Nucleotide sequence of the coat protein gene of pelargonium leaf curl virus and comparison of the deduced coat protein amino acid sequence with those of other tombusviruses

The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined. It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3' end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene. The cp gene was cloned into the expression vector pET8c and expressed in E. coli. The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses. The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions. Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.     

Nucleotide sequence of the coat protein coding region of the potyvirus tobacco vein-banding mosaic virus

The sequence of the 3' 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M(r) of 30,210. The 3' non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3' non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.     

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.     

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.     

Structure of Sesbania mosaic virus at 4.7 A resolution and partial sequence of the coat protein

Serum IgG subclasses and Serum IgA were studied in 43 infants with acute bronchiolitis and 20 healthy infants. IgG subclasses were determined by a capture ELISA and IgA was quantified by turbidimetry. IgG1 concentrations were significantly lower in infants with bronchiolitis than in normal infants. The other IgG subclasses and IgA did not differ between the groups. The subgroups of infants with bronchiolitis who had previously suffered from otitis media or bronchitis, had significantly lower IgG2 than the other infants with bronchiolitis. The same was found for infants with bronchiolitis who had suffered from three or more lower respiratory tract infections. In infants who had suffered from upper or lower respiratory infections before the acute bronchiolitis, IgA was significantly higher than in infants without previous respiratory infections. Ten infants with bronchiolitis (23%) had IgG1 deficiency, that is values below the lower reference limit calculated in a population of healthy Norwegian infants. No healthy infants had any IgG1 deficiency. No infant with bronchiolitis had IgG2 or IgG3 deficiency. The low IgG1 values found in infants with acute bronchiolitis, may be one cause for infants to be more susceptible to RS virus infections.     

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.     

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver

The 3-years-old patient presents bilateral exophthalmia and papillar stasis at both eyes. The cranial examination shows the frontal boss at the level of the anterior fontanelle and the hypoplasia of the inferior facial floor. The cranial radiography shows digital impressions on the entire projection surface of the cap. The child also presents mental retardation. The affection integrates in the category of craniofacial dysostosis described by Crouzon, maladies which are transmitted autosomal with great penetrance.     

Primary structure of horse erythrocyte glycophorin HA. Its amino acid sequence has a unique homology with those of human and porcine erythrocyte glycophorins

The complete amino acid sequence of the major sialoglycoproteins of horse erythrocyte membranes, glycophorin HA, was determined by manual sequencing methods, using tryptic, chymotryptic, and cyanogen bromide fragments. Glycophorin HA is a polypeptide chain of 120 amino acid residues and contains 10 oligosaccharide units attached to the amino-terminal side of the molecule. Its amino terminus is pyroglutamic acid. All of the oligosaccharides are linked O-glycosidically to threonine or serine residues. The amino acid sequence is consistent with the transmembrane orientation of glycophorins. There is no significant homology between the glycosylated domains of horse, human, and porcine glycophorins, but there is a considerable homology between the hydrophobic domains of the three glycophorins, which interact with the lipid bilayer of the erythrocyte membrane.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EIIMan) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EIIMan showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EIIGlc). Similar homology was shown between the C-terminal sequence of EIIMan and the E. coli glucose-specific enzyme III (EIIIGlc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EIIMan contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIIIGlc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

The nucleotide sequence of the coat protein genes of satsuma dwarf virus and naval orange infectious mottling virus

The sequence of the 3'-terminal 4320 and 2409 nucleotides were determined for RNA2 of satsuma dwarf virus (SDV) and navel infectious mottling virus (NIMV). Both sequences contained a part of a long open reading frame which encodes larger and smaller coat proteins (CPs) at the 3'-terminus followed by a 3'non-coding region upstream of a poly (A) tail. Amino acid sequence identity for larger and smaller CPs ranged 81-84% and 68-78%, respectively, among SDV, NIMV and the previously sequenced citrus mosaic virus (CiMV). No significant sequence similarity was found between the CPs of SDV or NIMV and those of the como-, nepo- or other viruses. The nucleotide sequence identity of the 3' non-coding region of RNA2 were 68%-78% among SDV, CiMV and NIMV. These results suggest that SDV, CiMV and NIMV are distinct, though related, viruses. They may be assigned as members of the new genus, which is close to the genera of Comovirus and Nepovirus.     

Molecular cloning of a novel human receptor gene with homology to the rat adrenomedullin receptor and high expression in heart and immune system

Lipopolysaccharide was isolated from strain LMG 6999 of Burkholderia vietnamiensis. Degradative and NMR spectroscopic studies established the presence of two polymeric fractions based on the following trisaccharide repeating units: I:-->3)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc- (1-->; II:-->3)-alpha-D-GalpNAc-(1-->3)-beta-D-GalpNAc-(1-->4)- alpha-L-Rhap-(1-->. The same polymers have previously been found together in lipopolysaccharide from the reference strain for Burkholderia cepacia serogroup O4 and, individually, in those from B. cepacia serogroups C (I) and A (II).     

Determination of the primary structure of alfalfa mosaic virus (strain S) coat protein. II. Complete sequence of the protein (author's transl)

The alfalfa mosaic virus protein was submitted to the action of cyanogen bromide. Four peptides were isolated. Study of these peptides allowed us to determine the order. Then protein was submitted, after S-carboxymethylation or S-aminoethylation, to the action of different proteolytic enzymes: trypsin, chymotrypsin, thermolysin and papain. The peptides issued from these different hydrolysis were separated on Dowex 50 X4 and Dowex 1 X2, and their amino acid composition was determined. The use of classical methods of sequence determination, of mass spectrometry and for one case the use of a sequencer, lead to the obtention of the primary structure of all the tryptic peptides. The studies of chymotryptic, thermolytic and papainic hydrolysates, and of cyanogen bromide rupture, allowed us to isolate the overlapping peptides which were necessary for the reconstitution of the complete proteic chain.     

The nucleotide sequence of the 3'-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production

A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA.