DNA指纹技术在生物鉴种中的应用

通过DNA指纹技术可以看到同种生物DNA序列的微小差异。使用限制性核酸内切酶酶切DNA,将有差异DNA片断通过电泳,与样本DNA比对,以判断出哪个是需要的样本。本文利用PCR分析,鉴别未知物种种类,18cDNA测序,并进行BLAST比对,就可以观察到不同物种DNA非保守序列间差异,从而鉴定物种的种类及其在进化树上所处的生态位置。在珍稀物种保护等方面有一定指导意义。     

寡聚核苷酸诱导突变法改造φ×174噬菌体A基因蛋白的DNA识别序列

 为了进一步研究φX174噬菌体A基因蛋白的复制功能与其所识别的30核苷酸保守序列的关系,我们采用寡聚核苷酸诱导的定点突变法成功地改造了这30核苷酸保守序列。将此保守序列重组到M_(13)mp9噬菌体后,以其单链为模板,在14或16寡聚核苷酸的诱导下,合成共价闭环DNA。经转化到E.coli JM103菌株,用点印迹(Dot blot)杂交法筛选,得到两种重组突变株。一种突变株其30核苷酸保守序列正链的第22碱基由A改为G。另一突变株为其第10碱基A改为C,第11碱基T改为A。突变效率约为5％。制备了此突变株单链及双链DNA,分别做了双脱氧末端终止法及Maxam和Gilbert法序列分析鉴定。     

DNA保守序列识别算法的并行化和MPI集群环境构建

DNA序列中保守序列的识别需要较大的计算量。开发了一个转录因子结合位点识别的并行算法,能够从多条DNA序列中识别指定长度的序列模式。算法使用概率模型进行序列模式保守性的度量,利用迭代过程实现保守序列的搜索。使用C编程结合MPI消息传递模型开发了相应的程序,并在Windows平台下构建了一个3节点的集群环境,利用20个长度均为200的序列数据集进行测试,实现了模体识别工作,结果表明并行算法使模体识别的效率得到提高。     

马铃薯块茎组织特异性启动子的克隆及序列分析

利用PCR技术从马铃薯陇薯3号基因组DNA中扩增出长度约为1.0 kb的DNA片段,经与T载体连接,测序表明,克隆到的DNA片段大小为969 bp,该序列与GenBank中已公布的patatin启动子序列同源性为97.94％;采用植物顺式调控元件数据库PLACE和PlantCare进行序列分析,结果表明,该片段含有启动子的保守序列TATA-box和CAAT-box,且在CAAT-box上游有8 bp的增强子.此外,还具有马铃薯块茎蛋白patatin基因启动子保守调控序列的蔗糖效应元件(SURE)4个及马铃薯储藏物特异结合调控patatin蛋白表达位点(B-box)2个,而这些特异序列可能是基因特异表达所必须的.     

平菇产植酸酶菌株的筛选及植酸酶基因区域的克隆

从平菇（Pleurotus ostreatus）8个菌株中筛选出3株高产植酸酶菌株,并根据GenBank中植酸酶基因的保守区设计并合成一对特异性引物,以平菇菌丝的总DNA为模板,通过PCR扩增,获得了一条长约920 bp的片段.DNA序列测定结果表明,该片段长度为919 bp.采用blast进行序列比对,结果表明：该片段与曾报道的源于Trametes pubescens的植酸酶phyA（GenBank Accession：AJ310700）基因相比较,其DNA序列同源性为93%.该片段含有3个内含子,含有植酸酶基因的活性位点保守序列（Active-site sequence）RHGARYPT.     

利用序列比较寻找胰岛素基因表达启动区与DNA结合蛋白的结合位点

NDFl、IPFl和HNF4是与胰岛素基因表达有关的DNA结合蛋白,通过比较SWISSPROT蛋白质数据库中人类、小鼠、大鼠这三种核蛋白氨基酸一级序列、模体和结构域,发现其结构十分相似,根据蛋白质结构和功能的关系,推测这些DNA结合蛋白与胰岛素基因结合的核苷酸序列相似;从GenBanl(核酸数据库中获得人类、小鼠、大鼠胰岛素DNA序列,用ClustalW比较三者Promoter区的核苷酸序列,显示有一段核苷酸序列较为相似,同时搜索TRANSFAC基因转录数据库中NDFl、IPFl和NHF4蛋白核苷酸结合位点,发现核酸比对保守的部分序列与TRANSFAC数据库中这三个转录因子的DNA结合位点一致,另外一些核酸保守序列可能为其他未知DNA结合蛋白的结合位点。这种核酸序列比对设计为分子生物学实验寻找和验证胰岛素DNA结合蛋白与核苷酸的结合位点提供了简单而实用的方法。     

用微型计算机在TSr(Bg1Ⅱ)-1核苷酸顺序中寻找相似序列

<正> 为了在TSr(Bgl Ⅱ-1)核苷酸顺序中寻找有无类似α—顺序的冷点区顺序,我们在微型电子计算机上编制了查找相似序列顺序(WSS程序)。本程序的特点是运行速度快,无重复扫描,可自行选择欲查找的相似百分比和百分比精度,它不仅适用于寻找内切酶的酶切点,而且可以在相当长度的已测序DNA顺序中快速准确地检出碱基位置和数量发生随机变异的DNA相似片段,并直接计算、打印出相似百分比值。     

蒙古绵羊 tnp1基因cDNA全编码区的克隆及序列分析

过渡蛋白1基因(tnp1)是圆形精子细胞特异表达的基因.绵羊tnp1基因的DNA序列至今尚未报道.为了开展绵羊圆形精子细胞标记基因的研究,根据其他物种tnp1基因cDNA的保守序列设计引物,从成年蒙古绵羊睾丸中提取总RNA,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp1基因cDNA全编码区.该基因cDNA 长246 bp,包含一个168 bp的ORF,编码含有54个氨基酸的多肽链.DNA序列测定结果与牛的核苷酸序列比对,同源性为94.0%.绵羊tnp1基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础.     

南丰蜜橘β-胡萝卜素羟化酶基因的克隆和序列分析

以南丰蜜橘[Citrus reticulata Blanco var.kinokuni(Tanaka)H.H.Hu]基因组DNA为模板,根据已报道的柑橘β-胡萝卜素羟化酶基因保守序列设计4对引物,进行PCR扩增,得到4条长度为470、761、294和991 bp的片段.将这些片段克隆到pMD18-T载体,并进行测序.测序结果拼接成1条2 326 bp的序列.分析发现该序列含6个内含子,7个外显子.内含子两侧有典型的GT-AG保守序列.该序列中预测的编码序列与温州蜜柑、拟南芥等植物的β-胡萝卜素羟化酶基因序列保守性达80%以上,表明该序列确实为β-胡萝卜素羟化酶基因.该基因编码了1个含311个氨基酸的蛋白.将该序列递交到GenBank数据库,序列号为AM408552.     

玉米特异DNA通过有性杂交导入小麦DH后代的分子证据

构建了玉米的随机基因组文库,筛选了近100个玉米基因组特异的重复DNA克隆,用它们作探针分别对2个来自小麦×玉米的小麦DH群体进行RFLP分析.结果发现,玉米的MR64克隆导入到2个小麦群体的各1株后代中,即在普通小麦DH系的18号株和波斯小麦DH系的15号株中检测到强杂交信号,这个结果首次从DNA水平上证明某些玉米特异的DNA序列,可通过受精作用以很低的频率转移到小麦DH后代的基因组中.测序分析证实,克隆MR64的插入片段长度为695Pb,A+T含量为58%,用多种酶切玉米基因组进行RFLP分析表明它是一个带1～3个主串联重复单位的散布重复序列.同时还对它的序列结构、甲基化图谱、拷贝数和在玉米染色体上的分布以及在其它小麦品种和禾本科种基因组中的序列同源性情况进行了详细的研究.     

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain. Comparison of predicted amino acid sequences from G-protein alpha-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively.     

Localization of mRNAs encoding the alpha-subunits of signal-transducing G-proteins within rat brain and among peripheral tissues

The sequence of the mRNAs which encode the alpha-subunits of the signal-transducing G-proteins Gs, Go and two forms of Gi (termed Gi1 and Gi2) have recently been reported. Based on rat sequences we prepared oligodeoxynucleotide probes for measurement of these mRNAs in rat brain and peripheral tissues. The relative abundance of these mRNA species in brain was Gs greater than Go approximately Gi2 greater than Gi1. The Gs and Gi2 mRNAs had somewhat lower levels in heart, kidney and liver than in brain, and Go and Gi1 mRNAs were not detected in the peripheral tissues. Using in situ hybridization we localized each of these mRNAs within slices of the rat brain. The patterns of distribution of Gs and Gi2 mRNA were very similar, but very different from that of Go and Gi1 mRNA. These data illustrate that receptor-effector coupling G-proteins are regionally specialized in their expression. This regional specialization may reflect a selective coupling of individual G-proteins with the various neurotransmitter receptors and effector pathways.     

Web-based programs for the display and analysis of transmembrane alpha-helices in aligned protein sequences

We developed novel programs for displaying and analyzing the transmembrane alpha-helical segments (TMSs) in the aligned sequences of homologous integral membrane proteins. TMS_ALIGN predicts the positions of putative TMSs in multiply aligned protein sequences and graphically shows the TMSs in the alignment. TMS_SPLIT (1). predicts the positions of TMSs for each sequence; (2). allows a user to select proteins with a specified number of TMSs, and (3). splits the sequences into groups of TMSs of equal numbers. TMS_CUT works like TMS_SPLIT, but it can cut sequences with any combination of TMSs. The BASS program similarly allows comparison of protein repeat elements, equivalent to TMS_SPLIT plus IC, but it provides the comparison data expressed in BLAST E values. These programs, together with the IntraCompare program, facilitate the identification of repeat sequences in integral membrane proteins. They also facilitate the estimation of protein topology and the determination of evolutionary pathways.     

Quantitation of inhibitory G-proteins in fat cells of obese and normal-weight human subjects

Antisera were raised in rabbits against synthetic peptides corresponding to sequences of the guanine nucleotide binding proteins Gi1, Gi2, Gi3 and Go. These and previously described antisera were used to identify different G-proteins in Western blots of human adipocyte plasma membranes and to quantify them using purified recombinant α subunits as standards. Go was shown to be absent or ⪡ 15 pmol/mg of protein. A band stained by a previously characterized Go antiserum is suggested to be due to nonspecific staining of Gi1. Gi1 and Gi2 were the major G-proteins. Gi1 was present at concentrations of 52 and 18 pmol/mg of protein in lean and obese subjects, respectively, and the concentration was negatively correlated with the body mass index. Gi2 concentrations averaged 64 pmol/mg of protein and there was no correlation to the body mass index. Gi3 levels were much lower (⪡ 13 pmol/mg of protein) and the presence of this protein could not be demonstrated with certainty. The concentrations of Gi₁ and Gi₂ are thus over two orders of magnitude higher than those of the receptors whose effects they mediate. The low concentration of Gi1 in adipocyte plasma membranes of obese subjects could in part explain the attenuated inhibitory responses of adenylate cyclase in isolated fat cells in obesity.     

Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system

The selectivity of D2 dopamine receptor-guanine nucleotide-binding protein (G protein) coupling was studied by reconstitution techniques utilizing purified D2 dopamine receptors from bovine anterior pituitary and resolved G proteins from bovine brain, bovine pituitary, and human erythrocyte. Titration of a fixed receptor concentration with varying G protein concentrations revealed two aspects of receptor-G protein coupling. First, Gi2 appeared to couple selectively with the D2 receptor with approximately 10-fold higher affinity than any other tested Gi subtype. Second, the G proteins differed in the maximal receptor-mediated agonist stimulation of the intrinsic GTPase activity. Gi2 appeared to be maximally stimulated by agonist-receptor complex with turnover numbers of approximately 2 min-1. The other Gi subtypes, Gi1 and Gi3, could be only partially activated, resulting in maximal rates of GTPase of approximately 1 min-1. Agonist-stimulated GTPase activity was not detected in preparations containing Go from bovine brain. The differences in maximal agonist-stimulated GTPase rates observed among the Gi subtypes could be explained by differences in agonist-promoted guanyl nucleotide exchange. Both guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding and GDP release parameters were enhanced 2-fold for the Gi2 subtype over the other Gi subtypes. These results suggest that even though several types of pertussis toxin substrate may exist in most tissues, a receptor may interact discretely with G proteins, thereby dictating signal transduction mechanisms.     

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.     

Isoprenylation of C-terminal cysteine in a G-protein gamma subunit

The predicted amino acid sequences for the Gi alpha 1 and G gamma 6 subunits of brain heterotrimeric G-proteins both contain C-terminal Cys-A-A-X elements (A is an aliphatic residue and X is any amino acid). This domain represents the site of Cys thioether modification by isoprenoids in p21ras, nuclear lamins, and fungal mating factors. We now show that G gamma 6, translated in reticulocyte lysate, is efficiently labeled with the isoprenoid precursor, [3H]mevalonate. Alteration of the sequence of G gamma 6 so that a Gly was substituted for Cys in the C-terminal Cys-A-A-X element rendered the protein incapable of undergoing isoprenoid modification. In contrast to G gamma 6, the Gi alpha 1 subunit did not appear to undergo isoprenylation when translated in reticulocyte lysate. Transient expression of the protein in COS cells, which were able to isoprenylate the p21 product of transfected H-ras, also failed to demonstrate isoprenylation of Gi alpha 1. The modification of the gamma subunit by a hydrophobic moiety may have important implications for the assembly of the brain G-protein beta gamma complexes into the cell membrane.     

Clustering cDNA sequences

A set of programs has been written to quantify the similaritiesbetween large numbers of cDNA sequences. This information isused to cluster similar sequences together. The main programcan cluster thousands ofcDNA sequences per day using a novel,computationally inexpensive algorithm. The clustering informationis kept in a small index file so that disk storage requirementsare negligible. Using this index file, subsidiary programs createvarious views and statistical summaries of the entire cDNA sequencecollection.     

DePIE: Designing Primers for Protein Interaction Experiments

Several primer prediction and analysis programs have been developed for diverse applications. However, none of these existing programs can be directly used for the design of primers in protein interaction experiments, since proteins may have transmembrane domains (TMDs) and/or a signal peptide that must be excluded from experiments. Furthermore, it is frequently the case that a short restriction sequences must be added to each primer in order to clone PCR products into a given destination vectors for expression. DePIE, a web-based primer design tool, was developed to address these deficiencies. The program takes as input NCBI protein accession numbers and returns primer information including nucleotide sequences, thermodynamic melting temperature of the nucleotide sequences and the target positions. DePIE is implemented in JAVA, PERL and PHP and has proven to be very efficient in designing primers for our interaction experiments. DePIE services can be accessed at the web site: http://biocore.unl.edu/primer/primerPI.html.