Isolation and cloning of a new gene encoding maize initiation factor

It was demonstrated that parental gene expression of maize in hybrid genetic background was different from that in their inbred lines. RT-PCR revealed a cDNA overexpressed in hybrid maize. The cDNA was reamplified and used to screen a corresponding cDNA library. A full length of a cDNA insert ZH01 was isolated and shown to have an open reading frame encoding 414a. a. Sequence comparison shows that ZH01 has extremely high similarity with the previously reported plant initiator factor elF-4A and distinct difference from another maize elF-4A gene MIF4A. Ovcrexpression of ZH01 in hybrid may involve vigorous growth of hybrid maize seedlings.     

胚胎性横纹肌肉瘤中下调表达基因HumCyr61的分？…

分离和克隆胚胎性横纹肌肉瘤中下调表达基因ＨｕｍＣｙｒ６１。方法：利用计算机辅助的同源扩增策略，成功克隆一个长１８８７ｂｐ ｃＤＮＡ，并与ＧｅｎＢａｎｋ核酸数据库比较。结果：该基因与鼠生长因子诱导早期表达的Ｃｙｒ６１基因同源度为８２％，在蛋白质水平上的同源度高达９２％，其中对高级结构有重大贡献的半胱氨酸和脯氨酸等氨基酸残基高度保守。查新结果还显示，它包括在胚胎性横纹肌肉瘤细胞系ＲＤ中表达呈下调表达的     

利用CODEHOP设计简并引物克隆红色红曲霉丝氨酸羧肽酶基因片段和序列分析

利用CODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primers)软件设计了红色红曲霉丝氨酸羧肽酶基因片段的简并引物,选取1对简并引物进行逆转录-聚合酶链式反应(RT-PCR),得到348 bp的聚合酶链式反应产物,经pMD18-T载体克隆转化至大肠杆菌DH5α中,测序后进行BLASTX比对,发现此DNA产物与其他丝氨酸羧肽酶基因序列有相似性,推断所克隆的产物即为红色红曲霉的丝氨酸羧肽酶基因片段.     

1株脂肪酶产生菌的筛选鉴定及其脂肪酶基因的克隆表达

从武汉市郊蓖麻地土壤中分离到1株产脂肪酶菌株,结合形态观察和生理生化鉴定,扩增16S rDNA并测序,运用Blast比对构建了进化树,发现该菌与粘质沙雷氏菌(Serratia marcescens)的同源性高达99％,初步鉴定该菌属于沙雷氏菌属(Serratia),命名为Serratia sp.HS-L5.克隆了脂肪酶...     

Isolation of cDNA fragment of fertility-related gene in photoperiod-sensitive genic male sterile rice

The photoperiod_sensitive genic male sterile rice (PGMR) is particularly useful to take advantage of heterosis in rice. mRNA differential display was used to isolate the fertility_relative genes in rice. After establishing an optimized mRNA differential display system, one of the differential cDNA fragments that maybe related to the development and maturation of rice panicle was cloned from a PGMR Nongken 58S.     

Isolation of a gene from Drosophila by complementation in yeast

Transformation of mutant yeast cells by cloned genomic DNA from a higher eukaryote has made it possible to isolate a Drosophila DNA sequence that complements a yeast adenine-8-mutation. A 0.8-kilobase poly(A)-containing RNA is transcribed from the cloned Drosophila segment in transformed yeast cells and can account for functional expression of the gene.     

白蚁肠道木质素分解菌PY12的分离鉴定及lip基因的克隆

利用苯胺蓝平板法从白蚁肠道中分离到一株木质素分解高效菌株PY 12,经形态观察、生化鉴定和16 SrRNA鉴定为肠杆菌属的霍氏肠杆菌(Enterobacterhormaechei).根据已报道的lip(木质素过氧化物酶,Lignin Peroxidase)基因序列设计特异性引物,克隆该菌的lip基因,将其克隆入pMD19-T载体,获得的核苷酸序列为918 bp,并与GenBank中已知的lip序列进行同源性对比,同源性不高,但该核苷酸翻译后的氨基酸序列比对结果发现,其与假单胞杆菌Pseudomonas sp.编码蛋白质的氨基酸序列同源性为88.5%.     

T载体的构建及对小麦差异显示片段的克隆

提取克隆转化后蓝白斑菌落中蓝色菌落的质粒用于自行构建T载体,再将小麦差异显示片段克隆到该T载体中进行检验．结果显示,该T载体对PCR产物克隆效率在90％以上,而且使用方便,价格低廉．     

甜菜硝酸还原酶活性分析及基因片段的克隆

为了明确缺氮条件下甜菜NR的活性,用活体测定法检测了经缺氮胁迫不同时间后甜菜叶中NR活性．结果表明,随着缺氮处理时间的延长,甜菜叶中的NR活性逐渐降低,在缺氯处理1h后,其活性下降较快．用50mmol／1．KNO3溶液处理的甜菜幼苗总RNA,通过RT—PCR分离得到了硝酸还原酶基因片段,长度为471bp,Blast分析表明,其与Genbank中硝酸还原酶基因部分序列具有高度同源性．     

亚洲黑熊活化素βA亚基基因克隆及分子进化分析

借鉴互联网中已克隆的活化素βA亚基基因的序列.设计并合成一对兼并引物,首次扩增和克隆到亚洲黑熊βA亚基基因的357 bp片段,以水作为阴性对照排除污染,多次重复实验获得一致稳定结果.序列分析显示此片段在处于不同进化程度的物种之间,仍然具有高度的保守性,亚洲黑熊βA亚基基因与人相比有29个核苷酸差异,一致性高达91.6%;与亲缘关系较近的大熊猫、小熊猫和马来熊相比,分别有4、20、3个核苷酸差异,一致性分别为98.9%、94.4%、99.2%.系统发育分析和酶切图谱分析显示亚洲黑熊、大熊猫和马来熊具有较近的亲缘关系,而它们与小熊猫的亲缘关系较远.     

Metallothionein gene cluster is split by chromosome 16 rearrangements in myelomonocytic leukaemia

The metallothioneins (MTs) are a family of proteins of low relative molecular mass which bind heavy-metal ions. MTs exist in several molecular forms (MT-I, MT-II) and are encoded by a multi-gene family containing at least 14 closely related genes and pseudogenes. These proteins function in the regulation of trace-metal metabolism, the storage of these ions in the liver, and as a protective mechanism against heavy-metal toxicity. Somatic cell hybridization has shown that most MT genes, including the functional MT genes (MT1A, MT1B, MT2A), lie on human chromosome 16. Using in situ hybridization, we have now localized the MT genes to band q22 of chromosome 16. This chromosomal band is also a breakpoint in two specific rearrangements, the inv(16)(p13q22) and t(16; 16)(p13;q22) rearrangements, found in a subgroup of patients with acute myelomonocytic leukaemia (AMML). Hybridization of a MT probe to malignant cells from two patients with an inv(16) showed labelled sites on both arms of the inverted chromosome, indicating that the breakpoint at 16q22 splits the MT gene cluster. Similar results were obtained when this probe was hybridized to metaphase cells from two patients with a t(16; 16). These results suggest that the MT genes or their regulatory regions may function as an 'activating' sequence for an as yet unidentified cellular gene located at 16p13.     

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or 'uncoupling' of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.     

β-1,4-葡聚糖内切酶基因的克隆及在大肠杆菌中的表达

利用PCR方法从一株短小芽孢杆菌H9克隆了编码葡聚糖内切酶的基因（该序列已经被已收录于GeneBank,登录号为EF620915）,序列分析表明该基因读码框为1980bp,编码659个氨基酸。预测的酶由两个不连续的结构域组成,其一为N-端催化结构域,由糖基水解酶家族9组成,第二个结构域为C-端底物结合结构域,由碳水化合物绑定结构域家族3组成。将基因构建在大肠杆菌表达载体pET20b中得到重组质粒pET20b-EglA,将重组载体转化至大肠菌株BL21(DE3)菌株,基因在大肠杆菌中得到了良好的分泌表达, SDS-电泳图谱表明酶的分子大小约为73KD。     

Molecular cloning and characterization of a cotton phosphoenolpyruvate carboxylase gene

Phosphoenolpyruvate carboxylase (PEPC) plays diverse physiological functions during plant development. In this study, a new phosphoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton (Gossypium hirsutum cv. zhongmian 35) by RACE-PCR. The cloned cDNA of GhPEPC2 is 3,364 bp in length, and has an open reading frame of 2,913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110.6 kD and pI of 5.56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs. Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome. The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cotton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers. In addition, the recombinant GhPEPC2 has been purified by expressing it in E. coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C₃ PEPC with a higher K_m (83.6 μM) and lower V_max (8.0 μmol min^-1 mg^-1) compared with the C₃ PEPCs previously reported.