Tissue-specific expression of human CD4 in transgenic mice.

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines.

Analyses of linked beta-globin genes suggest that nondeletion forms of hereditary persistence of fetal hemoglobin are bona fide switching mutants.

The analysis of nondeletion forms of hereditary persistence of fetal hemoglobin (ndHPFH) has led to the identification of cis-acting elements, located in the promoter regions of the fetal genes, that appear to be involved in the process of fetal to adult hemoglobin switching. Individuals with these disorders demonstrate elevated levels of fetal hemoglobin and lowered levels of adult hemoglobin during adult life. This phenotype could be either the result of an abnormality in the switching process or the result of two independent mutations: one mutation increasing the level of fetal (gamma) gene expression and another mutation decreasing the level of adult (beta) globin gene expression. Here we demonstrate that the adult beta genes linked to two different forms of ndHPFH, G gamma beta + HPFH and Greek ndHPFH, produce normal levels of correctly processed mRNA in transient-expression systems. We also report that the nucleotide sequences of the beta genes are normal. These results indicate that these gamma gene promoter mutations are linked to functionally normal beta-globin genes and are consistent with the hypothesis that these mutations interfere with the normal switching process.

Mitochondrial protein synthesis in interspecific somatic cell hybrids.

The fusion of an oligomycin (OLI)-resistant mutant of mouse LM(TK-) cells to a chloramphenicol (CAP)-resistant mutant of AK412 Chinese hamster cells resulted in a series of interspecific somatic cell hybrids. Hybrids selected in HAT medium retained only mouse mitochondrial genomes while hybrids selected in HAT plus CAP and OLI retained both hamster and mouse mitochondrial genomes in approximately equal amounts. Nuclear-coded mitochondrial proteins from both parental species were incorporated into mitochondria in all of the hybrids. However, the mitochondrially coded proteins of three individually isolated hybrid cell lines were predominantly mouse-specific, with only trace amounts of hamster protein detected.

Transcription and translation of mitochondrial DNA in interspecific somatic cell hybrids.

We examined the mitochondrial transcription and translation products of somatic cell hybrids constructed by the fusion of Chinese hamster and mouse cells. The hybrid cell lines OAC-k, OAC-l, and OAC-m contain approximately equal amounts of hamster and mouse mitochondrial DNA and produced mitochondrial rRNA from both parental species in the same ratio. Cell lines OAC-k, OAC-l, and OAC-m also produced poly(A)+ mouse mitochondrial RNA transcripts comparable in complexity and quantity to poly(A)+ RNA from the mouse parent. However, the overall level of poly(A)+ hamster mitochondrial RNA from these hybrids was significantly reduced compared with that from the hamster parent. The hybrid cells also lacked several poly(A)+ RNA species found in the hamster parent, but contained additional minor transcripts. The mitochondrially coded proteins of the OAC-k, OAC-l, and OAC-m cells were predominantly encoded by the mouse mitochondrial DNA.

Retention of both parental mitochondrial DNA species in mouse-Chinese hamster somatic cell hybrids.

Interspecific somatic cell hybrids were isolated following the fusion of an oligomycin-resistant derivative of LM (TK-) mouse cells to a chloramphenicol-resistant derivative of AK412 Chinese hamsters cells. Hybrids were selected in either HAT medium, HAT plus chloramphenicol (CAP), HAT plus oligomycin (OLI), or HAT plus chloramphenicol and oligomycin. Cytogenetic analysis of the hybrids indicated that their karyotype reflected the sum of the parents. Hybrids selected in HAT medium alone or HAT plus OLI retained primarily mouse mitochondrial DNA while those selected in HAT plus CAP, or HAT plus CAP plus OLI retained both species of mitochondrial DNA. There was no evidence for mitochondrial DNA recombination, despite the continued growth of these hybrids in CAP plus OLI. Hybrids that were removed from dual antibiotic selection for over three months retained both species of mitochondrial DNA in approximately equal amounts with no detectable loss or rearrangement.