DE3)中经IPTG诱导表达,纯化后对产物进行生物活性测定。结果:从菌株S.solfataricus中克隆出目的基因,该基因的编码框由375个碱基组成,编码的蛋白质由124个氨基酸组成。含该质粒的大肠杆菌经诱导表达了一个与预期理论值相符的约14kDa的蛋白,利用亲和层析和凝胶柱分离纯化了重组蛋白。试验证明纯化后的重组SsHsp14.1具有分子伴侣活性,重组蛋白在体内表达时能提高E.coli细胞的耐热性。结论:成功克隆SsHsp14.1基因并表达出蛋白,并明确了其分子伴侣活性,为该热激蛋白的研究和应用奠定基础。     

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.     

Construction of Rhodococcus expression vectors and expression of the aminoalcohol dehydrogenase gene in Rhodococcus erythropolis

NADP⁺-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 produces double chiral aminoalcohols, which are used as pharmaceuticals. However, the genetic manipulation of Rhodococcus strains to increase their production of such industrially important enzymes is not well studied. Therefore, I aimed to construct Rhodococcus expression vectors, derived from the Rhodococcus–Escherichia coli shuttle vector pRET1102, to express aadh. The plasmid pRET1102 could be transformed into many actinomycete strains, including R. erythropolis. The transformation ef?ciency for a species closely related to R. erythropolis was higher than that for other actinomycete strains. Promoters of various strengths, hsp, 1200rep, and TRR, were obtained from Gram-positive bacteria. The activity of TRR was stronger than that of hsp and 1200rep. The aadh-expressing plasmid pRET1172 with TRR could be transformed into many actinomycete strains to increase their AADH production. The Rhodococcus expression vector, pRET11100, constructed by removing aadh from the pRET1172 plasmid may be useful for bioconversion.     

Heterologous expression of alcohol oxidase in Saccharomyces cerevisiae: properties of the enzyme and implications for microbody development

Alcohol oxidase (AO) expressed in transformed oleic acid-grown Saccharomyces cerevisiae, accumulated into microbodies to up to 8% of the total protein content of the organelles. This led to a small increase in volume fraction of the organelles, but not in their number. Most of the AO protein was present in large aggregates in the cytosol. The AO synthesized was inactive, irrespective of its subcellular localization and did not contain FAD. When the same AO gene was expressed in fused protoplasts of transformed S. cerevisiae and Hansenula polymorpha, the enzyme was properly assembled and activated in H. polymorpha microbodies.     

Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.     

人HCN2真核表达载体的构建及在HEK293细胞中的表达

目的：构建含有人HCN2基因的真核表达载体,并观察在人胚胎肾细胞（HEK293）中的表达情况。方法：对人HCN2基因全序列进行分析,进行oligo设计,通过PCR,扩增HCN2全长cDNA,通过双酶切（XhoI和BamHI）装入真核表达载体pIRES2-EGFP中,脂质体法转染入HEK293细胞中,利用真核表达载体中带有绿色荧光蛋白GFP报告基因,对转染效率进行监测,采用反转录-聚合酶链反应检测HCN2 mRNA表达,全细胞膜片钳技术检测HCN2通道电流。结果：测序及酶切结果表明HCN2基因正确,荧光显微镜下,转染细胞观察到绿色荧光,反转录-聚合酶链反应检测到HCN2 mRNA表达,膜片钳检测到hHCN2基因编码的通道电流。结论：成功地构建了HCN2真核表达载体并进行了起搏通道HCN2基因的异源性表达。     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

Somaclonal variation in wheat: genetic and cytogenetic characterisation of alcohol dehydrogenase 1 mutants

Summary The progeny of 551 regenerants of the hexaploid wheat cultivar Millewa were analysed for somaclonal mutants at the threeAdh-1 loci in hexaploid wheat. Seventeen regenerants gave rise to progeny having altered ADH1 zymograms. Progeny with altered zymograms in 13 of these regenerants were aneuploid. The remaining 4 regenerants gave rise to euploid progeny with altered ADH1 zymograms. The genetics of three of these somaclonal mutants is described in detail. These regenerants were interpreted to possess a 4A isochromosome, a 3BS/4A translocation and a 7BS/4A translocation, respectively.This research was partly supported by the Rockefeller Foundation and a Reserve Bank of Australia Rural Credit Development Fund research grant     

人HCN2 真核表达载体的构建及在HEK293 细胞中的表达

目的:构建含有人HCN2基因的真核表达载体,并观察在人胚胎肾细胞(HEK293)中的表达情况。方法:对人HCN2基因全序列进行分析,进行oligo设计,通过PCR,扩增HCN2全长cDNA,通过双酶切(XhoI和BamHI)装入真核表达载体pIRES2-EGFP中,脂质体法转染入HEK293细胞中,利用真核表达载体中带有绿色荧光蛋白GFP报告基因,对转染效率进行监测,采用反转录-聚合酶链反应检测HCN2 mRNA表达,全细胞膜片钳技术检测HCN2通道电流。结果:测序及酶切结果表明HCN2基因正确,荧光显微镜下,转染细胞观察到绿色荧光,反转录-聚合酶链反应检测到HCN2 mRNA表达,膜片钳检测到hHCN2基因编码的通道电流。结论:成功地构建了HCN2真核表达载体并进行了起搏通道HCN2基因的异源性表达。     

pPSX: a novel vector for the cloning and heterologous expression of antitumor antibiotic gene clusters

A cosmid cloning vector has been constructed that demonstrates high levels of segregational stability in Escherichia coli K12. pPSX is a 14-kilobase vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumor antibiotic violacein. The incorporation of the lambdacos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36kb. The inclusion of a lacZalpha multiple cloning site (MCS) allows blue/white screening. pPSX cosmids can be extracted from the host cell with commercial plasmid extraction kits facilitating downstream analysis, sequencing and sub-cloning. pPSX can be transferred to a variety of heterologous hosts by either electroporation or mobilization from E. coli S17-1. While it is unstable in non-E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts.     

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α₁-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc.     

Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum

We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904 bp long open reading frame contained six introns located either close to the 5′- or 3′-end of the ORF. The corresponding cDNA of 2487 bp was cloned into the expression vector pPICZαB to achieve inducible heterologous expression and secretion of the recombinant flavocytochrome in the methylotrophic yeast Pichia pastoris. Transformants were selected on media with normal and 10-fold increased zeocin concentration, and selected clones were tested for inducible extracellular production of the recombinant oxidoreductase. The maximally obtained volumetric activity was 0.25 U/ml in YPM (rich) medium and 2.15 U/ml in production stage (minimal) medium in a fed-batch fermentation. Recombinant CDH was purified in two consecutive chromatographic steps leading to a final specific activity of up to 7.4 U/mg protein at 40 °C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 °C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.     

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.     

Development of an inducible gene expression system for Lactobacillus sakei

AIM: To develop an inducible gene expression system for Lactobacillus sakei, based on the regulatory system of sakacin A production. METHODS AND RESULTS: A Lactobacillus/Escherichia coli shuttle vector; pKRV3, was constructed including the signal transducing system genes of the bacteriocin sakacin A. The gusA gene fused to PsapA promoter, cloned in this vector allowed for inducible beta-glucuronidase expression in L. sakei and L. plantarum following the addition of the sakacin A inducing peptide. PsapA appeared to be a strong and tightly controlled promoter when compared with known promoters. CONCLUSION: The pKRV3 system can be used as an inducible gene expression system in lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel, inducible gene expression system has been developed for lactic acid bacteria relevant in food fermentations.     

Genetics of gene expression characterizes response to selective breeding for alcohol preference

Numerous selective breeding experiments have been performed with rodents, in an attempt to understand the genetic basis for innate differences in preference for alcohol consumption. Quantitative trait locus (QTL) analysis has been used to determine regions of the genome that are associated with the behavioral difference in alcohol preference/consumption. Recent work suggests that differences in gene expression represent a major genetic basis for complex traits. Therefore, the QTLs are likely to harbor regulatory regions (eQTLs) for the differentially expressed genes that are associated with the trait. In this study, we examined brain gene expression differences over generations of selection of the third replicate lines of high and low alcohol‐preferring (HAP3 and LAP3) mice, and determined regions of the genome that control the expression of these differentially expressed genes (_deeQTLs). We also determined eQTL regions (_rveQTLs) for genes that showed a decrease in variance of expression levels over the course of selection. We postulated that _deeQTLs that overlap with _rveQTLs, and also with phenotypic QTLs, represent genomic regions that are affected by the process of selection. These overlapping regions controlled the expression of candidate genes (that displayed differential expression and reduced variance of expression) for the predisposition to differences in alcohol consumption by the HAP3/LAP3 mice.