Biomedical subjects
Publications and source records attributed to C A Thomas.
A new procedure has been worked out to establish restriction maps. The method is fast, does not in general require labeled DNA and has been applied to map the linear palindromic rDNA of Physarum with the restriction enzyme Bst EII.
Despite the extensive clinical use of corticosteroids (CS) in the treatment of immune-mediated diseases, relatively little is known concerning the effects of CS on B cell function as measured by in vitro assays. The effects of single-dose vs several days of in vivo CS therapy on the spontaneous and mitogen-induced Ig production by human peripheral blood B cells are reported here. Spontaneous Ig production by individual B cells was enhanced by in vivo CS as measured by an in vitro plaque-forming cell (PFC) assay. The same increased response was also observed with a brief in vitro exposure of the B cells to CS, which suggests that a mere brief exposure to an active CS analogue is all that is required to produce the enhanced response. The immunoregulatory effects of in vivo CS on mitogen-induced Ig production are more complex. Pokeweed mitogen-induced PFC responses were suppressed 4 to 5 hr after a single in vivo pharmacologic dose of CS, with complete recovery by 24 hr. In contrast, after a 5-day course of CS, the suppressed PFC response did not recover until 60 hr after the last dose. Moreover, several mechanisms of suppression were operative. Ten hours after completing the 5-day course of CS, there was a relative enrichment in the peripheral blood compartment of lymphocytes bearing the OKT8 suppressor/cytotoxic T cell phenotype that coincided with a depressed PFC response. At 36 hr after the last dose, the T lymphocyte profile returned to normal while B cell function remained suppressed. The complex, multifaceted modulation of the immune response, resulting from redistribution of cell subsets as well as altered cell functions, vary with time-dose parameters of in vivo CS administration. These observations should provide additional insights into the heterogeneity of CS-induced therapeutic effects.
Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme. The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments. This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.
Drosophila melanogaster DNA (Dm) was sequentially cleaved by BamHI and EcoRI and separated by two-dimensional gel electrophoresis. Six different prominent bands, which are derived primarily from the cleavage of long sequences that are repeated 20-100 times per genome, were recovered from the gel and cloned in pBR322. Hybridization and restriction analysis of the cloned Dm segments showed that three of these bands are mainly derived from the ribosomal and histone gene repeating units. Segments cloned from the other three bands are not homologous to any known repeating elements that we have tested. They represent long repetitive sequences of moderate multiplicity that appear not to have been hitherto described. These segments have been restriction-mapped and hybridized to cDNA prepared from poly(A)RNA from adult flies. While two minority segments did hybridize to this probe, the majority failed to hybridize. The arrangement of genomic sequences homologous to each plasmid was tested by restriction analysis and Southern hybridization. The results indicate that the repetitive element is largely conserved intact although occupying numerous different positions in the genome. The DNAs from four different strains of D. melanogaster and two of D. simulans produced restriction patterns having some segment lengths in common and some showing clear differences, a fact that indicates that these sequences can move about to occupy different genomic locations in different strains.
This study uses quantitative cultures and immunofluorescent demonstration of antibody-coated bacteria to differentiate colonizing from infecting bacteria in lower respiratory secretions obtained by fiberoptic bronchoscopy. The fiberoptic bronchoscope was passed transnasally without the use of a nasotracheal tube, and a single-sheath cytology brush dipped in lower respiratory secretions served as inoculum for quantitative cultures. Secretions were also collected by aspiration through the bronchoscope side channel for fluorescent examination. None of 60 control patients had greater than 4,000 colony-forming units (CFU) per brush of a single bacterium on quantitative culture. In contrast, more than 4,000 CFU per brush were isolated from 29 of 33 episodes of clinically defined lower respiratory infection (p less than 0.001). Only 1 of 60 control patients had antibody-coated bacteria in their lower respiratory secretions, but antibody coating was demonstrated in association with 24 of 33 episodes of infection (p less than 0.001).
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Methylation of DNA in normal mouse cultured 3T3 cells and in their virally or chemically transformed derivatives was studied. DNA methylation was studied by restriction with HpaII, MspI, or HpaII plus MspI. DNA from the chemically transformed cells was cleaved about twice as often with HpaII than was the DNA of normal and virally transformed cells. Digests with MspI and HpaII plus MspI were identical in all cell lines studied. Densitometry of the restriction patterns allowed an estimate of total DNA methylation from the weight average lengths. The chemically transformed cell line showed 25% reduction in methylation compared to the other cell lines. Southern blot hybridization using satellite DNA showed that these sequences followed a pattern of modification similar to that of total DNA.
A dual parameter flow microfluorometric technique for accurately measuring Fc gamma receptor (FcR) expression on defined subsets of cells within a heterogeneous cell sample was developed. The FcR distribution of human peripheral blood mononuclear cells consists of three distinct peaks. By analyzing cells fluorescently labeled with the 3Al, an anti-T cell hybridoma antibody (using a green-emitting fluorophore) and for FcR (with a red-emitting fluorophore), and by using cell isolation procedures, it was shown that the cells lying within the peak with intermediate FcR density are mainly monocytes, while cells lying within the peaks with highest and lowest (i.e. negative) FcR densities are predominantly T cells. The FcR+ T cells (T gamma cells) express higher levels of the 3Al antigen than other T cells, thus demonstrating the utility of the 3Al hybridoma antibody as a marker for T gamma cells.
Ribosomal DNA (rDNA) methylation was studied in various strains of mice. We used restriction enzymes that are sensitive to methylation and a cloned probe containing the transcribed spacer and part of the 18S and 28S gene. Strains C3H/He3, C57/B6-3, and AKR/J were found to have less than 9% of the rDNA methylated. In sharp contrast, Balb/c mice showed 30-50% of the Hpa II and Hha I sites to be methylated. Further study of the Balb/c DNA showed that there are three groups of rDNA sequences. In the first group, all the Hpa II and Hha I sites are almost completely unmethylated; in the second group these sites are all methylated (greater than 30 sites for each enzyme); in the third group most sites are methylated, but there are discrete hypomethylation sites. These hypomethylation positions are at similar sites for both Hpa II and Hha I and show a tissue-specific pattern. Comparison of AKR/J with Balb/c copy level showed that AKR/J had about 60% fewer rDNA genes. The rDNA methylation level might thus be correlated directly with the number of rDNA genes. Finally, analysis of F1 mice from a cross between Balb/c and AKR/J showed both low copy number and low methylation levels.
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When chromatin is extracted from nuclease-digested nuclei by stepwise salt elution, two different classes of newly replicated chromatin can be distinguished. Nascent DNA eluted from nuclei under conditions of low to moderate ionic strength (0.1-0.3 M NaCl) exhibits nucleosomal periodicity and is found in particles which have the same electrophoretic mobility as bona fide H1- or high mobility group protein-containing mononucleosomes. Thus, factors believed to be involved with both the higher order coiling and transcriptionally active state of chromatin are rapidly complexed with newly synthesized DNA and may be retained on parental nucleosomes throughout replication. In contrast, approximately 40% of new DNA is resistant to extraction with solutions of moderate ionic strength. Most of this material is eluted from nuclei by 0.4-0.6 M NaCl. While bulk chromatin that is extracted by 0.4-0.6 M NaCl is organized into nucleosomes, most of the newly replicated "chromatin" from the same fractions lacks subunit structure, as determined by DNA size analyses in polyacrylamide gels, thereby distinguishing this nascent material from newly replicated chromatin eluted at lower ionic strength. Within 15 min all newly synthesized chromatin matures and exhibits the solubility and nucleosomal periodicity characteristics of bulk chromatin. The unusual properties of the "nonnucleosomal" fractions may reflect the structure of newly synthesized DNA prior to its assembly into nucleosomes.
A convenient, preparative scale, nonlytic separation of mouse T lymphocytes into Lyt2.2+ and Lyt2.2- populations is reported. Immunoglobulin-negative (Ig-) spleen cells, Ig- lymph node cells, and peanut lectin-unagglutinated (PNA-) thymocytes were incubated under sterile conditions at 0 degree C with monoclonal mouse antibody to the Lyt2.2 T cell differentiation antigen. The antibody-treated cells were washed and placed in polystyrene tissue culture dishes that had been precoated with antibody to mouse Ig. Nonadherent populations were depleted to Lyt2.2+ cells and were essentially devoid of cytotoxic T lymphocyte precursors (CTLp), but contained helper activity for in vivo T-dependent IgM, IgG and IgA antibody formation. Adherent cell populations were enriched for Lyt2.2+ cells and for CTLp. The graft-vs.-host activity of the separated, adherent (Lyt2.2+) and nonadherent (Lyt2.2-) cells in the Simonsen spleen assay in neonatal (C57BL/6 x BALB/c)F1 mice was less than of unfractionated cells, but the activity of remixed Lyt2.2+ plus Lyt2.2- cells was higher than the sum of the contributions of these cells tested separately, and equal to that of the unfractionated cells. PNA- thymocytes were also separated into Ly2.2+ and Lyt2.2- populations by fluorescence-activated cell sorting. Nonlytic separation allows the recovery of the Lyt1+2+ population, which is lost in cytotoxic elimination experiments. Under the conditions described for the plate separation, the purity of the separated cells and recovery of activity approaches that of cells separated by sorting. Therefore, the plate separation offers a convenient alternative to fluorescence-activated cell sorting when large numbers (i.e. up to 5 x 10(7) positively selected cells) are needed, as in studies of in vivo cell-mediated immune reactions.
Two-dimensional restriction analysis has been applied to terminally labeled restriction segments from adult male and female flies that were the progeny of ten successive single-pair, brother-sister matings. Many of the 2500-4000 different bands that can be seen on autoradiographs are of widely different density, a fact that suggests the superposition of multiple restriction segments of similar sequence. The number of bands observed is significantly fewer than expected. Nearly all of the bands seen in male-DNA displays appear equivalent to those seen in female-DNA displays, but a few sex differences (male-dense and female-dense bands) can be identified--much fewer than expected. When the HpaII-MspI test was applied to fractionated BamHI fragments, between 900 and 1800 discrete C-C-G-G sites were found to be cleaved in a completely equivalent fashion by each isoschizomer.
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Various cultured cell lines of Drosophila melanogaster contain 10 to 13 discrete double-stranded RNAs ranging in length from 1 to 4 kilobases. These RNAs were characterized by nuclease susceptibility, density, solubility in LiCl, thermostability, electron microscopy and gel electrophoresis. These RNAs, which are similar to Reo virus RNA could not be detected in adult or embryonic tissues.
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