Exaggerated triglyceride accretion in human preadipocyte-murine renal line hybrids composed of cells from massively obese subjects.

To learn about adipose differentiation of precursors from postnatal adipose tissue of lean and massively obese subjects, human omental adipocyte precursor-murine renal adenocarcinoma cell (RAG) hybrids were formed by fusion with polyethylene glycol, and cultured selectively with 50 microM ouabain in hypoxanthine aminopterin thymidine (HAT) medium. Under conditions in which the parent cells did not differentiate, a number of hybrids, which were cloned, revealed morphologic and biochemical evidence of differentiation. In addition to activation of human genes within the common nucleus of the hybrids, murine cytoplasmic activators are probably also involved because heterocaryons (fused cells with two interspecific nuclei) revealed the same phenomenon. Hybrids composed of precursors from massively obese subjects disclosed more frequent and prominent differentiation. Since these hybrids, in contrast to those from the lean, recapitulate this phenomenon in subcultures, they provide the potential system for mapping the human gene(s) responsible for adipose differentiation and its exaggeration in massive obesity.

Hemophilia B (Christmas disease) variants and carrier detection analyzed by DNA probes.

We have used two strategies to study 14 hemophilia B families from 11 kindreds for possible carrier detection and prenatal diagnosis. First, we sequentially used the Factor IX probes (sequentially with restriction enzymes Taq I, Xmn I, and Dde I), and the linked probes p45h (Taq I), p45d (Pst I), and 52a (Taq I) for restriction fragment length polymorphism (RFLP) analysis. Second, we searched for useful variant Taq I digestion fragments using the Factor IX complementary DNA. Two separate new Taq I variants in exon VIII were identified. Using both strategies, 11 of 14 families (from 9 of 11 kindreds) were informative for further studies. In five kindreds studied in detail, the carrier status of all 11 at risk females was determined and prenatal diagnosis could be offered to the offsprings of each of the six carriers identified. Thus, in this study, we have identified a higher proportion of informative families than has previously been reported.

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

Hemopoietic origin of factor XIII A subunits in platelets, monocytes, and plasma. Evidence from bone marrow transplantation studies.

Factor XIII A subunit (FXIIIA) is found in plasma, platelets, and monocytes. The hemopoietic contributions to FXIIIA in these components were studied in patients transplanted with marrows from donors with different FXIIIA phenotypes. In three patients with successful engraftment (by DNA genotyping, red cell phenotyping, and cytogenetic studies) platelet and monocyte FXIIIA changed to donor phenotypes with hematologic recovery. Thus, FXIIIA in platelets and monocytes is synthesized de novo and/or from their progenitor cells. Plasma FXIIIA phenotype change after transplantation was more complex. Patient I changed from phenotype 1-1 (one electrophoretically fast band) to 1-2 (three bands) in 115 d; patients 2 and 3 did not change completely from phenotype 1-2 to 1-1 in up to 458 d, but did show enrichment of the fastest band. Thus, while there is a definite contribution of donor hemopoiesis to plasma FXIIIA, another source of recipient FXIIIA appears to be present to delay or prevent the phenotype change.

Mental retardation, distinct facial changes, short stature, obesity, and hypogonadism: a new X-linked mental retardation syndrome.

We describe a 3-year-old boy and his 2 maternal uncles with moderate to severe mental retardation, short stature, mild obesity, hypogonadism, a low total finger ridge count, and a distinctive face characterized by bitemporal narrowness, almond-shaped palperbral fissures, depressed nasal bridge, anteverted nares, short and inverted-V-shaped upper lip, and macrostomia. Two other males in this family who had similar facial anomalies and developmental delay died in early infancy and midchildhood. This apparently new disorder is reminiscent of, but distinct from, the Prader-Willi syndrome, and is likely inherited as an X-linked recessive trait. Preliminary studies with DNA probes are consistent with an X-linked locus and permit exclusion of distal Xp and Xq regions as the site of this mutation.

DNA banking: the effects of storage of blood and isolated DNA on the integrity of DNA.

Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from -70 degrees C to +65 degrees C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37 degrees C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45 degrees C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at -70 degrees C for at least 2 months or at 23 degrees C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at -20 degrees C for 7 years appeared to be intact.

Tentative assignment of gene for oto-palato-digital syndrome to distal Xq (Xq26-q28).

Detailed physical mapping of oto-palato-digital (OPD) syndrome gene on the X-chromosome was attempted on a family of 3 generations with 2 affected men. Although the result remains statistically non-significant, it indicates that the OPD-I gene might be located on the distal Xq.

Successful paternity of twins following bone marrow transplantation with busulfan, melphalan and cyclophosphamide conditioning.

A 33-year-old man who had received previous chemotherapy with cytarabine, daunorubicin and mitoxantrone followed by an autologous marrow transplant after conditioning with busulfan, melphalan and cyclophosphamide, fathered sex-mismatched fraternal twins approximately 6 years post-transplant. HLA and DNA analyses showed the probability of paternity to be in excess of 99% for each twin. To our knowledge this represents the first documented case of paternity following conditioning with this combination of marrow ablative agents and the first report of twin paternity following autologous marrow transplantation.

Neurofilament gene expression following beta,beta'-iminodipropionitrile (IDPN) intoxication.

beta,beta'-Iminodipropionitrile (IDPN) is an agent that produces a disorganization of the axonal cytoskeleton with massive accumulation of neurofilaments in the proximal axon. Abnormalities in axonal transport of neurofilament proteins and in their phosphorylation occur in this model. In this study we evaluated the gene expression of neurofilament and other cytoskeletal components at an early, intermediate and late stage of intoxication to determine whether this neuropathy is directly due to or secondarily affects the expression of these components. Specific cytoskeletal mRNA expression was evaluated in the spinal cords of rats treated with IDPN for varying durations using Northern analysis and in situ hybridization. Our results show no qualitative or quantitative alteration in the mRNA expression of the neurofilament triplet, alpha-tubulin, alpha-actin or glial fibrillary acidic protein. We conclude that abnormalities at various stages of cytoskeletal processing such as the early disorganization of the cytoskeleton, the impairment of neurofilament transport, and the long-term redistribution of neurofilaments along the axon are not directly due to, nor do they affect the gene expression of cytoskeletal components in IDPN neuropathy.

Micronucleus assay in human fibroblasts: a measure of spontaneous chromosomal instability and mutagen hypersensitivity.

By comparing fibroblast strains derived from individuals exhibiting chromosome instability and/or mutagen hypersensitivity (Cockayne syndrome, ataxia telangiectasia, and Fanconi anemia) with strains derived from healthy donors, the fibroblast micronucleus assay has been established as a reproducible measure of the genotypic variation in spontaneous or mitomycin C (MMC)-induced chromosomal instability. The patient strains that were moderately or exquisitely sensitive to MMC, whereas the mildly sensitive strain (Cockayne syndrome) overlapped with the control range. The reproducibility of the assay was evaluated within and between experiments. Paired comparison analyses between duplicate cultures and between repeat experiments failed to show any significant differences between micronucleus frequencies within strains, whereas a significant differences in the spontaneous micronucleus frequencies between strains was observed. In addition to its value as a test system for genotoxins, the fibroblast micronucleus assay may be useful for investigating genetically determined hypersensitivity to mutagens, elevated spontaneous chromosomal breakage, and chromosome segregation errors.

Assessment of the sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies.

The effectiveness of sephadex sperm filtration on altering the sex ratio of human sperm was tested using three different methods. For each ejaculate the ratio of X- and Y-bearing sperm was analysed before and after sephadex filtration using three different methodologies: sperm chromosome analysis after fusion of human sperm with hamster oocytes, deoxyribonucleic acid analysis using the Y-preferential probe pS4 and the fluorescent Y-body test. In 182 sephadex-treated sperm analysed by sperm chromosomes the sex ratio (55.5%X) did not differ significantly from 226 untreated sperm (54.7%X) from the same semen samples. The DNA and Y-body tests demonstrated variability in both degree and pattern of enrichment for replicates but no consistent enrichment for X-chromosome-bearing sperm was observed for any one fraction after sephadex gel filtration.

Host-cell reactivation of UV-irradiated adenovirus in Cockayne syndrome.

Measurements of the host-cell reactivation (HCR) of mutagen-treated virus provides a very sensitive tool for detecting abnormal DNA repair. The best example of the utility of HCR studies in the examination of the DNA-repair capacity of human cells has come from studies of cells from the UV-sensitive repair-deficient xeroderma pigmentosum (XP) patients. We have examined the HCR of UV-treated adenovirus type 5 (Ad5) and type 2 (Ad2) in cells from patients with Cocayne syndrome (CS), another sun-sensitive syndrome whose cells also exhibits UV-sensitivity in culture. Comparisons with obligate heterozygotes and normal controls failed to reveal an abnromality in the HCR capacity of the CS cells. As the abnormality in DNA metabolism in CS appears to be in a late step in excision repair, a bypass mechanism may exist in these cells for circumventing the defect in the repair of viral DNA.

Kinetochore immunofluorescence in micronuclei: a rapid method for the in situ detection of aneuploidy and chromosome breakage in human fibroblasts.

We have developed a rapid and simple immunodetection assay for the in situ identification of aneuploidy in mitotic fibroblasts. Kinetochore (centromere)-containing micronuclei can be detected easily and rapidly by immunofluorescence. The action of colchicine and its derivatives on the mitotic spindle apparatus of mammalian cells induces chromosome lag and aneuploidy. The treatment of normal human fibroblasts with Colcemid resulted in increased levels of micronuclei. Using an immunofluorescence stain (scleroderma CREST antiserum, biotinylated goat antihuman IgG and streptavidin-Texas Red) to detect the presence of kinetochores, it was observed that 90% of the Colcemid-induced micronuclei contained one or more fluorescent bodies (kinetochores). Cultured skin fibroblasts from a patient with ataxia telangiectasia (AT), which is a chromosome breakage syndrome, were used as a control. The AT fibroblasts exhibited elevated levels of spontaneous micronuclei when compared with normal fibroblasts, and 85% of these micronuclei were kinetochore-negative. This finding supports the hypothesis that the majority of spontaneous micronuclei in AT cells arise from chromosome breakage. The spontaneous micronucleus frequencies for 8 strains of human fibroblasts were in the order of 0.5-2%. Spontaneous levels of kinetochore-positive micronuclei were measured for these 8 strains; in 5 of the strains, about 25% of the micronuclei were kinetochore-positive, and in the other 3 strains approximately 50% of the micronuclei were kinetochore-positive. These data suggest that genetic factors may play a role in the control of the spontaneous levels of chromosome breakage and/or segregation errors which result in aneuploidy.

Kinetochore analysis of micronuclei allows insights into the actions of colcemid and mitomycin C.

We have induced micronuclei in two strains of diploid human fibroblasts with a known aneugen, colcemid, and a known clastogen, mitomycin C. Using immunofluorescence to detect the presence of kinetochores in micronuclei, we were able to demonstrate a 26.8-fold increase in fluorescence-positive micronuclei (aneuploidy) in colcemid-treated cells. However, colcemid also induced an increase in kinetochore-negative micronuclei. Our findings support previous reports that suggest colcemid may induce chromosome breakage in addition to its major aneugenic effect. The frequency of kinetochore-negative micronuclei (chromosome breakage) in mitomycin C-treated cells rose an average of 7.9-fold in the two test strains, a clear reflection of its clastogenic action. However, a 4-fold increase in the kinetochore-positive fraction was seen. We conclude that the fibroblast micronucleus assay, coupled with kinetochore immunofluorescence, provides a useful screening approach for genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the rates of chromosomal breakage or lag may require more detailed analysis.

Molecular diagnosis.

Recombinant DNA technology, one of the major controversial areas of biological research in the late 1970s, is now rapidly providing new avenues for diagnosis and treatment. With the early recognition that extensive DNA variation exists in human populations, molecular genetic diagnosis of a variety of common hereditary diseases has become a reality. Recent identification of the location of the gene (or genes) for cystic fibrosis and adult polycystic kidney disease, and characterization of the region of the Duchenne muscular dystrophy gene will lead us towards a better understanding of the basic defects in these diseases. The identification of large multi-generation families with genetic diseases that are useful for identifying gene locations will require the co-operative participation of clinicians, medical geneticists and molecular biologists.

Familial mutagen sensitivities: are these the hallmarks of meiotic or mutator mutants in humans?

Mutagen hypersensitivity (MHS) has been found to be associated with abnormalities in DNA metabolic processes in many prokaryotic and eukaryotic organisms. The study of fibroblasts derived from humans with genetic diseases believed to have altered DNA metabolism, has also revealed patterns of MHS. In this paper results are presented that suggest MHS patterns unrelated to obvious disease can be inherited in a dominant fashion. As these individuals exhibiting MHS patterns have been observed in families with poor reproductive history, new syndromes, or ontogenetic problems (including malignancies) there may be a causal relationship between these events and the MHS. These events which may have a genetic basis appear as maternal or paternal effect mutants as the consequences are observed in reduced reproductive fitness or abnormal progeny. Since these effects are similar to the events precipitated by the meiotic or mutator mutants in Drosophila, it is speculated that the MHS patterns may be the hallmarks of such mutants in man.

Genetic evidence for subunits in the fertility repressor produced by F-like R factors.

Repressor-negative mutants derived from R 100 were tested for genetic complementation. The results indicate that the active repressor is a multimer. The properties of an unusual repressor mutant are also discussed.

Fertility regulation in F-like resistance transfer factors.

Mutants of the R factor R100 have been isolated that mediate high-frequency transfer of the R factor during conjugation. Complementation tests revealed two classes of mutants, operator-constitutive and repressor-negative. Some of the latter class were suppressible by amber and ochre suppressors. The results support a simple model of regulation for the control of R-factor-mediated piliation.

Primate evolution of a human chromosome 1 hypervariable repetitive element.

The clone designated hMF #1 represents a clustered DNA family, located on chromosome 1, consisting of tandem arrays displaying a monomeric length of 40 bp and a repetition frequency of approximately 7 x 10(3) copies per haploid genome. The sequence hMF #1 reveals multiple restriction fragment length polymorphisms (RFLPs) when human genomic DNA is digested with a variety of 4-6-bp recognition sequence restriction enzymes (i.e., Taq I, Eco RI, Pst I, etc.). When hamster and mouse genomic DNA was digested and analyzed, no cross-species homology could be observed. Further investigation revealed considerable hybridization in the higher primates (chimpanzee, gorilla, and orangutan) as well as some monkey species. The evolutionary relationship of this repetitive DNA sequence, found in humans, to that of other primates was explored using two hybridization methods: DNA dot blot to establish copy number and Southern DNA analysis to examine the complexity of the RFLPs. Homology to the hMF #1 sequence was found throughout the suborder Anthropoidea in 14 ape and New and Old World monkey species. However the sequence was absent in one species of the suborder Prosimii. Several discrepancies between "established" evolutionary relationships and those predicted by hMF #1 exist, which suggests that repetitive elements of this type are not reliable indicators of phylogenetic branching patterns. The phenomenon of marked diversity between sequence homologies and copy numbers of dispersed repetitive DNA of closely related species has been observed in Drosophila, mice, Galago, and higher primates. We report here a similar phenomenon for a clustered repeat that may have originated at an early stage of primate evolution.

DNA repair in Cockayne syndrome.

Cockayne syndrome (CS) is a rare recessive genetic disease characterized in part by premature ageing and photosensitive skin. Because of the latter characteristic, this syndrome was considered to be an example of a UV-sensitive DNA repair-defective human disorder. We demonstrated normal levels of UV-induced unscheduled DNA synthesis (UDS) in four unrelated CS patients that show hypersensitivity to both UV and Mitomycin C (MMC). At low UV exposure, CS DNA shows a dose-dependent decrease in size. By contrast, heterozygotes appear to have a threshold below which there is little change in size of single strand DNA. Immediately following UV or MMC treatment, CS DNA is deficient in high molecular weight species, but undergoes a normal transition to larger DNA during a chase interval in the presence or absence of caffeine. This suggests a defect in replication or excision repair and no defect in post-replication repair (PRR). Pulse studies performed in the presence of hydroxyurea (HU) also reveal a deficient production of large DNA, suggesting the defect is in repair. As these cells have normal UDS and normal PRR, the basis for their UV sensitivity must be distinct from that observed in xeroderma pigmentosum (XP).