Detection of potential positive regulatory motifs of transcription in yeast introns by comparative analysis of oligonucleotide frequencies

We conducted a comparative statistical analysis of tetra- through hexanucleotide frequencies in two sets of introns of yeast genes. The first set consisted of introns of genes that have transcription rates higher than 30 mRNAs/h while the second set contained introns of genes whose transcription rates were lower than or equal to 10 mRNAs/h. Some oligonucleotides whose occurrence frequencies in the first set of introns are significantly higher than those in the second set of introns were detected. The frequencies of occurrence of most of these detected oligonucleotides are also significantly higher than those in the exons flanking the introns of the first set. Interestingly some of these detected oligonucleotides are the same as well known "signature" sequences of transcriptional regulatory elements. This could imply the existence of potential positive regulatory motifs of transcription in yeast introns. (C) 2003 Elsevier Ltd. All rights reserved.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide Ligation assay on DNA chips

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates. (C) 2003 Elsevier B.V. All rights reserved.

Oligonucleotide-stabilized Ag nanoclusters as novel fluorescence probes for the highly selective and sensitive detection of the Hg2+ ion

Fluorescent oligonucleotide-stabilized Ag nanoclusters are demonstrated as novel and environmentally-friendly fluorescence probes for the determination of Hg2+ ions with a low detection limit and high selectivity.

Studies on the Interaction between Single-strand Oligonucleotide and Ginsenosides by Electrospray Ionization Mass Spectrometry

Oligonucleotide from SARS virus was selected as a target molecule in the paper. The noncovalent complexes of ginsenosides with the target molecule were investigated by electrospray ionization mass spectrometry. The effects of experimental conditions were examined firstly on the formation of noncovalent complexes. Based on the optimized experimental conditions, the interaction of different ginsenosides with the target molecule was researched, finding that the interaction orders are relative with the structure of aglycons, the length and terminal sugar types of saccharide chains in the ginsenosides. There are certain rules for the interaction between the ginsenosides and DNA target molecule. For different type ginsenosides, the interaction intensity takes the orders 20-S-protopanaxatriol > 20-S-protopanaxadiol, and panaxatriol ginsenosides > panaxadiol ginsenosides. For the ginsenosides with the same type aglycone, tri-saccharide chain > di-saccharide chain > tetra-saccharide chain and single-saccharide chain > panaxatriol. For the ginsenosides with the same tetra-saccharide chain, the ginsenosides with smaller molecule masses > the ginsenosides with larger molecule masses.

Diluting thiol-derivatized oligonucleotide monolayers on Au(111) by mercaptohexanol replacement reaction

Surface replacement reaction of thiol-derivatized, single-stranded oligonucleotide (HS-ssDNA) by mercaptohexanol (MCH) is investigated in order to reduce surface density of the HS-ssDNA adsorbed to Au(111) surface. Cyclic voltammograms (CVs) and scanning tunneling microscopy (STM) are employed to assess the composition and state of these mixed monolayers. It is found that each CV of mixed self-assembled monolayers (SAMs) only shows a single reductive desorption peak, which suggests that the resulted, mixed SAMs do not form discernable phase-separated domains. The peak potential gradually shifts to negative direction and the peak area increases step by step over the whole replacement process. By analyzing these peak areas, it is concluded that two MCH molecules will replace one HS-ssDNA molecule and relative coverage can also be estimated as a function of exposing time. The possible mechanism of the replacement reaction is also proposed. The DNA surface density exponentially reduces with the exposing time increasing, in other words, the replacement reaction is very fast in the first several hours and then gradually slows down. Moreover, the morphological change in the process is also followed by STM.

玉米 rbcL 基因与兰藻 rbcS 基因定向融合及它们在宿主大肠杆菌中的表达

二磷酸核酮糖羧化酶/氧化酶(简称Rubisco, EC, 4.1.1.39)是绿色植物光合作用中参与固定CO2的关键酶。在高等植物，该酶是由8个分子量为55KD的大亚基(LSU)和8个分子量为14KD的小亚基(SSU)构成的16聚体。每个大亚基有四个活性中心，具有双向催化功能，其编码基因位于叶绿体基因组大单拷贝区;小亚基功能还不清楚，它由核基因组编码且有几个拷贝;未成熟小亚基N端有一段transit peptide，靠它的定向跨越叶绿体膜。迄今为止，取自几种植物材料的这二种亚基的氨基酸顺序和编码基因的核苷酸顺序分析业已完成。为该酶的遗传操作奠定了必要的基础。 由于Rubisco与人类利用太阳能和提高作物产量直接相关，所以成为通过生物技术进行改造的重大项目。 巢状假囊细菌(Anacyslis nidulans) R2是一种不含限制性内切酶的单细胞原核生物，能营光合作用，其Rubisco大亚基的氨基酸顺序与玉米的LSU同源性高达80%，但是第四个活性部位(Leu 456位)与玉米不同(Sys, 459位)，由此导致其对CO2的亲合力降低。另一方面，其rbcL与rbcS仅相隔93个bp，且同属一个操纵子。这意味着有可能用同源DNA片段等位交换的办法来改造其rbcL基因。 根据现有的资料，设计出玉米rbcL与兰藻rbcS定向重组于pUC119的兰图：先从pANP1155中切出0.7kb含蓝藻rbcS的PstI-HindIII片段，克隆进pUC119的lacZ启动子下游得pTAS28，采用Reverse primer作引物进行核苷酸顺序分析，确认蓝藻rbcS基因座落在pTAS28正链上。随后从pZmc460中切出包含玉米rbcL基因1.7kb的BglII-HincII片段，将它插入pTAS28的HincⅡ-BamHⅠ双酶切位点，得到pTMN3;为了比较，在另一个质粒pTMN7于1.7kb片段之前加进0.1kb的PstI-HaeIII蓝藻DNA。根据玉米rbcL基因核苷酸顺序（1218-1251）合成一个Oligonucleotide probe，对这三个质粒的总RNA抽提物进行Northern Blot，得到明显的杂交斑点;接着用菌体总蛋白冻干品进行了Western分析，并以新鲜的玉米和烟草叶片为对照，得到阳性结果。显然这二种基因重组之后仍能在宿主E. coli中正常表达。 真正的挑战应是下一步用上述二种质粒转化兰藻A. nidulans R2，考查其能否整合进基因组并表现出较低的氧化酶活性。

植物高赖氨酸蛋白质的分离和纯化及其N端序列核苷酸探针的合成

禾谷类作物水稻和小麦是人们的主要植物性食物来源．而这些作物种子蛋白质中人类不能合成的必需氨基酸含量不平衡，造成了优质蛋白质的缺乏和人体对蛋白质利用的极大浪费．大约有二分之一的谷类种子蛋白质和四分之一的大豆蛋白质不能被合理地利用。大多数禾本科作物包括水稻和小麦的种子蛋白质中第一限制性氨基酸是赖氨酸，纠正其不平衡现象可大大提商蛋白质的营养价值。本研究在高赖氨酸植物种的筛选、高赖氨酸种子储藏蛋白质的纯化及其基因的分离等方面开展了工作。 选用与禾本科亲缘关系较远的8个植物种为研究材料，它们分别属于榛科，十字花科、胡桃科、豆科、胡麻科和松科。氨基酸组分分析确定豆科和十字花科的三个植物种赖氨酸含量在5.5%以上，其中豆科植物四棱豆(Pso phocarpus tetragonolobus)种子全蛋白赖氨酸含量达7.9%．用5种提取液提取了四棱豆种子的清蛋白、球蛋白和全蛋白。经测定发现0.025M Tris.HCl(pH7.4)提取液提取的清蛋白赖氨酸含量高最．通过自然胶电泳，SDS-PAGB电泳，非变牲IEF和变性IEF／SDS双向电泳，对四棱豆种子清蛋白进行了定性研究。用变性IEF/ SDS双向电泳分析出60多种蛋白质和蛋白质亚基及多肽。研究中改进了等电聚焦电泳纯化蛋白质的方法，经处理的胶板显现出清晰的蛋白质带型，不需染色即可确定带的位置，从切下的胶条中洗脱的蛋白质，其纯度达到双向电泳纯和HPLC纯。用三种电溶方法(SDS-PAGE非变性IEF，变性IEF)纯化出三十一种蛋白质或多肽分子。分别进行了分子量确定和氨基酸组分分析，发现了一个赖氨酸含量高达11.4%的蛋白质，其分子量为18KD，并制备了该蛋白质的抗体，测定了18KD蛋白质N端30个氨基酸残基的顺序，根据这一顺序设计合成了一组17个核苷酸的基因探针．经鉴定单链DNA探针的纯度和总量达到了设计要求。用尿素法与CTAB法结合提取了四棱豆幼苗核基因组DNA，其分子量在50Kb以上，达到了构建GenormicDNA文库的要求．用bamHI EcoRI和HindⅢ三种酶切割提取的DNA，得到了分子量大小不同的片段。 对四棱豆种子蛋白质的定性、高赖氨酸蛋白质的纯化、18KD蛋白N端序列分析及寡核苷酸探针的合成以及GcnomicDNA的提取与酶切，尚未见有资料报道．这些工作为克隆高赖氨酸基因打下了良好的基础，对改良禾本科作物蛋白质品质意义深远．

A COMPARATIVE-STUDY OF KARYOTYPES OF MUNTJACS BY CHROMOSOME PAINTING

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific ''paints'' from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4', 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.

Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree

We have made a set of chromosome-specific painting probes for the American mink by degenerate oligonucleotide primed-PCR (DOP-PCR) amplification of flow-sorted chromosomes. The painting probes were used to delimit homologous chromosomal segments among human, red fox, dog, cat and eight species of the family Mustelidae, including the European mink, steppe and forest polecats, least weasel, mountain weasel, Japanese sable, striped polecat, and badger. Based on the results of chromosome painting and G-banding, comparative maps between these species have been established. The integrated map demonstrates a high level of karyotype conservation among mustelid species. Comparative analysis of the conserved chromosomal segments among mustelids and outgroup species revealed 18 putative ancestral autosomal segments that probably represent the ancestral chromosomes, or chromosome arms, in the karyotype of the most recent ancestor of the family Mustelidae. The proposed 2n = 38 ancestral Mustelidae karyotype appears to have been retained in some modern mustelids, e.g., Martes, Lutra, ktonyx, and Vormela. The derivation of the mustelid karyotypes from the putative ancestral state resulted from centric fusions, fissions, the addition of heterochromatic arms, and occasional pericentric inversions. Our results confirm many of the evolutionary conclusions suggested by other data and strengthen the topology of the carnivore phylogenetic tree through the inclusion of genome-wide chromosome rearrangements. Copyright (C) 2002 S. KargerAG, Basel.

Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules

We have made a complete set of painting probes for the domestic horse by degenerate oligonucleotide-primed PCR amplification of flow-sorted horse chromosomes. The horse probes, together with a full set of those available for human, were hybridized onto metaphase chromosomes of human, horse and mule. Based on the hybridization results, we have generated genome-wide comparative chromosome maps involving the domestic horse, donkey and human. These maps define the overall distribution and boundaries of evolutionarily conserved chromosomal segments in the three genomes. Our results shed further light on the karyotypic relationships among these species and, in particular, the chromosomal rearrangements that underlie hybrid sterility and the occasional fertility of mules.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Localization of Sry gene on Y chromosome of Muntjac munticus vaginalis

The chromosomes 1, Y-1, Y-2 of Muntjac munticus vaginalis were isolated by fluorescence activated chromosome sorting and amplified by degenerate oligonucleotide primed-polymerase chain reaction ( DOP-PCR). A primer pair within human Sry HMG box was design

Transcription rates of yeast genes are influenced by the distribution of introns

A comparative analysis on the intron sequence oligonucleotide usages in two sets of yeast genes with higher and lower transcription frequencies, respectively, has shown that the intron sequence structures of the two sets of genes are different. There are more potential binding sites for transcription factors in the introns of the genes with high transcription frequencies. So it is speculated that introns regulate the transcription of genes. But more evidences are needed to favor this speculation. The detailed comparative analyses on the distribution ( length and position) of introns and exons in the two sets of gene sequences also show that there is an obvious boundary between the lengths of the two sets of introns. There is no boundary between the lengths of the two sets of exons, although the means of their lengths are of discrepancy. The situation of the gene lengths ( length of intron and exon) is similar to exon lengths. As far as the relative position, the introns in two sets of genes all have a bias toward the 5' ends of genes. But as the actual position is considered, more introns in high transcription genes have a tendency to be located toward the 5' ends of genes, some even located at 5'-UTR. These results suggest that the gene transcription rates are related to the length of intron, but not to the lengths of exons and genes sequences. The positions of introns may also influence the transcription rates. The transcriptional regulation of introns may be correlative with the transcriptional regulation of the upstream of genes, or be its continuous action.