Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non-overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene. The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析。结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%。RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定。利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp prote-ase proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性。     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Cloning and sequence analysis of membrane-bound alkaline phosphatase cDNA of the silkworm, Bombyx mori.

The nucleotide sequence (1974 bp) of cDNA coding for membrane-bound alkaline phosphatases (m-ALP) of Bombyx mori was isolated. The cDNA clone contained an open reading frame encoding a polypeptide (547 amino acids), which contains a hydrophobic signal peptide of 36 amino acids and the mature protein of 511 amino acids (Mr = 56,163). We found a highly hydrophobic domain presumed to be a membrane anchoring region at the C-terminus. Comparing analysis between Bombyx m-ALP and mammalian and Escherichia coli ALPs suggested an evolutionary relationship of sharing a common ancestral gene.     

Characterization of fcp4 and fcp12, Two Additional Genes Encoding Light Harvesting Proteins of Cyclotella cryptica (Bacillariophyceae) and Phylogenetic Analysis of this Complex Gene Family

Abstract: Two additional cDNA clones containing genes which encode fucoxanthin chlorophyll a/c binding proteins (Fcps) of the centric diatom Cyclotella cryptica have been sequenced. The first cDNA clone containing fcp12 had an insert size of 871 base pairs (bp). The open reading frame (ORF) of 693 bp corresponds to a precursor protein of 231 amino acids with a molecular weight (Mr) of 25 200. For the mature Fcp12, a protein of 196 amino acids with a Mr of 21 700 is proposed. The second cDNA clone contained the fragmentary fcp4 with an insert of 805 bp. The ORF of 492 bp corresponds to a polypeptide of 164 amino acids with a Mr of 18 050. Phylogenetic analyses revealed that the proteins Fcp1, Fcp2, Fcp3 and Fcp5 are closely related to the Fcps of other diatoms, whereas Fcp6, Fcp7 and Fcp12 share the highest homology to the Fcp of the haptophyte Isochrysis galbana and to the light inducible proteins LI818r3 and LI818 of Chlamydomonas reinhardtii and Chlamydomonas eugametos. The subunit Fcp4 revealed some homology with the red algal LH subunits LhcaR1 and LhcaR2 of Porphyridium cruentum.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Reactivity of E. coli-derived trans-activating protein of human T lymphotropic virus type III with sera from patients with acquired immune deficiency syndrome

Randomly sheared DNA fragments from HTLV-III proviral DNA were cloned into an E. coli open reading frame (ORF) expression vector. The inserted ORF DNA was expressed in E. coli transformants as a polypeptide fused to the lambda CI protein at the amino terminus and to beta-galactosidase at the carboxyl terminus. The reactivity of the recombinant peptides with antibodies from sera of AIDS patients was determined by the Western blot technique. The coordinates of the DNA inserts of the immunoreactive clones were then determined by DNA sequencing. A clone, ORF 628, was found to contain a short DNA segment located between the sor and env genes (nucleotide positions 5367 to 5597), a region previously thought to be noncoding. Inspection of the DNA sequences of this clone and of other HTLV-III isolates revealed the presence of a small ORF located between nucleotide position 5411 and 5625, capable of encoding a polypeptide of 72 amino acids. The biosynthesis of the polypeptide of ORF 628 initiates from an ATG codon within the HTLV-III insert. The fusion protein of ORF 628 was partially purified by affinity chromatography on CH Sepharose 4B coupled to a beta-galactosidase ligand, and tested against a panel of sera from AIDS patients by Western blot analysis. Approximately 35% of the sera from patients with AIDS or ARC contained antibodies reactive with the peptide. The DNA region spanned by ORF 628 is now thought to be the major functional element of the trans-activator gene, tat.     

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing

Summary Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.     

Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.

Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.     

Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein.

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.     

Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena

A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA. The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein.     

Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.

Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids. To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein. This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells. To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells. The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis. Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons.     

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.     

水稻矮缩病毒第11号组分基因序列和编码蛋白的功能分析

水稻矮缩病毒(Rice Dwarf Virus-RDV)广泛分布于中国、日本及东南亚地区,侵染水稻和禾本科其它一些作物,是造成水稻减产的主要原因之一,对农作物危害极大。RDV属于呼肠孤病毒科(Re-oviridae)中的植物呼肠孤病毒属(Phytoreovirus)成员,其病毒粒子直径70nm,为20面体,有双层     

Molecular cloning and characterization of a cysteine-rich 16.6-kDa prolamin in rice seeds

An alcohol-soluble storage protein, a 16.6-kDa prolamin found in rice seeds, was purified from both the total protein body and purified type I protein body fractions. The partial amino acid sequences of three tryptic peptides generated from the purified polypeptide were analyzed. A part of the 16.6-kDa prolamin cDNA was amplified from developing seed mRNA by the reverse transcribed polymerase chain reaction using an oligo (dT) primer and a primer which was synthesized based on the partial amino acid sequence. The amplified product was used to isolate the full-length cDNA clone (lambda RP16) from a developing seed cDNA library. The cDNA has an open reading frame encoding a hydrophobic polypeptide of 149 amino acids. The polypeptide was rich in glutamine (20.0%), cysteine (10.0%), and methionine (6.9%). The cysteine content was higher than those of most other rice storage proteins. Messenger RNA of the 16.6-kDa prolamin was detected in seeds, but not in other aerial tissues.     

Primary structure of human thromboxane synthase determined from the cDNA sequence.

Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase-I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450.     

Structure and expression of cysX, the second gene in the Escherichia coli K-12 cysE locus

Protein products of the cysE region at 81 min on the chromosome of Escherichia coli K-12 (1) were analyzed by the maxicell method. Two kinds of polypeptides of molecular weight 33 K and 16 K were the products. The open reading frame (ORF) of the 33 K polypeptide consisted of 273 amino acids (Mr = 29,261). On the other hand, the 16 K ORF was overlapped by the opposite 33 K ORF and specified an extremely basic protein of 130 amino acids (Mr = 15,233). The gene coding for the 16 K polypeptide was named cysX. The expression of cysE and cysX in vivo was confirmed further by constructions of the cysE'-'lacZ and cysX'-'lacZ hybrid genes.     

Cloning, expression in yeast, and functional characterization of CYP76A4, a novel cytochrome P450 of petunia that catalyzes (omega-1)-hydroxylation of lauric acid

A cDNA clone of a novel cytochrome P450, CYP76A4, was isolated from Petunia hybrida. The cDNA clone contained an open reading frame (ORF) encoding a predicted 510 amino acid polypeptide. The CYP76A4 cDNA was expressed in yeast Saccharomyces cerevisiae AH22. Recombinant yeast microsomes containing the CYP76A4 hemoprotein were found to catalyze (omega-1)-hydroxylation of lauric acid.