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Biomedical subjects

S A Latt

Publications and source records attributed to S A Latt.

At least 73 records · Page 4Linked to original sources
PubMed1983

Identification and isolation of transcribed human X chromosome DNA sequences.

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.

Animals↗
PubMed1983

High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells.

A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechst plus near UV light followed by S1 nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells.

Animals↗
PubMed1983

Maturation rates and transition probabilities of cycling cells.

The kinetics of cell cycling are analyzed in terms of equations incorporating a DNA synthesis rate continuously varying along S phase and a portion of G1 phase through which the cells advance in a probabilistic fashion. These equations are solved analytically for several steady state as well as nonsteady state populations of cells. Experiments are described illustrating how these results can be applied to obtain both the rate of DNA synthesis at each point along S-phase and the probability of transition through the nondeterministic portion of G1.

Animals↗
PubMed1983

Transposition and amplification of oncogene-related sequences in human neuroblastomas.

We have cloned a 2.0-kb EcoRI fragment of human genomic DNA (NB-19-21) which has homology to the v-myc oncogene but is distinct from the classical c-myc gene. This sequence is amplified from 25- to 700-fold in eight of nine tested human neuroblastoma cell lines which contain either homogeneously staining regions or double minutes (HSRs or DMs), the caryological manifestations of amplified genes. In the remaining line, the c-myc proto-oncogene is amplified approximately 30-fold. NB-19-21 hybridizes to a 3.2-kb cytoplasmic, poly(A)+ RNA species that is abundant only in lines in which the sequence is amplified. We propose that the gene encoding the NB-19-21-related RNA species may represent a new oncogene, which we call N-myc. NB-19-21 derives from chromosome 2; but in the five HSR-containing lines that have amplified this sequence, none has HSRs on chromosome 2. NB-19-21 is associated with DMs in a DM-containing line. A second, randomly cloned, amplified DNA segment from the HSR of one of the neuroblastoma lines is amplified in a subset of the lines in which NB-19-21 is amplified. In addition, this probe identifies a novel joint in the amplification unit of one line relative to that of the others. We suggest that, in the eight lines which have amplified NB-19-21, the amplification units are overlapping, but not identical, and that transposition of the common sequences may occur prior to amplification.

Base Sequence↗
PubMed1983

Cloning and analysis of reverse transcript P160 genomes of Abelson murine leukemia virus.

Circular duplex reverse transcripts of the genome of a strain of Abelson murine leukemia virus that encodes a 160,000-molecular-weight protein were isolated, cleaved with HindIII restriction endonuclease, and cloned into the unique HindIII site of lambda phage Charon 21A. Recombinant phage clones, some of which were infectious in transfection assays, were found to contain a 789-base-pair region specific for Abelson murine leukemia virus; this region is not found in other strains of this virus. The extra sequence was localized by restriction endonuclease and electron microscopic heteroduplex analysis. Sequence analysis showed no homology at the ends of the extra sequence, implying that it was deleted by an event that did not utilize sequence homology. The sequence of this unique region has an open reading frame through its entirety.

Abelson murine leukemia virus↗
PubMed1983

Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.

The transcriptional promoter of the Harvey sarcoma virus long terminal repeat has been used to construct a biologically active dihydrofolate reductase chimera. The construction placed the long terminal repeat at the 5' end of a dihydrofolate reductase cDNA. This chimera mediated methotrexate resistance when introduced into wild-type NIH3T3 mouse cells by transfection. The chimeric sequences were expressed in the form of polyadenylated RNA and dihydrofolate reductase protein and were amplified when the methotrexate-resistant transfectants were selected to grow in increasing methotrexate concentrations. This chimera was dominant acting and able to confer a methotrexate-resistant phenotype on wild-type NIH3T3 cells. It has been used in cotransfection experiments with DNA from human tumor cells to obtain foci of methotrexate-resistant transformed NIH3T3 cells resulting from uptake of exogenous DNA. The transfected methotrexate-resistant cells carried double minute chromosomes that appeared to contain DNA acquired during transfection.

Animals↗
PubMed1983

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.

Animals↗
PubMed1982

Regional localization on the human X of DNA segments cloned from flow sorted chromosomes.

Fluorescence activated sorting of chromosomes from 49,XXXXY human lymphoblasts has been used to obtain DNA enriched for the human X. This DNA was cloned in lambda phage Charon 21A to obtain a library of approximately 60,000 pfu. Phage inserts free of human highly repeated DNA sequences are localized to different regions of the human X by two independent hybridization analyses. The first utilized comparative hybridization to rodent-human hybrid cell DNA samples containing all or known portions of the human X, while the second was based on hybridization dosage to DNA samples from human cell lines differing in the number of X chromosomes or X chromosome segments. Of five unique sequence inserts tested, three were X chromosome specific and were localized to regions Xpter leads to Xcen, Xql leads to Xq22 and Xq24 leads to Xqter, respectively. The library presented here represents a highly enriched source of human X chromosome-specific DNA sequences.

Animals↗
PubMed1982

Isolation of mouse x-chromosome specific DNA from an x-enriched lambda phage library derived from flow sorted chromosomes.

A lambda phage library enriched in X(7) chromosomal material has been constructed from flow sorted chromosomes isolated from mice carrying the Cattanach translocation T(X;7)1Ct. The flow sorted fraction that was cloned contained 40% X(7) chromosomes, so that the resulting lambda phage library should be more than 10-fold enriched for X chromosomal DNA. Approximately 100,000 lambda phage clones were obtained; of these, at least 80% were recombinant. Three quarters of recombinants were positive for mouse repetitive DNA as detected either by phage plaque filter hybridization or by Southern blotting. Recombinant DNA inserts were prepared from some of the remaining nonrepetitive phage fraction. The X-chromosome specificity of cloned DNA inserts was tested by hybridization to DNA from mouse-hamster somatic cell hybrids that had retained all or most of the mouse X as the only mouse chromosome and by comparison of the extent of hybridization to DNA from male and female mice. Out of nine cloned unique sequence segments successfully examined thus far, two were presumably derived from the X. Possession of phage library highly enriched for mouse X DNA should facilitate molecular studies of the control of X chromosome gene expression.

Animals↗
PubMed1982

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment.

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.

Alkylating Agents↗
PubMed1982

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange.

C57Bl/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regenerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57Bl/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.

Animals↗
PubMed1982

Cytogenetic and flow cytometric studies of cells from patients with Fanconi's anemia.

Cells from patients wtih Fanconi's anemia are unusually sensitive to agents which are capable of crosslinking DNA. This increased sensitivity can be detected both by cytogenetic and flow cytometric methods. An elevated frequency of chromosome aberrations, which is further exaggerated by exposure of cells to DNA crosslinking agents, is a general feature of Fanconi's anemia. Information about the formation of sister chromatid exchanges in this disease is less consistent. Cytogenetic analysis of cells from patients with Fanconi's anemia can be compromised by a low mitotic index. This is reflected in an accumulation of cells In the G2 phase of the cycle, after exposure to the bifunctional alkylating agent, mitomycin C. New methods for differentiating individuals with Fanconi's anemia from unaffected individuals should be of empirical use and might also facilitate mechanistic studies of this disease.

Anemia, Aplastic↗
PubMed1981

Sister-chromatid exchange induction in Chinese hamster ovary cells by 8-methoxypsoralen and brief pulses of laser light: assessment of the relative importance of 8-methoxypsoralen--DNA monoadducts and crosslinks.

Brief (less than or equal to 20 nsec) 5-15 mJ pulses of near-ultraviolet laser light were employed to promote, in a controlled manner, the reaction between 8-methoxypsoralen and the DNA of Chinese hamster ovary (CHO) cells. According to Johnston et al. [21], a single brief laser pulse should induce only monoadducts between 8 methoxypsoralen and DNA, while multiple laser pulses are needed to induce 8-methoxypsoralen--DNA crosslinks. In the present study, a single laser pulse was found sufficient to cause a significant elevation in the sister-chromatid exchange (SCE) frequency in CHO cells exposed to 0.75-1.2 X 10(-5) M 8-methoxypsoralen. Additional laser pulses caused less than proportional further increments in SCEs, although the total yield of 8-methoxypsoralen adducts in similarly exposed solutions of isolated DNA increased linearly. Higher numbers of laser pulses induced detectable amounts of 8-methoxypsoralen--DNA crosslinks and distorted the DNA flow histogram of CHO cells in a manner consistent with retarded cell cycle traverse. It is concluded that 8-methoxypsoralen--DNA monoadducts are sufficient to induce SCEs in CHO cells, while 8-methoxypsoralen--DNA crosslinks might have relatively little overall effect on SCE frequencies. These results, together with previous data correlating SCE induction and mutagenesis [10] are discussed in the context of strategies for evaluating psoralen derivatives for their suitability in the phototherapy of skin disorders.

Animals↗