No abstract

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), liver-type (L), and platelet or fibroblast-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign the human PFKP locus to a specific human chromosome, in this study, we have examined ten human X rodent somatic cell hybrids for the expression of human P subunits using a mouse anti-human P subunit-specific antiserum in an active-enzyme-immunoprecipitation technique. In nine of ten hybrids studied, the expression of the PFKP locus segregated concordantly with chromosome 10 and none other, indicating that PFKP is located on chromosome 10; the discordancy rates for all the other chromosomes were 0.2 or greater. In the one discordant hybrid, only the long arm of chromosome 10 was retained and PFKP was not expressed. Human fibroblasts from a patient with duplication of the short arm of chromosome 10 consistently exhibited PFK activity values 180% of normal. These data indicate that human PFKP is located on the short arm of chromosome 10, and that a gene dosage effect is demonstrable in fibroblasts with a duplication of 10p. The use of rodent antihuman antibody combined with immunoprecipitation aided by staphylococci-bearing protein A may find general application in mapping human enzyme genes, when human and rodent gene-products are not distinguishable by other means.

In experimental studies of bone in rats, two morphometric indices reflecting bone density have been proposed, the bone robusticity index and bone weight/ bone length index. In rats, the bone mineral content (BMC) and bone mineral density (BMD) of a selected bone can be determined using dual-energy X-ray absorptiometry (DXA); bone volume can be measured by histomorphometry and other techniques. This study was undertaken to compare two morphometric indices (bone robusticity and bone weight/bone length) with the results of DXA and histomorphometry. Forty female Wistar rats (100 days old, mean weight 239 ± 12 g) were studied: 20 controls and 20 ovariectomized rats (OVX). The morphometric indices and BMD differed significantly (Friedman test) in the overall group of rats; no differences were observed in the control group, but significant differences were apparent in the OVX group (p < 0.0001). The morphometric indices correlated more closely with BMC than with BMD; the femur length/ femur weight index had closer correlations than the robusticity index. Nonetheless, both morphometric indices differed significantly from BMD determined by DXA under abnormal conditions, which makes them unreliable for use in these circumstances.

Fibroblast cell strains derived from a normal individual and from eight patients with various genetic mutations were transformed by a small-plaque variant of simian virus 40 (SV40, strain 776), cloned and studied after long-term in vitro maintenance. Seven of the cultures continued to express the mutant phenotype. Cultures derived from a patient with phosphoglycerate kinase I deficiency exhibited reappearance of normal enzyme activity after transformation. Compared to untransformed controls, all transformed cultures displayed decreased population doubling times, an increase in the relative number of cycling cells and increased saturation density on solid substrates, and did not show evidence of cellular senescence after long-term cultivation. Unlike previous studies on wild-type SV40-transformed human fibroblasts, the majority of cultures transformed by the small-plaque variant of SV40 did not exhibit signs of crisis. The cells also exhibited a decreased dependence on serum and were able to grow in semi-solid medium. The different transformed cultures expressed variable levels of SV40 large T-antigen, synthesized some infectious SV40 virus, and contained both unique arrangements and quantities of covalently integrated and episomal SV40 DNA. No correlation was observed between the rate of growth and synthesis of infectious virus in the different transformed clones. These studies indicate that this small-plaque variant of SV40 can be used effectively to generate long-lived human cultures, which generally retain their mutant phenotype. Transformation with this SV40 variant permits the generation of large quantities of clonal cell cultures for the biochemical and molecular analysis of their genetic defects.