Cloning of an endo-1,4-beta-xylanase gene from Penicillium canescens and construction of multicopy strains

The complete gene xylA that encodes endo-1,4-beta-xylanase secreted by Penicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven-eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30-50% of the total secreted protein.     

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.     

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.     

Sucrose Phosphate Synthase Genes in Plants Belong to Three Different Families

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date. Received: 17 April 2001 / Accepted: 27 August 2001     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

番石榴实蝇和瓜实蝇的线粒体L-rRNA基因测序及双翅目昆虫L-rRNA基因的结构特征与系统发育关系分析

RNA二级结构比一级结构包含更多的信息, 在物种系统发育分析中更能反应真实情况。因而, 本研究对番石榴实蝇Bactrocera correcta和瓜实蝇B. cucurbitae的L-rRNA全基因进行了测序, 构建了双翅目L-rRNA基因二级结构的模式图, 并分析了其结构特征, 而后基于H45~H47茎环结构参数和结构序列及L-rRNA结构序列对双翅目13科的系统发育关系进行了初步分析。结果表明： 双翅目昆虫具有保守的L-rRNA二级结构; 不同结构区碱基组成和分布不均匀, Ⅳ区和Ⅴ区的完全保守碱基含量最高, GC含量最高, 绝大部分科Ⅵ区的AT斜率均小于0; 科内特有碱基和科间少数保守碱基大部分为G或C。瘿蚊科与双翅目其他科具有较远的系统发育关系, 而丽蝇科、 寄蝇科和食蚜蝇科有相近的系统发育关系; 虻科与网翅虻科同在一个小分支; 蠓科与蚊科同在一个大的系统发育分支。利用单方面参数不易得到理想的进化结果, 准确系统发育分析需要结合多方面的参数。     

Isolation and characterization of extracellular α-galactosidases from Penicillium canescens

Two alpha-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH- and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding alpha-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both alpha-galactosidases from P. canescens could be successfully used as feed additives. alpha-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and alpha-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder.     

人Boule基因启动子区结合蛋白的生物信息学分析

目的：对精子发生RNA结合蛋白Boule基因启动子区结合蛋白进行生物信息学分析。方法：从基因参考序列数据库获取Boule基因启动子区序列,使用TFSEARCH程序对启动子序列中的转录因子结合位点进行预测。结果：成功获得长度为2kb的人Boule基因启动子区序列。该启动子区Thresholdscore〉90的共有60个转录因子结合位点,涉及sox家族、GATA结合蛋白家族、热休克因子家族、锌指蛋白Kruppel家族、POU家族、runt家族、同源异型框基因家族、TALE类同源结构蛋白家族、转录因子螺旋环螺旋家族、IKAROS家族、FOX家族11个家族的转录因子和3个TATAbox。结论：Boule基因表达的调控是在一定时间或空间上、一种或多种调节蛋白作用的复杂过程。调控Boule基因表达的转录因子绝大部分与胚胎发育、性别决定、个体生长密切相关。     

Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate

An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ～70 mgg(-1); xylitol to ～18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1).     

Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities.

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.     

Abundant variations of <Emphasis Type="Italic">MC4R</Emphasis> gene revealed by Phylogenies of Yak (<Emphasis Type="Italic">Bos grunniens</Emphasis>) and other mammals

MC4R gene was proved to play important roles in body weight regulation in many mammals and exhibit higher homology among different species. The mutations MC4R significantly correlated to the restricted feeding weight, fat deposition and energy balance. In this work, ORF sequences of MC4R gene of Bos grunniens were cloned and phylogenetic relationships of yak and other mammals were analyzed on the basis of MC4R genes. Totally 290 variable sites were examined in 25 sequences from 22 different mammals, and 23 haplotypes were defined with a haplotype diversity of 0.9900. All the sequences were clustered into phylogenetic clades representing different orders or families. The individuals of Bos grunniens, Bos taurus and Ovis aries which belonged to the family of Bovidae were more divergent from the other orders or families and bovid animals may have branched out from the phylogenetic tree earlier than other mammals analyzed during 450 million years of vertebrate evolution. Amino acid sequences inferred from MC4R genes exhibited 54 variable sites, while high conservation of MC4R was observed within the same order or family. We concluded that coding region of MC4R gene displayed abundant variations among different mammal phylogenetic clades, whereas, the conservation of MC4R within order or family could be explained that MC4R gene may have been subjected to substantial constraints or strong purifying selection during several million years of mammal evolution.