A human heavy chain variable region subgroup III pseudogene (HV3.3) was isolated, characterized, and sequenced. When HV3.3 was hybridized to Southern blots of human DNA, two potentially informative polymorphic bands, resulting from 2.7 kb Hind III (HH2.7) and 7.3 kb Eco RI (HE7.3) fragments, were detected with frequencies of 0.553 and 0.606, respectively. These polymorphic bands showed Mendelian segregation in families and appeared to be in tight linkage disequilibrium with each other (chi 2(1) = 24.91, P less than 0.001). Evidence from sibling-pair data indicated linkage of the Hind III polymorphic band to constant region allotypic and restriction fragment length polymorphism markers. Bands representing alternative forms of the polymorphic restriction sites were not detected for either HH2.7 or HE7.3. This indicates either that the alternative fragments comigrate with homologous fragments resulting from conserved restriction sites, or that the polymorphism is due to a gene duplication or deletion. No band segregating with HH2.7 was found in separate digests using eight other enzymes. Although this indicates that a major deletion or unlikely, it does not exclude the possibility of a gene deletion or duplication affecting the intergenic region(s) of one or more homologous genes. Whatever the precise explanation, these findings support the hypothesis that there is polymorphic variation of VH gene repertoires in man.
A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.
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