CHARACTERIZATION OF RAD4 GENE REQUIRED FOR ULTRAVIOLET-INDUCED EXCISION REPAIR OF Saccharomyces cerevisiae PROPAGATED IN Escherichia coli WITHOUT INACTIVATION

The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae (Yoon et al., 1985, Korean J. Genetics 7, 97-104) appeared to propagate in Escherichia coli and yet retained its complementing activity to rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg1II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli.     

aprE基因表达的分子生物学和微量热法分析

利用分子生物学方法(SDS-PAGE和酶活检测法)未检测到所克隆的aprE基因在大肠杆菌中的表达产物(碱性蛋白酶), 而微量热法检测结果发现: 重组菌株的生长代谢产热曲线之间存在明显的差异. 根据这些差异, 分析了该基因的上、下游调控序列对该基因在大肠杆菌中表达的重要作用, 从而进一步对该基因进行了亚克隆, 得到了生物学方法可检测到的表达产物. 由此推测, 微量热技术有可能为检测外源基因表达及其调控, 以及为指导进一步基因工程操作提供一种新的快速灵敏的技术和方法.     

CLONING AND SEQUENCE ANALYSIS OF 3''''-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se     

产甘油基因工程菌的构建

利用途径工程的基本原理,在大肠杆菌中构建一条产甘油的新代谢途径。从酿酒酵母Sacchdromyces cerevisiae INVSc1菌株总DNA克隆3-磷酸甘油脱氢酶基因(gpdl)和3-磷酸甘油酯酶基因(hot2),构建由两个杂合启动子trc启动基因的双表达盒的重组质粒pGEM-Cgpd1-Chor2,后者转入E．coli JM109菌株,构建的重组菌株就具有一条直接将葡萄糖转化为甘油的新代谢途径,将该重组菌株以葡萄糖为底物进行摇瓶发酵,甘油产率为1．18g／L。该研究结果为进一步构建生产1,3-丙二醇工程菌打下了基础。     

Cloning and Sequencing of Trichosanthin Gene and Its Expression in Escherichia coli and Tobacco Plant

A Trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature Trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a highlevel expression plasmid (pJLA502) of E. coli under the control of a P_RP_L promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the Trichosanthin gene has been     

Construction of yeast xylulokinase mutant by recombinant dna techniques

Applied Biochemistry and Biotechnology - ASaccharomyces cerevisiae xylulokinase mutant was constructed by using the cloned yeast xylulokinase gene, XYK-Sc, and the gene disruption technique. TheS....     

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered saccharomyces yeast capable of cofermenting glucose and xylose

Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.     

毛白杨环氧水解酶的异源表达及其在催化拆分手性环氧化物中的应用

对已公布的全基因组进行检索发现,杨树(Populus tomentosa)至少含有24个预测为可溶性环氧水解酶的基因.从中选取了7个可能的环氧水解酶基因进行克隆,通过扩增得到其中5个毛白杨(Populus tomentosa Carr)环氧水解酶基因.序列分析显示,它们与已克隆的巨大芽胞杆菌环氧水解酶的同源性仅为24%～26%.对该系列基因进行了在E.coli中的异源表达,并将得到的5个环氧水解酶(PTEH1～5)用于缩水甘油苯基醚和对硝基苯乙烯氧化物的酶促水解反应.结果发现,其中3个重组酶具有显著的环氧水解酶活性.进一步研究表明,PTEH1和PTEH2对于缩水甘油苯基醚显示了一定的反常规的(R)-选择性,而PTEH5则优先水解(S)-构型的缩水甘油苯基醚.因此,毛白杨中环氧水解酶表现出多样性.     

Genotype-phenotype correlation of SMN locus genes in spinal muscular atrophy patients from India

Spinal muscular atrophy has been classified into four groups based on the age of onset and clinical severity of the disease. Homozygous deletion in SMN1 gene causes the disease but the clinical severity may be modified by copy number of homologous gene SMN2 as well as the extent of deletion at SMN locus. In the view of scarcity of genotype and phenotype correlation data from India, this study has been undertaken to determine that correlation in SMA patients by using the SMN and NAIP genes and two polymorphic markers C212 and C272 located in this region. Two to four alleles of the markers C212 and C272 were observed in normal individuals. However, majority of Type I patients showed only one allele from both markers whereas in Type II and III patients, 2-3 alleles were observed. The SMN2 copy number in our type III patients showed that patients carry 3-5 copies of SMN2 gene. Our results suggest that extent of deletions encompassing H4F5, SMN1, NAIP and copy number of SMN2 gene can modify the SMA phenotype, thus accounting for the different clinical subtypes of the disease.     

Construction of a Recombinant <Emphasis Type="Italic">S. cerevisiae</Emphasis> Expressing a Fusion Protein and Study on the Effect of Converting Xylose and Glucose to Ethanol

Gene XYL1 from Candida shehatae and gene XYL2 from Pichia stipitis were amplified by polymerase chain reaction (PCR), and the two genes were both placed under the strong promoter of alcohol dehydrogenase (ADH) of plasmid pAD2 to produce the recombinant expression vector pAD2-P12. Because the amplified XYL1 fragment lacks the stop codon UAA, the polypeptide expressed in yeast cells should be a fusion protein, which is a fusion of xylose reductase and xylitol dehydrogenase. Subsequently, the pAD2-P12 vector was transformed into Saccharomyces cerevisiae YS58 to produce a recombinant S. cerevisiae YS58-12. It was indicated that S. cerevisiae YS58-12 has the ability of metabolizing xylose to produce ethanol by fermentation experiment. The result of cofermentation of glucose and xylose by using this recombinant S. cerevisiae YS58-12 showed a relatively satisfactory result. The highest percentage of xylose consumption rate reached 81.3% and the ethanol yield was equal to 67.14% of the ideal value.     

固定化酵母非水相催化羰基不对称还原反应的研究

用海藻酸钙包埋法对增殖培养的酵母细胞进行了固定化,并用于催化有机溶剂中乙酰乙酸乙酯的不对称还原反应。考察了固定化时所用缓冲溶液的pH、催化剂颗粒大小与用量、辅助底物种类、底物浓度、以及重复利用批次等因素对反应产物（S）-3-羟基丁酸乙酯的浓度和光学纯度的影响。结果表明,固定化时应采用pH为7的Tris-HCL缓冲溶液,颗粒的直径以2mm左右为较佳;反应时应以正已烷为溶剂,正已醇为辅助底物,固定化酵母颗粒的最适用量为6g/20ml反应液;底物的初始浓度以100mmol/L为佳,浓度过高对反应有一定的抑制作用;固定化细胞重复利用三次对映选择性基本保持不变。     

A ROLE FOR STEROLS IN THE PORPHYRIN MEDIATED PHOTOSENSITIZATION OF YEAST

The yeast Saccharomyces cerevisiae was used as a model system to determine the role of sterols in the porphyrin mediated photosensitization of yeast. A sterol auxotroph, RD5-R, was grown on sterols with different levels of unsaturation and assayed for photosensitivity in the presence of either protoporphyrin IX or hematoporphyrin (both at 100 micrograms/ml). Cells grown on the completely saturated sterol (stanol), cholestanol, were substantially more resistant to the photosensizing effects of the porphyrin. We hypothesize that this resistance arises from the inability of the porphyrin to mediate the oxidation of the membrane sterol. Our results indicate that photodegradation of the native yeast sterol, ergosterol, can account for substantial losses of cell viability.     

Cloning of the histidine transport genes from Salmonella typhimurium and characterization of an analogous transport system in Escherichia coli

The genes for the well-characterized high-affinity histidine transport system of S typhimurium have been cloned in lambda gt4. Genetic and physiological analyses of the analogous transport system of E coli were undertaken in order that available lambda vectors, recombinant DNA techniques, and a genetic selection for transport function might be used to isolate the Salmonella genes. The presence of the transport genes on a 12.4 Kb cloned DNA fragment has been confirmed 1) genetically, by complementation studies; 2) physiologically, by the rates of histidine uptake by bacteria containing this DNA; and 3) by demonstrating that the cloned DNA codes for the previously identified transport proteins J and P. The isolated fragment carries the entire transport operon, the argT gene and the ubiX locus, but neither the purF gene nor the ack/pta loci.     

POSSIBLE INVOLVEMENT OF MEMBRANE DAMAGE IN THE IN ACTIVATION BY BROAD-BAND NEAR-UV RADIATION IN Saccharomyces cerevisiae CELLS

Multiple cellular effects of near-UV radiation (300-380 nm) on inactivation, disruption of the permeability barrier and induction of gene conversion at the trp 5 locus were simultaneously measured in the same culture of a diploid strain of the yeast Saccharomyces cerevisiae in order to assess the critical lethal damage. Inactivation of exponential phase cells in water appeared to be closely related to the disruption of the permeability barrier. Inactivation and membrane damage were remarkably oxygen dependent, whereas the induction of genetic changes was very low and dependent much less on oxygen. The dependence on the temperature for inactivation and membrane damage was both low, conforming with the expectation that the processes are mainly photochemical and not enzymatic. These features are very contrasted with the characteristics of far-UV radiation effects. Possible involvement of membrane damage in near-UV inactivation of exponential phase yeast cells is discussed.     

STRUCTURE OF THE MITOCHONDRIAL URFA6L GENE AND tRNA~(Lys) GENE FROM CARP

The carp mitochondrial URFA6L gene consists of 165 base pairs. The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene. Their nucleotide sequences exhibit 68% homology. The carp URFA6L gene encodes a protein of 54 amino acids. The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids(proline, tryptophan, leueine, isoleueine and tyrosine). A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function asATPase8. The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in cloverleaf secondary structure. Similar to amphibian and mammalian mitochondrial tRNA~(Lys) genes, the carp mitochondrial tRNA~(Tys) gene also has some unusual structural features as compared with its cytoplasmic counterpart     

Expression of Cu,Zn-superoxide dismutase gene from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its resistance to oxidative stress

The gene for the Cu, Zn-superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of the yeast P. pastoris. The SOD was purified from the cultured yeast by ammonium sulfate precipitation and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overexpressed SOD protein was shown to have immunologically biologic activity and to be enzymatically active. The yeast overexpressing Cu, Zn-SOD appeared to be more resistant to oxidative stress such as paraquat, menadione, and heat shock.