华葵根瘤菌nifA基因的克隆和功能分析

华葵根瘤菌(Mesorhizobium huakuii即R.astragali)159的nifA基因的序列分析表明,该基因全长1227bp,编码分子量为44734D的Nif A蛋白。与其它NifA蛋白的序列比较发现,华葵根瘤菌NifA蛋白也存在保守的中间结构域和C末端DNA结合结构域,但其氨基端缺失。Tn5定点突变得到的突变体是Nif-表型。构建了nifA基因组成型表达的质粒,此质粒在大肠杆菌中对华葵根瘤菌nifHlacZ有激活作用。     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR 克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHAR dehydroascorbate reductase 基因的 cDNA 与 DNA 全长序列,命名为BoDHAR。并利用双链接头介导 PCR 的染色体步行技术（genome walking）克隆了其上游 644bp 的5′端序列。所获的BoDHAR基因全长 1486bp,存在两个内含子,DNA 编码区序列633bp,编码210个氨基酸;序列分析表明：BoDHAR与同源基因AT1G195701cDNA 序列有 82.3% 的一致性,推导的氨基酸序列有 79.6% 的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明：BoDHAR 在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。     

甜菜银叶病菌的PCR检测

本研究用16S23S rDNA间的ITS 序列通用引物L1（5′AGTCGTAACAAGGTAGCCGT3′）和L2 （5′ GTGCCAAGGCATCCACC3）扩增甜菜银叶病菌（Curtobacterium flaccumfaciens pv. betae,Cfb）和其它相近细菌的基因组DNA;并对其PCR产物进行回收、克隆和测序,将所获序列和其它已报道的细菌内源转录间隔区(Internally Transcribed Spacer,ITS)序列进行多重比较后设计出Cfb的特异性引物B1（5′GGCCTCGTGTTGTCCCTTATC3′）和B2 （5′GTCACCAATCAACAACCCGAG3′）。此引物可以从Cfb中扩增出387bp 的特异性片段,而其余参试的21个细菌PCR反应结果均为阴性。该方法可以应用于病害防治工作中的Cfb快速、可靠的检测。     

浑球红细菌谷氨酸合酶大亚单位基因(gltB)的序列分析

测定了浑球红细菌(Rhodobacter sphaeroides)谷氨酸合酶大亚单位基因(gltB)及其5'端和3'端的序列,全长为5510bp。序列分析表明,R. sphaeroides gltB基因全长为4636bp。从核苷酸序列推测其蛋白质分子量约为164kD。R. sphaeroides gltB基因与Azospirillum brasilense和Escherichia coli的gltB基因DNA序列有很高的同源性。其蛋白质氨基酸序列与A. brasilense gltB基因产物GltB也具有很高的同源性。此外,还对R. sphaeroides GltB的各可能功能区进行了分析,发现它们具有很高的保守性。     

钝齿棒杆菌天冬氨酸激酶基因的克隆和序列分析

运用PCR方法,从野生型钝齿棒杆菌株(Corynebacterium crenatum)AS1542及具有AEC抗性的突变株CD945染色体上分别扩增出天冬氨酸激酶(AK)基因(ask),构建了重组质粒。核苷酸序列分析表明,C.crenatum AS1542AK基因与C.crenatum CD945相比,第1199位的碱基由T变为C,引起酶蛋白β亚基第80位氨基酸从亮氨酸变成脯氨酸。该氨基酸的突变在蛋白结构上位于ACT结构域内,该区受赖氨酸调控。C.crenatum AS1542的AK基因的编码区核苷酸序列与C.glutamicum\,C.flavum及B.lactofermentum相比,同源性分别为97.23%、97.55%和97.62%,酶蛋白氨基酸序列的同源性分别为99.76%、99.52%和99.76%。但在AK基因的启动子上游序列部分与其它棒杆菌相比有较大差异。     

交替假单胞菌(Pseudoalteromonas sp.)DY3菌株产内切葡聚糖酶的性质研究及基因克隆

从深海样品ES0109中分离到一株具有高内切葡聚糖酶活力的细菌DY3, 16ＳrDNA序列分析表明该菌与交替假单胞菌属(Pseudoalteromonas sp.)的Pseudoalteromonas citrea和Pseudoalteromonas elyakovii的同源性为99%。PCR扩增DY3的内切葡聚糖酶基因cel-X全长1479bp,编码一个492AA的蛋白质。酶的氨基酸序列分析表明CelX与Pseudoalteromonas haloplanktis的内切葡聚糖酶CelG有95%的相似性,包括一个糖基水解酶家族5的催化结构域,一个连接序列和位于C端的的CBM5结构域。对酶性质的初步研究发现,CelX的最适温度为40 ℃,酶的最适pH在6～7之间。     

镰形扇头蜱两个组织蛋白酶L-样半胱氨酸蛋白酶新基因的克隆与序列分析

蜱是动物常见的外寄生虫,并且传播多种人和动物的疾病,严重危害畜牧业发展和人类健康。为了寻找基因工程疫苗候选抗原基因,根据半胱氨酸蛋白酶的保守性氨基酸序列及镰形扇头蜱氨基酸密码子偏好设计引物,PCR扩增、测序并分析得到2个镰形扇头蜱的半胱氨酸蛋白酶基因片段cysA和cysB,再通过RACE的方法得到全长基因序列。cysA全长168bp,编码332个氨基酸;cysB全长1153bp,编码335个氨基酸。经过分析, CysA和CysB均与其他蜱种或物种的组织蛋白酶L样半胱氨酸蛋白酶有高度同源性,两者均含有半胱氨酸蛋白酶活性位点处的保守性氨基酸序列, 因此cysA, cysB均为镰形扇头蜱两个新的组织蛋白酶L-样半胱氨酸蛋白酶基因。RT-PCR分析表明,CysA和CysB在镰形扇头蜱的不同发育阶段表达情况不一。     

苏云金芽胞杆菌鲇泽亚种HD-133cry1D的表达调控

利用PCR扩增基因cry1D启动子及上游区片段,在测序的基础上构建含cry1DlacZ融合基因穿梭质粒,导入不同遗传背景的苏云金芽胞杆菌菌株中,并以cry1AblacZ融合基因为对照测定β半乳糖苷酶活性,检测启动子上游区的作用。结果表明,cry1DlacZ和cry1AblacZ融合基因在不同遗传背景的菌株中表达完全不同,也许一些宿主专一性的因子参与了转录调控;而在同一菌株中Ccry1DlacZ和cry1AblacZ的表达差异是由于上游区的不同以及竞争有限的σ因子所致。利用PCR定点诱变技术突变其SD序列GGGGA为GGAGG后,cry1DlacZ融合基因的表达提高了1.0～1.6倍。表明GGAGG是苏云金芽胞杆菌合适的SD序列,也揭示了不合适的SD序列是cry1D表达量低的原因之一。     

一株多环芳烃降解菌的鉴定及GST基因克隆和序列分析

由石油污染土壤中分离到一株能以多环芳烃（菲、芴、萘）为唯一碳源的细菌,经形态观察、生理生化（BiologGN）和 G+C mol%分析,鉴定该菌为少动鞘氨醇单胞菌(Sphingomonas paucimobilis)。与16S rDNA序列同源性的比较进一步确证了鉴定结果。经菲诱导后的细菌谷胱甘肽S转移酶（Glutathione Stransferase, GST）酶活明显高于未诱导前,表明谷胱甘肽S转移酶可能与多环芳烃的降解有关。根据该酶基因的同源性序列设计引物,PCR扩增出编码谷胱甘肽S转移酶基因片段,进一步证实在该菌中有GST的存在。测序后基于编码GST的基因所进行的系统发育分析表明,该多环芳烃降解菌与其它多环芳烃降解菌在进化上亲缘关系较近。     

菊粉酶酶源菌株筛选及其基因克隆

筛选出一株产菊粉酶酶源菌株AF10,鉴定为黑曲霉(Aspergillus niger)。PCR扩增AF10的内切菊粉酶基因inuA1并进行核苷酸序列分析。结果表明inuA1全长1551bp,没有内含子,所编码的氨基酸序列中,存在4个潜在的N糖基化位点,其中存在菊粉酶的保守序列WMNEPN。以pUC118为克隆载体,以E.coli JM109为受体菌株,获得菌粉酶基因克隆。     

An ABC transporter homologous to TAP proteins.

Polymerase chain reaction amplification of cDNA from rat intestine revealed the expression of a novel ABC transporter, TAPL (TAP-like). Subsequently, the protein sequence was deduced from the nucleotide sequence of cDNA carrying the entire coding region. TAPL is transcribed ubiquitously in various rat tissues. The protein, with 762 amino acid residues, has potential transmembrane domains, and an ATP-binding domain in its amino and carboxyl terminal regions, respectively, and is highly homologous to TAP1 and TAP2 (transporters associated with antigen presentation/processing): pairwise comparisons with TAPL demonstrated 39 and 41% of the residues are identical, respectively. These numerical values are essentially the same as that for TAP1 and TAP2 (39%), and the hydropathy profiles of TAPL, TAP1 and TAP2 are quite similar. The similarity among these three proteins suggests that they could be derived from a common ancestral gene. Furthermore, we found that there is a potential splicing isoform, sharing the amino terminal 720 amino acid residues of TAPL.     

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.     

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.     

Mutagenesis study on the zebra fish SOX9 high-mobility group: comparison of sequence and non-sequence specific HMG domains

A unique class of proteins, containing high-mobility group (HMG) domain(s), recognizes unusual DNA structures and/or bends specific to AT-rich linear double-stranded DNA. The DNA binding feature of these proteins is exhibited in the HMG domain(s). Although the sequence specific and non-sequence specific HMG domains exhibit very high degrees of sequence similarity, the reasons for the difference between their DNA recognition mechanisms are unclear. A series of zebra fish SOX9 HMG domain mutants was prepared in an effort to elucidate the importance of various residues on protein stability and DNA binding. This study is the first of a comprehensive mutagenesis study on a sequence specific HMG domain. Comparing how various residues influence sequence specific and non-sequence specific HMG domains helps us to rationalize their mode of action. Positively charged amino acids concentrated at the surface of sequence specific HMG domains recognize specific, linear AT-rich DNA segments. After the negative charges at the surface of the DNA are neutralized, the hydrophobic residues of the protein may intercalate DNA. Phenylalanine at position 12 plays a crucial role in the sequence specific HMG domain. The differences in pI values, the instability index, and DNA contact regions between sequence and non-sequence specific HMG domains are associated with their functional modes.     

Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene

We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.     

A murine sequence-specific DNA binding protein shows extensive local similarities to the amyloid precursor protein.

Microinjection experiments suggested previously that protein binding to the DNA nucleotide sequence GTCACATG, identical to the CDEI element of the yeast centromere, plays an important role in the early development of the mouse. We established from a series of overlapping mouse cDNA clones the sequence of a candidate CDEI-binding protein. Synthesis in Escherichia coli of a fusion protein which binds specifically the CDEI box in vitro confirmed its identification. On the other hand, the translated 511 amino acid sequence shows two regions with high degrees of similarity to the protein precursor (APP) of the beta-protein (amyloid) that accumulates in the brain and blood vessels of Alzheimer patients. A continuous stretch of 195 amino acids includes 133 residues identical to part of the extracellular domain of APP, and 48 of the 70 C-terminal residues of the open reading frame are identical to the APP transmembrane and cytoplasmic domains.     

Characterization of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni

To identify and characterize Schistosoma mansoni proteins that are recognized by infected hosts, we have used a pool of sera from infected humans to screen cDNA libraries constructed from poly(A)+ mRNA of adult S. mansoni. The deduced amino acid sequences of the three isolated clones showed a high degree of similarity to the large subunit of calcium-activated neutral proteinase (CANP) from humans and chicken. These overlapping clones, which include a nearly full-length clone with an open reading frame of 758 amino acid residues, together encode the entire large subunit of CANP. The deduced sequence of this S. mansoni protein can be divided into four domains (I-IV) that include the two domains characteristic of other large subunits of CANP: a thiol-protease domain (II) and a calcium-binding domain (IV) containing EF hand motifs. However, the schistosome protein is unique in having only three EF hand motifs in the calcium-binding domain and in having an additional EF hand motif that is shared between domains II and III. We have shown that these EF hand motifs are capable of binding 45Ca2+. Furthermore, the large subunit is S. mansoni contains an NH2-terminal sequence of 28 residues that is absent from the mammalian CANPs and has a high degree of similarity to the presumed receptor binding sequence of colicin Ia and Ib.