Examination of potato virus X proteins synthesized in infected tobacco plants.

Nucleotide sequence analysis of potato virus X (PVX) genomic RNA predicts five open reading frames (ORFs). Previous analysis of total RNAs from PVX-infected leaf tissue suggested that six subgenomic RNAs are synthesized during infection. However, the proteins encoded by the genomic RNA, the subgenomic RNAs, or the predicted ORFs have not been identified in vivo. To characterize the coding properties of the viral RNA, particularly to determine whether the five predicted ORFs function in vivo, total protein extracts prepared from PVX-infected leaf tissue were analyzed by using antibodies raised against virus-specific synthetic peptides and against the virus capsid protein. Dot blot analyses showed that these antibodies reacted to PVX-infected extracts, indicating in vivo expression of the five predicted ORFs. In addition, Western blot (immunoblot) analysis of the extracts showed that ORF 1, 2, 3, and 4 peptide antisera and coat protein antiserum detect predominantly a single protein.     

木薯HSP21基因的电子克隆与生物信息学分析

HSP21是植物响应高温胁迫的关键基因,在防止蛋白变性、保护细胞结构和维持植物正常生长发育等方面发挥重要作用,克隆HSP21基因是揭示木薯抵抗高温胁迫分子机制的基础。为得到木薯HSP21同源基因及分析其表达蛋白的性质,文中采用电子克隆技术对新基因进行组装和衍生,并使用生物信息学分析方法,对预测蛋白的一级至高级结构、亲水性/疏水性、信号肽、蛋白同源性和系统进化等进行全面解析。结果表明,HSP21基因含有969个碱基对,其开放阅读框有705个碱基,预测蛋白含234个氨基酸。预测蛋白是非跨膜蛋白,具有碱性和亲水性,主要定位于叶绿体内。通过多重序列比对和系统进化分析发现,木薯与巴西橡胶树、蓖麻和麻疯树等植物的HSP21蛋白同源性较高。结果可为该基因的克隆和转化提供参考依据。     

棉花抗逆相关基因GhDr1的克隆及生物信息学分析

通过TAIL-PCR的方法从棉花中克隆到未知功能的新基因GhDr1,生物信息学分析结果表明,GhDr1的预测蛋白含有酪氨酸激酶和蛋白酶C磷酸化位点、潜在的锌指类功能模块、MAPK磷酸化位点及MAPK相互作用识别位点;与GhDr1同源基因位于同一基因簇的基因多为参与逆境信号转导的蛋白。推测GhDr1基因可能在逆境信号转导途径的磷酸化级联反应中被调节,参与棉花逆境应答的生理过程。     

Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.     

Prediction of transmembrane topology of F0 proteins from Escherichia coli F1F0 ATP synthase using variational and hydrophobic moment analyses.

The a subunit, a membrane protein from the E. coli F1F0 ATP synthase has been examined by Fourier analysis of hydrophobicity and of amino-acid residue variation. The amino-acid sequences of homologous subunits from Vibrio alginolyticus, Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans, Schizosaccharomyces pombe and Candida parapsilosis were used in the variability analysis. By Fourier analysis of sequence variation, two transmembrane helices are predicted to have one face in contact with membrane lipids, while the other spans are predicted to be more shielded from the lipids by protein. By Fourier analysis of hydrophobicity, six amphipathic alpha-helical segments are predicted in extra-membrane regions, including the region from Glu-196 to Asn-214. Fourier analysis of sequence variation in the b- and the c-subunits of the Escherichia coli F1F0 ATP synthase indicates that the single transmembrane span of the b-subunit and the C-terminal span of the c subunit each have a face in contact with membrane lipids. On the basis of this analysis topographical models for the a- and c-subunits and for the F0 complex are proposed.     

Rapid repression of quiescence-specific gene expression by epidermal growth factor, insulin, and pp60v-src.

We isolated a cDNA for p20K, a secreted protein preferentially synthesized in nonproliferating cells. p20K mRNA and protein levels declined rapidly following treatment with various mitogens. DNA sequence analysis of the p20K cDNA predicted a novel protein distantly related to alpha 2 mu-globulin and plasma retinol-binding protein.     

Identification of a 40S Ribosomal protein (S17) that is differentially expressed between the macroschizont and piroplasm stages of Theileria annulata.

The nucleotide and protein sequence of the 40S ribosomal protein S17 (RibS17) of the protozoan parasite Theileria annulata has been determined. Southern blot analysis showed the gene was single copy and comparative sequence analysis revealed that the predicted polypeptide had high sequence homology with the RibS17 from other organisms. Northern blot analysis showed that there was a 3-fold increase in the level of RibS17 RNA between the macroschizont and the piroplasm stage of the lifecycle, whereas, there was no difference in expression between the sporozoite and the macroschizont stages. Antisera to the purified fusion protein, corresponding to the terminal 50 amino acids of the protein sequence, were raised in rabbits. Western analysis detected a polypeptide of the predicted size that was more abundant in the piroplasm stage compared with the macroschizont stage. Immunofluorescence analysis with the same antisera revealed a strong signal in the macroschizont and piroplasm stages, but the antiserum did not cross-react with the bovine host cells. The antisera did, however, cross-react with Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. The possible functional significance of the stage related increase in abundance of a ribosomal protein is discussed.     

Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p. Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA. Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p. Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum. This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP. Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein O-glycosylation. We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins.     

The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1

The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.     

睾丸特异抗原C_2B细胞抗原决定簇的定位

免疫性不育病人的血清（ＩＰＳ）能１００％地抑制人体外受精．用该血清筛选人睾丸ｃＤＮＡ基因表达文库,发现了一种新的睾丸特异抗原（称作Ｃ２）．通过ＤＮＡ顺序研究,并与基因库中的有关同源性基因数据进行比较,证实Ｃ２是一个新的特异蛋白．Ｃ２基因仅与睾丸组织的ｍＲＮＡ杂交．该克隆在大肠杆菌中所表达的融合蛋白能够被３个不同的不育病人血清识别．利用嵌套缺失和Ｗｅｓｔｅｒｎ印迹的方法研究其抗原决定簇定位,发现Ｂ细胞抗原决定簇在羧基端２９个氨基酸范围内．用ＧＣＧ软件分析该抗原的氨基酸的亲水性和疏水性以及其存在于蛋白表面的可能性,确定其中的１５个氨基酸为抗原决定簇所必需,并合成了多肽     

Use of covariance analysis for the prediction of structural domain boundaries from multiple protein sequence alignments

Current methods for identification of domains within protein sequences require either structural information or the identification of homologous domain sequences in different sequence contexts. Knowledge of structural domain boundaries is important for fold recognition experiments and structural determination by X-ray crystallography or nuclear magnetic resonance spectroscopy using the divide-and-conquer approach. Here, a new and conceptually simple method for the identification of structural domain boundaries in multiple protein sequence alignments is presented. Analysis of covariance at positions within the alignment is first used to predict 3D contacts. By the nature of the domain as an independent folding unit, inter-domain predicted contacts are fewer than intra-domain predicted contacts. By analysing all possible domain boundaries and constructing a smoothed profile of predicted contact density (PCD), true structural domain boundaries are predicted as local profile minima associated with low PCD. A training data set is constructed from 52 non-homologous two-domain protein sequences of known 3D structure and used to determine optimal parameters for the profile analysis. The alignments in the training data set contained 48 +/- 17 (mean +/- SD) sequences and lengths of 257 +/- 121 residues. Of the 47 alignments yielding predictions, 35% of true domain boundaries are predicted to within 15 amino acids by the local profile minimum with the lowest profile value. Including predictions from the second- and third-lowest local minima increases the correct domain boundary coverage to 60%, whereas the lowest five local minima cover 79% of correct domain boundaries. Through further profile analysis, criteria are presented which reliably identify subsets of more accurate predictions. Retrospective analysis of CASP3 targets shows predictions of sufficient accuracy to enable dramatically improved fold recognition results. Finally, a prediction is made for geminivirus AL1 protein which is in full agreement with biochemical data, yielding a plausible, novel threading result.     

Protein disorder is positively correlated with gene expression in Escherichia coli

We considered, on a global scale, the relationship between the predicted fraction of protein disorder and the RNA and protein expression in Escherichia coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins.     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明, 模式植物拟南芥叶绿体中含有大约4 000多种蛋白质, 目前只分离得到1 000多种, 其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5'端长178 bp的DNA片段, 与绿色荧光蛋白(GFP)基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察, GFP只在叶绿体中特异表达。实验结果表明, AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。     

cDNA Cloning, Tissue Distribution and Chromosomal Localization of the Human ID4 Gene

A cDNA encoding the human Id4 protein has been isolated froman astrocytoma library. The predicted protein product shares98% identity with the mouse Id4 protein and is markedly differentfrom that already reported. By FISH analysis, the human ID4gene was more precisely mapped to chromosome 6p22.3-p23. Northernblot analysis showed that ID4 is mainly expressed in thyroid,brain and fetal tissue and in some nervous system tumor celllines.     

The NII2 gene of Hansenula polymorpha is involved in nitrite assimilation

To establish a basis for genetic and molecular studies of nitrite assimilation in the methylotrophic yeast Hansenula polymorpha, we isolated and characterised six nitrite-negative mutants still capable of growing on nitrate. Gene isolation work yielded the NII2 gene, encoding a membrane protein homologous to the Saccharomyces cerevisiae Pho86p. Sequence analysis revealed an ORF of 860 bp encoding a 286-amino-acid protein with a predicted molecular mass of 32.8 kDa. This protein is shorter than its S. cerevisiae homologue, and is predicted to lack an ER-retention signal. Cell suspension work revealed that the null mutant is unable to take up nitrite from the medium.     

Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis

To discover germ cell-specific genes, we used in silico subtraction and identified testis expressed gene 14 (Tex14). Mouse Tex14 contains an open reading frame encoding a 1450-amino-acid protein, which shares 64% amino acid identity with the predicted human TEX14 protein. The predicted TEX14 amino acid sequence consists of three ankyrin repeats, a protein kinase domain, and a leucine zipper dimerization motif. Northern blot analysis and in situ hybridization show that Tex14 mRNA is expressed specifically in the testis, with highest levels observed in pachytene, diplotene, and meiotically dividing spermatocytes. Two 5' splice variants of mouse Tex14 were discovered by sequencing 5'-RACE polymerase chain reaction products. TEX14 is predicted to be localized to the nucleus, suggesting that it may play a key role in regulating gene expression or modulating nuclear events during mammalian spermatogenesis.