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Biomedical subjects

D E Comings

Publications and source records attributed to D E Comings.

At least 163 records · Page 9Linked to original sources
PubMed1977

Optical Studies of the interaction of 4'-6'-diamidino-2-phenylindole with DNA and metaphase chromosomes.

The optical absorption and fluorescence characteristics of 4'-6-diamidino-2-phenylindole (DAPI) with DNA and chromosomes were studied. There is a decrease in extinction coefficient and chift in the absorption spectra to a higher wavelength when the dye binds to DNA. The fluorescence of DAPI is enhanced by both A-T and G-C base-pairs. The enhancement by A-T rich is significantly greater than by G-C rich DNA. The chromosomes and the constrictions of human chromosomes 1 and 16; these regions are known to contain A-T rich DNA and show dull fluorescence when treated with quinacrine. This dye may be useful for identifying A-T rich region in chromosomes. The fluorescence of DAPI bound to polynucleotides or chromosomes is partially quenched by the introduction of BrdU. This suppression of dye fluorescence allows optical detection of sister chromatid exchanges and chromosome region containing DNA with an unequal distribution of thymidine between polynucleotide chains after BrdU incorporation.

Adenine Nucleotides↗
PubMed1976

Histones and G banding of chromosomes.

Polylysine, polyarginine, and histones H1, H2A, H2B, and H3 inhibit Giemsa staining and chromosome banding by binding to DNA and preventing side stacking of the positively charged thiazine dyes to the negatively charged phosphate groups on DNA. This is a nonspecific effect and does not of itself provide evidence for a role of histones in G banding. The question of whether histones are involved in chromosome banding is reviewed.

Arginine↗
PubMed1976

Mechanisms of chromosome banding. IX. Are variations in DNA base composition adequate to account for quinacrine, Hoechst 33258 and daunomycin banding?

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.

Adenine Nucleotides↗
PubMed1976

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.

Actins↗
PubMed1976

Fine structure of the heterochromatin of the kangaroo rat Dipidomys ordii, and examination of the possible role of actin and myosin in heterochromatin condensation.

Biochemical studies have suggested that some actin and myosin may be present in the nucleus. This raises the possibility that heterochromatin condensation might be the result of an actin-myosin rigour type complex. Since ATP dissociates actin and myosin, this possibility could be examined by determining the effect of ATP on heterochromatin condensation. Thin-section electron microscopy showed large amounts of condensed constitutive heterochromatin in the kidney nuclei and somewhat less in the liver nuclei of the kangaroo rat, Dipidomys ordii. Surprisingly, there were some nuclei in the brain which contained no condensed heterochromatin despite the fact that this genome is composed of 50% satellite DNA. Although washing kidney nuclei with solutions of 10 mM Tris-ATP caused marked decondensation of the heterochromatin, when they were washed with Mg-ATP the heterochromatin was more condensed than in the controls. This suggests the decondensation by Tris-ATP is due to its ability to chelate divalent cations and provides no support for condensation of heterochromatin being the result of myosin-actin interaction. Despite being decondensed, the chromatin fibres of heterochromatin were distinct from those of euchromatin. The heterochromatin formed rod-like 19-5 nm fibres, the euchromatin formed random coils of 11-0-nm fibres.

Actins↗
PubMed1975

Fibroblasts in Huntington's disease.

We investigated the growth of skin fibroblasts in tissue culture from 10 patients with Huntington's disease and eight healthy, unrelated controls. The patients' ages ranged from 34 to 56 years (mean 48.5), and the mean duration of their clinical illness was 12.4 years. The controls' ages ranged from 32 to 64 years (mean, 42.3). No statistically significant differences were observed between the two groups in out-growth of cells from the biopsies nor during subsequent routine culturing of fibroblasts. This contradicts the finding of earlier investigators that skin fibroblasts from patients with Huntington's disease grow poorly in tissue culture. Matched pairs of Huntington's disease and control cultures grew at the same rate, but Huntington's disease cells grew to a significantly higher maximal density (P less than 0.02). This may indicate a genetically determined change in the cell surface or metabolic difference in Huntington's disease.

Adult↗
PubMed1975

Mechanisms of chromosome banding. VIII. Hoechst 33258-DNA interaction.

The interaction of Hoechst 33258 with DNA has been examined to help clarify the mechanisms of banding. 1. In agreement with previous studies Hoechst fluorescence is enhanced to a greater degree in AT-rich compared to GC-rich DNA. 2. Hoechst causes an increase in the DNA Tm which is greater at the higher AT content of the DNA. 3. There is a decrease in extinction coefficient and shift in the adsorption spectra to a higher wavelength when Hoechst binds to DNA. 4. DNA is completely precipitated at a ratio of one dye molecular per base pair, and this precipitation is not affected by salt. 5. There is no increase in viscosity or change in the circular dichroism of DNA when bound to Hoechst. These findings suggest Hoechst does not bind to DNA by intercalation or by ionic interaction with the phosphate groups, but rather binds by an attachment to the outside of the double DNA helix by interacting with the base pairs. This type of binding allows greater sensitivity to the base composition than occurs with intercalating agents. In this respect its binding is similar to that of dibutyl proflavine (Muller et al., 1973).

Adsorption↗
PubMed1975

Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC-banding).

Hoechst 33258 fluorescent staining of bromodeoxyuridine substituted chromosomes provided a high resolution technique for following the segregation of replicated chromosomal DNA (Latt, 1973). Modifications have produced the same results after Giemsa staining (Wolff and Perry, 1975). Since this does not necessarily require Hoechst (Korenberg and Freedlander, 1975), we call this bromodeoxyuridine-Giemsa banding (BG-banding). We here describe a further modification which allows one to follow the T-rich strand of the AT-rich satellite DNA of C-band heterochromatin. We call this TC-banding. This technique was used to examine metacentric marker chromosomes found in mouse L-cells that contain many interstitial blocks of centromeric-type heterochromatin in each arm plus the usual two blocks of centromeric heterochromatin. One of the advantages of this technique for such chromosomes is that it is possible to distinguish first from second cell cycle sister chromatid exchange and unambiguously detect centromeric sister chromatid exchange. We found some chromosomes to have high rates of centromeric sister chromatid exchange. After one cycle in bromodeoxyuridine we could examine the satellite polarity of the heterochromatic DNA. Since there was no change in satellite polarity in any of the heterochromatic blocks, marker chromosomes could not have been formed by paracentric inversions, inverted insertions or inverted translocations. These results allow the formulation of several rules of chromosome organization.

Animals↗
PubMed1975

Mechanisms of chromosome banding. VII. Interaction of methylene blue with DNA and chromatin.

The binding of methylene blue to DNA and chromatin treated in various ways was examined by equilibrium dialysis. The maximum r value (moles of bound dye/mole of nucleotide) was 1.0 for DNA, 0.6 for unfixed chromatin, and 0.83 for chromatin fixed in methanol-acetic acid. When fixed chromatin was treated with saline-citrate at 60 degrees C for 3 hours, as used for G-banding chromosomes, the r value decreased from 0.83 to 0.55. When unfixed chromatin was treated as for R-banding the r values also dropped. Equilibrium dialysis indicated there was no disproportionate increase of dye binding as the concentration of DNA increased. -- These results, and others, suggest that some of the Giemsa negative regions of G- and R-banded chromosomes are due to the denaturation of non-histone proteins so that they more effectively cover the DNA and prevent side binding of the thiazin dyes.

Animals↗
PubMed1975

Mechanisms of chromosome banding. IV. Optical properties of the Giemsa dyes.

A thorough understanding of the mechanisms of R-, C- and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral absorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. - Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not necessary component for banding. - The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their absorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.

Aniline Compounds↗