Chemiluminographic detection of von Willebrand factor multimeric composition.

Diagnosis of von Willebrand's disease (vWD) requires quantitation of von Willebrand factor (vWF) in plasma plus qualitative assessment of the vWF multimers according to molecular size ranges. Characterization of vWF multimeric size distributions is typically done using sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) followed by immunoblotting in the gel with radiolabeled antibody against vWF and autoradiographic exposure. We applied a western blot technique to vWF multimeric analysis. It included SDS-AGE, electroblotting onto a membrane, and chemiluminescent detection using rabbit anti-human vWF as primary antibody and goat anti-rabbit IgG as secondary antibody conjugated with horseradish peroxidase. Using this method, 18 to 20 vWF multimers were regularly resolved in normal plasma with exposure times of 2 to 4 sec compared to 4 hr or longer by autoradiography. Sensitivity of detection was at least 4-fold enhanced by chemiluminescence compared to radiolabel. Specificity of the assay was confirmed by analysis of plasma samples known to be deficient to different degrees in the larger vWF multimers. The chemiluminographic assay for vWF multimers is superior to the autoradiographic one because it is more sensitive, avoids use of radioactivity, and has shorter total assay time (under 2 days versus five radiolabel).

Gaucher disease: gene frequencies in the Ashkenazi Jewish population.

DNA from over 2,000 Ashkenazi Jewish subjects has been examined for the four most common Jewish Gaucher disease mutations, which collectively account for about 96% of the disease-producing alleles in Jewish patients. This population survey has made possible the estimation of gene frequencies for these alleles. Eighty-seven of 1,528 individuals were heterozygous for the 1226G (N370S) mutation, and four presumably well persons were homozygous for this mutation. The gene frequency for the 1226G allele was calculated to be .0311, and when these data were pooled with those obtained previously from another 593 Jewish subjects, a gene frequency of .032 with a standard error of .004 was found. Among 2,305 normal subjects, 10 were found to be heterozygous for the 84GG allele, giving a gene frequency of .00217 with a standard error of .00096. No examples of the IVS2(+1) mutation were found among 1,256 samples screened, and no 1448C (L444P) mutations were found among 1,528 samples examined. Examination of the distribution of Gaucher disease gene frequencies in the general population shows that the ratio of 1226G mutations to 84GG mutations is higher than that in the patient population. This is presumed to be due to the fact that homozygotes for the 1226G mutation often have late-onset disease or no significant clinical manifestations at all. To bring the gene frequency in the patient population into conformity with the gene frequency in the general population, nearly two-thirds of persons with a Gaucher disease genotype would be missing from the patient population, presumably because their clinical manifestations were very mild.

Use of a portable ribosome-binding site for maximizing expression of a eukaryotic gene in Escherichia coli.

To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS). These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2). The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E. coli expression vectors, and TCGF production was measured. Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used. The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site. The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E. coli.

Isolation, characterization, and comparison of recombinant DNAs derived from genomes of human hepatitis B virus and woodchuck hepatitis virus.

The human hepatitis B virus (HBV) and the woodchuck hepatitis virus (WHV) are closely related by several criteria and belong to the same class of DNA viruses. The DNA genomes from these viruses are difficult to obtain in quantities required for biochemical analysis. We have, therefore, cloned these two DNAs in the vector lambda gtWES and subcloned into the kanamycin resistance plasmid pA01. Comparison of the recombinant DNAs with authentic viral DNAs by specific hybridization, size, and restriction enzyme analysis suggests that the recombinants contain the complete genome of each virus. The nominal size of the cloned HBV genome was 3150 base pairs, compared to 3200 base pairs for the cloned WHV genome. The small amount of nucleic acid homology previously reported between the HBV and WHV DNAs could be demonstrated between the cloned viral DNAs.

Serological relationship of woodchuck hepatitis virus to human hepatitis B virus.

Two antigenic systems of the woodchuck hepatitis virus have been identified. The relationship between viral antigens of the woodchuck hepatitis virus and the human hepatitis B virus was determined by using immunoprecipitation, hemagglutination, and immune electron microscopy techniques. Antigens found on the cores of the two viruses were cross-reactive. Lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments suggested that the major antigenic determinants of the viral surfaces are different; however, results of passive hemagglutination tests indicated that there are common minor determinants. Nucleic acid homology, as measured by liquid hybridization, was found to be 3 to 5% of the viral genomes. The results of this study provide further evidence that woodchuck hepatitis virus is the second member of a new class of viruses represented by human hepatitis B virus. Since virus-infected woodchucks may acquire chronic hepatitis and hepatocellular carcinoma, these antigens and their respective antibodies will be useful markers for following the course of virus infection in investigations of the oncogenic potential of this class of viruses. The nucleocapsid antigen described may be a class-specific antigen of these viruses and, thus, may be useful in discovering new members of the group.

A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks.

Particles with properties similar to those associated with human hepatitis B were found in serum from woodchucks with chronic hepatitis and hepatocellular carcinoma. It is suggested that woodchuck hepatitis virus is a second member of a novel class of viruses represented by the human hepatitis B virus.