Molecular cloning, sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (Spanish isolate AST/89).

We describe the cloning, nucleotide sequencing and expression in Escherichia coli of the major capsid component (VP60) from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). The sequence of the 3'-terminal 2483 nucleotides of the genome was found to be 95.4% identical to the German RHDV strain, showing ten changes in the deduced VP60 amino acid sequence. The gene coding for this structural polypeptide has been expressed in bacteria as a beta-galactosidase fusion protein or using a T7 RNA polymerase-based system. The VP60 fusion protein showed only partial antigenic similarity with native VP60 and did not confer protective immunity. The recombinant VP60 produced in the T7 RNA polymerase-based system was antigenically similar to the viral polypeptide as determined using polyclonal and monoclonal antibodies. When used to immunize rabbits the recombinant VP60 was able to protect the animals against a lethal challenge using purified RHDV.

LSTSQ: a module for reliable constrained and unconstrained nonlinear regression.

A software module for nonlinear regression analysis, based on the reliable Meyer-Roth algorithm (a modified damped least square algorithm), is presented. It allows both constrained and unconstrained optimization, and the use of a variety of weighting methods. Virtually any nonlinear function can be fitted, including those with several nondependent variables. The package has been thoroughly tested, and is available in several common computer languages (Pascal, Modula-2 and C). It is easy to use, and advanced knowledge of mathematics or computers is not essential. Standard test problems, and a fully working example of use on enzyme kinetics are included.

Predict7, a program for protein structure prediction.

We describe a program for protein sequence analysis which runs in IBM PC computers. Protein sequences are loaded from files in Mount-Conrad and Lipman-Pearson format. Seven features are analyzed: hydrophilicity, hydropathy, surface probability, side chain flexibility, antigenicity, secondary structure and N-glycosylation sites. Numeric results can be shown, printed or stored in files exportable to other programs. Graphics of up to four predictions can be displayed on the screen, printed out or plotted, with several definable options. This program has been designed to be fast, user-friendly and to be shared with the scientific community.

The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli.

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29,459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr 29,459 polypeptide and beta-galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.

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.

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 M, 47000, identical to the viral product, that reacted with a specific monoclonal antibody.

Mechanism of inactivation of UDP-glucose 4-epimerase from Saccharomyces cerevisiae by D-xylose and L-arabinose.

In a previous paper (Cármenes et al., 1984) we reported that UDP-glucose 4-epimerase from Saccharomyces was inactivated both in vivo and in vitro (crude extracts) by L-arabinose or D-xylose. In this paper, we report that pure epimerase requires the presence of UMP or UDP to be inactivated by sugars and that the inactivation is due to the reduction of the epimerase NAD+, which is essential for epimerase activity. The inactivation rate is directly proportional to epimerase and sugar concentrations and hyperbolically proportional to UMP concentration. In situ experiments made with permeabilized cells showed that epimerase is inactivated in the same way when it is inside the cell. In vivo studies showed that epimerase is inactivated to a smaller extent when 1% D-galactose is present in the culture medium than when 1% ethanol is the main carbon source.

L-Arabinose is not a gratuitous inducer of alpha-galactosidase from Saccharomyces carlsbergensis.

L-Arabinose has been described as a gratuitous inducer of the yeast alpha-galactosidase. This has been found to be an artefact resulting from galactose contamination of commercial samples of L-arabinose. The inactivation produced on UDP-glucose 4-epimerase by the pentose does not amplify the inducer activity of contaminating D-galactose.