ABSTRACT
The two human colony stimulating factors, interleukin-3 and granulocyte-macrophage colony stimulating factor, have been molecularly cloned and expressed as secreted proteins in yeast. In both cases, non-glycosylated and glycosylated forms of the molecules were produced. Removal of N-linked glycosylation sites from the genes by site-directed mutagenesis prevented addition of most of the sugar residues, but revealed a low level of residual O-linked glycosylation on a portion of the molecules. No difference in specific biological activity was found between the different forms of the proteins. It was found that a significant proportion of human granulocyte-macrophage colony stimulating factor was degraded by the yeast KEX2 protease that was cleaving after the dibasic sequence Arg-Arg at positions 23-24 of the mature protein. Site-specific mutagenesis was employed to change this sequence to Leu-Arg, and this change resulted in greatly increased expression levels of full length protein and biological activity.
Subject(s)
Colony-Stimulating Factors/genetics , Genes , Growth Substances/genetics , Interleukin-3/genetics , Mutation , Saccharomyces cerevisiae/genetics , Bone Marrow/drug effects , Bone Marrow Cells , Cloning, Molecular , Colony-Stimulating Factors/physiology , Granulocyte-Macrophage Colony-Stimulating Factor , Growth Substances/physiology , Humans , Interleukin-3/physiology , Recombinant Proteins/pharmacologyABSTRACT
Somatic cell hybridization of mouse erythroleukemia (MEL) cells and HEL cells, a human erythroleukemia line that produces fetal (gamma) but fails to express adult (beta) globin, was used to test whether the expression of the two human globin genes is regulated cis or trans. An experimental approach using anti-human globin monoclonal antibodies for detection, efficient cloning, and monitoring of hybrids of interest was employed. Further characterization of hybrids used isoelectric focusing for detection of human globins and S1 nuclease mapping. In contrast to the parental HEL line, all chromosome 11-retaining HEL-MEL hybrids expressed human beta-globin, suggesting that the HEL beta-globin genes (i) are transcriptionally competent, (ii) become activated in response to a positive trans-acting element within the MEL environment, and (iii) fail to express into the HEL environment because of either the absence of a positive trans-acting element or the presence of a trans-acting inhibitor of beta-globin gene expression. In addition to beta-globin, the primary HEL-MEL hybrids co-expressed gamma-globin; however, gamma-globin expression segregated by subcloning so that secondary and tertiary clones either expressed only beta-globin or co-expressed gamma- and beta-globin. The results of subcloning can be explained by assuming that gamma-globin gene expression is controlled by a HEL cell-derived transacting element encoded by a gene not syntenic to chromosome 11 or by postulating that the HEL gamma-globin genes become randomly modified during the continuous proliferation of hybrids.
Subject(s)
Gene Expression Regulation , Globins/genetics , Hybrid Cells/metabolism , Animals , Antibodies, Monoclonal , DNA Restriction Enzymes/metabolism , Endonucleases/metabolism , Female , Fetal Hemoglobin/genetics , Fluorometry , Humans , Isoelectric Focusing , Leukemia, Erythroblastic, Acute/metabolism , Mice , Pregnancy , Single-Strand Specific DNA and RNA EndonucleasesABSTRACT
A questionnaire was used to survey the methaqualone experiences of college student users, and psychological test data from these users and a control group of non-users were compared. On differential pattern found was that methaqualone users have experimented with a wider variety of psychoactive drugs than have non-users. However, the two groups were essentially indistinguishable on the psychological test variables assessed. A preliminary modal profile of the college student methaqualone abuser and abuse experience was generated.
