异源基因α1,3半乳糖转移酶与增强型绿色荧光蛋白融合对荧光蛋白表达的影响

目的 研究异源(猪)基因α1,3半乳糖转移酶(3GT)与增强型绿色荧光蛋白(EGFP)基因形成的融合蛋白对其荧光表达量的影响.方法 BamHI,EcoRI酶切pcDNA3.1-α1,3GT重组载体后,回收含α1,3GT的片段,与BamHI、EcoRI酶切回收的pEGFP-N1载体连接,并酶切、测序鉴定重组真核表达载体p...     

含非天然氨基酸定点突变的MLL3SET蛋白表达与纯化

在组蛋白H3K4甲基转移酶MLL3的催化结构域(MLL3SET)中定点引入非天然氨基酸N-炔丙基赖氨酸(N-propargyl-lysine,PrK),表达、纯化该突变蛋白(MLL3SET*),并评估突变蛋白的酶活,为后续进一步利用单分子荧光共振能量转移技术(smFRET)表征MLL3的作用机制奠定基础.将MLL3SE...     

无芒隐子草CsPEAMT基因克隆及表达特性分析

以无芒隐子草(Cleistogenes songorica)干旱胁迫下的cDNA文库中磷酸乙醇胺N-甲基转移酶(phosphoethanolamine N-methyltransferase,PEAMT)基因的EST序列为基础,采用RACE方法克隆该基因编码区序列,该序列全长为2 104bp,开放读码框1 506bp,编码501个氨基酸。无芒隐子草PEAMT蛋白编码的氨基酸序列与多种植物的PEAMT氨基酸序列有较高相似性,其中与高粱SbPEAMT、玉米ZmPEAMT的蛋白序列相似性最高(93%),说明PEAMT基因在植物进化中非常保守。采用实时定量RT-PCR分析无芒隐子草幼苗在干旱过程中CsPEAMT基因的表达结果显示,干旱胁迫诱导CsPEAMT基因在根和叶中大量表达,且在干旱第8天时CsPEAMT基因在叶和根中表达量分别是未干旱对照的43.35倍和13.25倍,复水后CsPEAMT基因的表达量开始下调。研究表明CsPEAMT基因可能是无芒隐子草抗旱性相关的基因。     

巴西橡胶树顺式异戊烯基转移酶基因cDNA克隆及其序列特征分析

以巴西橡胶树(Hevea brasiliensis)胶乳的RNA为Tester;叶片RNA为Driver,利用抑制消减杂交法(suppressive subtractive hybridization,SSH)构建了一个胶乳特异表达基因差减文库.通过反式Northern点杂交(reverse Northern dot blots)筛选到一个与顺式异戊烯基转移酶基因(橡胶生物合成的关键酶基因)高度同源的阳性克隆R363.采用RACE方法获得该克隆的全长cDNA(GenBank登陆号:AY461414).序列分析表明,该基因长1156 bp,含有873 bp的阅读框,编码290个氨基酸,分子量约为32.9 kD,等电点为7.2,含有N-端跨膜螺旋区.同源性分析表明R363编码的蛋白质具有异戊烯基转移酶家族的特征,含有cis-异戊烯基链转移酶的5个高度保守区,推测R363可能是一种新的顺式-异戊烯基转移酶基因.Northern blot分析显示,R363在胶乳中高度表达,在叶中不表达.乙烯处理前后表达强度一致,表明该基因表达不为乙烯所诱导.     

来源于大肠杆菌的NMN转移酶的克隆表达与酶学性质研究

在生物体内,NMN(烟酰胺单核苷酸)转移酶能够催化NMN生成NAD.本研究通过构建重组表达质粒pET30α(+)-Nmnat,成功实现来源于大肠杆菌的NMN转移酶基因(Nmnat)的原核表达.从大肠杆菌基因组中克隆得到的NMN转移酶基因长度为1 245 bp,所编码的重组酶分子量45 kDa.对重组酶的酶学性质进行分析,结果显示该酶最适反应温度和pH分别为37℃和7.5.4℃下,该酶的热失活半衰期可长达990.2 min.Mn2+、Fe2对该酶的酶活的激活作用显著,而EDTA对酶活能造成明显的抑制作用.酶动力学分析结果显示,该酶对底物NMN催化的Km和Vmax分别为16.89 mmol/L和2.46 μmol/(L·min).该NMN转移酶基因在大肠杆菌宿主中的成功表达,为NAD生物合成应用研究奠定了基础.     

对大肠杆菌中表达蛋白质精氨酸甲基转移酶5的几个缺失突变体的活性研究

蛋白质精氨酸甲基转移酶5(PRMT5)在细胞生长和信号转导方面是一个重要的调节因子,主要参与染色质重塑、RNA剪切、基因转录、细胞分化等过程.因此,对其结构和功能的研究就显得十分重要.通过大肠杆菌表达系统把全长基因PRMT5构建到pGEX-4T-1表达载体上,所得到GST标签的重组蛋白可溶性很低.为此,通过在其N端缺失不同氨基酸序列来增加其表达量,而且其中有一个缺失突变体的活性并没有发生改变.同时,还发现PRMT5 N端的前15个氨基酸对其甲基转移酶的催化活性很重要.     

甲醇利用菌SDM11中glyA基因的克隆及其在大肠杆菌中的表达

对甲醇降解菌Methylobacterium.sp SDM11中的glyA基因进行克隆及特性研究,以获得更多的丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)资源。根据GenBank中已报道的Methylobacterium extorquensAM1中的glyA基因序列(登录号:L33463)设计引物,以SDM11的基因组DNA为模板,PCR扩增glyA基因。利用pETblue-2载体将该基因在大肠杆菌BL21(DE3)中得到表达。PCR扩增到一个1.40 kb大小的DNA片段,经过blast软件比对分析,发现该片段与已报道的Methylobacterium extorquensAM1的glyA基因的序列相似性为95%,氨基酸序列的相似性为98%。该基因编码468个氨基酸,预测的分子量大小为52.2 kD,等电点为7.02,发现纯化后的目标蛋白具有SHMT酶活性,并初步测定了酶活力。     

牛樟芝中一个新型还原型聚酮合酶基因的克隆及表达分析

为了研究牛樟芝中PKS基因与化合物之间的关系,该研究通过对牛樟芝基因组分析获得牛樟芝聚酮合酶基因,以此序列为模板设计含有起始密码子和终止密码子的特异引物并以牛樟芝c DNA为模板克隆获得一个高度还原型PKS(HR-PKS)基因全长,命名为AcPKS2;对AcPKS2基因进行生物信息学分析,并比较该基因在不同培养基上的表达量。结果表明:AcPKS2全长7 842 bp,有24个内含子,其外显子共编码2 613个氨基酸,该蛋白的相对分子质量为293.5 kDa,理论等电点pI为5.78。用CDD分析其结构域显示,该基因属于HR-PKS,其结构域组织排列为KS-AT-DH-MT-ER-KR-ACP-TE,8个结构域其活性位点分别为β-酮基合成酶(DTACSSSL)、酰基转移酶(GHSIGETA)、脱水酶(RNDGSTSPL)、甲基转移酶(SFDIITAFDV)、烯酰还原酶(HAGVSSPAA)、酮基还原酶(GSPGQANYTAA)、酰基转移酶(YGLDSLTSVRL)、硫酯酶(KQPNGPY)。系统发育树显示AcPKS2与其他化合物未知的HR-PKS蛋白聚为一支,结构域和系统进化树分析显示该基因可能编码一种新的含TE结构域高度还原型聚酮合酶;表达分析结果显示葡萄糖和果糖能够诱导该基因的表达。     

金鱼草AmHMGS基因的克隆及表达特征分析

植物萜烯类化合物合成主要通过MVA和MEP途径,这些萜烯类化合物在植物生长、发育过程发挥着重要作用,萜烯类化合物在植物花香挥发成分中占有大比率.3-羟基-3-甲基戊二酰辅酶A合成酶基因(HMGS)是MVA途径中的关键酶基因,该酶作用主要是催化底物乙酰辅酶A和乙酰乙酰辅酶A生成3-羟基-3-甲基戊二酰辅酶A(HMG-CoA),是合成萜烯类化合物的前体限速酶.本研究以'马里兰'金鱼草(Antirrhinum majus'Maryland')为材料,克隆AmHMGS,基因全长1 383 bp,编码460个氨基酸,时空和组织特异性RT-qPCR表达分析表明,AmHMGS基因在花中表达量显著高于根茎叶;初开期的表达量最高;在盛花期的不同花器官中,AmHMGS基因在雄蕊和上瓣中表达量最高,与其它花器官中的表达量差异显著.用茉莉酸抑制剂不同浓度的菲尼酮处理盛开期金鱼草花瓣,RT-qPCR分析HMGS基因表达量结果表明,菲尼酮处理后能抑制该基因的表达.本研究的结果为明确AmHMGS基因在金鱼草中的表达模式,为今后研究该基因的功能及其在金鱼草花香释放中的信号作用奠定基础.     

杀粉蝶菌素A1生物合成基因簇中甲基转移酶PieB2的功能

【目的】研究杀粉蝶菌素A1产生菌中甲基转移酶基因pieB2的功能。【方法】利用接合转移和同源重组双交换的方法,构建pieB2基因缺失突变株,以及利用接合转移的方法,构建回补菌株。通过高保真PCR克隆pieB2基因到表达载体pET28a上,构建质粒pJTU5997,转化入大肠杆菌E.coliBL21(DE3)/pLysE中诱导表达。利用高效液相色谱检测PieB2的体外酶活。【结果】获得了pieB2基因缺失的双交换突变株。发酵结果显示,该突变株不再产生杀粉蝶菌素A1,而是积累了一种脱甲基产物。N-末端融合组氨酸标签的PieB2在大肠杆菌中获得可溶性表达,通过体外催化证明了PieB2甲基转移酶的功能。【结论】体内遗传实验和体外生化实验证明了PieB2作为甲基转移酶在杀粉蝶菌素A1合成中的作用。     

Isolation of BamHI variants with reduced cleavage activities

Derivation of the bamhIR sequence (Brooks, J. E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C. C., Mazzola, L. M., Slatko, B. E., and Benner, J. S. (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W. E. Jack, L. Greenough, L. F. Dorner, S. Y. Xu, T. Strezelecka, A. K. Aggarwal, and I. Schildkraut, submitted for publication). As expected, low-level constitutive expression of the bamhIR gene in E. coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell. We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities. Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities. Variants with residual cleavage activities induced the E. coli SOS response and thus are presumed to cleave chromosomal DNA in vivo. We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

Trimethylamine dehydrogenase of bacterium W3A1. Molecular cloning, sequence determination and over-expression of the gene.

The gene encoding trimethylamine dehydrogenase (EC 1.5.99.7) from bacterium W3A1 has been cloned. Using the polymerase chain reaction a 530 bp DNA fragment encoding a distal part of the gene was amplified. Using this fragment of DNA as a probe, a clone was then isolated as a 4.5 kb BamHI fragment and shown to encode residues 34 to 729 of trimethylamine dehydrogenase. The polymerase chain reaction was used also to isolate the DNA encoding the missing N-terminal part of the gene. The complete open reading frame contained 2,190 base pairs coding for the processed protein of 729 amino acids which lacks the N-terminal methionine residue. The high-level expression of the gene in Escherichia coli was achieved by the construction of an expression vector derived from the plasmid pKK223-3. The cloning and sequence analysis described here complete the partial assignment of the amino acid sequence derived from chemical sequence [1] and will now permit the refinement of the crystallographic structure of trimethylamine dehydrogenase and also a detailed investigation of the mechanism and properties of the enzyme by protein engineering.     

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.     

Cloning of human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG2) to 1p36.1----p35 and identification of a BamHI restriction fragment length polymorphism.

We have isolated a cDNA coding for the core protein of the large basement membrane heparan sulfate proteoglycan (HSPG) from a human fibrosarcoma cell (HT1080) library. The library was screened with a mouse cDNA probe and one clone obtained, with a 1.5-kb insert, was isolated and sequenced. The sequence contained an open reading frame coding for 507 amino acid residues with a 84% identity to the corresponding mouse sequence. This amino acid sequence contained several cysteine-rich internal repeats similar to those found in component chains of laminin. The HSPG cDNA clone was used to assign the gene (HSPG2) to the p36.1----p35 region of chromosome 1 using both somatic cell hybrid and in situ hybridization. In the study of the polymorphisms of the locus, a BamHI restriction fragment length polymorphism was identified in the gene. This polymorphism displayed bands of 23 and 12 kb with allele frequencies of 76 and 24%, respectively.     

Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.     

Liposomes for microcompartmentation of enzymes and their influence on catalytic activity

Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of phosphatidylcholine (PC), has an important role in regulating the acyl group of PC in mammalian cells. A 593bp cDNA coding for the 3(')-end of the CPT gene has been cloned from guinea pig liver using degenerative oligos based on the human CPT gene. It has 85% amino acid homology with the human CPT enzyme and amino acid variations were found to cluster at few points. Restriction enzyme polymorphisms were found particularly with respect to BamHI and NcoI. Hydrophobic and helix plot analysis of the sequence shows a similar pattern to human counterpart except for amino acid residues 142-179 and 173-179. PCR analysis suggested that a predominant pseudogene may be present in guinea pig and also the intronic sequences were much shorter when compared to the human CPT gene. We are the first to report on the C-terminal 195 amino acid residues of the CPT gene from any animal species alike in many aspects of cellular metabolism. The probable differences in genomic organization and its expression in different cancer cells have been discussed here having CPT as an important target for cancer drug development.     

Cloning and sequencing of pel gene responsible for CMCase activity from Erwinia chrysanthemi PY35

The phytopathogenic bacterium Erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. We cloned genomic DNA from Erwinia chrysanthemi PY35. One of the E. coli XL1-Blue clones contained a 5.1-kb BamHI fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning, we obtained a 2.9-kb fragment (pPY100) that contained the pel gene responsible for CMCase and pectate lyase activities. The pel gene had an open reading frame (ORF) of 1,278 bp encoding 425 amino acids with a signal peptide of 25 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of PelL of E. chrysanthemi EC16, we concluded that it belonged to the pectate lyase family EC 4.2.2.2, and we designated it PelL1. Sequencing showed that the PeIL1 protein contains 400 amino acids and has a calculated pI of 7.15 and a molecular mass of 42,925 Da. The molecular mass of PelL1 protein expressed in E. coli XL1-Blue, as analyzed by SDS-PAGE, appeared to be 43 kDa. The optimum pH for its enzymatic activity was 9, and the optimum temperature was about 40 decreased C.     

Isolation of novel herpes simplex virus type 1 derivatives with tandem duplications of DNA sequences encoding immediate-early mRNA-5 and an origin of replication.

Two naturally occurring variations of herpes simplex virus type 1 (Patton strain) with novel tandem DNA sequence duplications in the S component were isolated, and the DNA was characterized. These variants were identified among a number of plaque isolates by the appearance of new restriction enzyme fragments that hybridized with radiolabeled DNA from the BamHI Z fragment (map coordinates 0.936 to 0.949) located in the unique S region. One isolate, SP26-3, carried a 3.1-kilobase-pair duplication defined by recombination between a site in the BamHI Z fragment and a site near the origin of replication in the inverted repeat sequence of the S component carried by the EcoRI H fragment. The other isolate, SP22-4, carried a 3.5-kilobase-pair duplication defined by a recombination event between a tandem repeat array in the BamHI Z fragment and a site near the amino terminus of the Vmw175 gene in the S-region inverted repeat sequence contained in the EcoRI K fragment. Both duplicated segments contained the entire immediate early mRNA-5 coding region as well as the origin of replication located in the inverted repeat sequence of the S component. The DNA sequence of each duplication joint was determined.