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Biomedical subjects

I M Leffak

Publications and source records attributed to I M Leffak.

11 recordsLinked to original sources
PubMed1984

Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair.

In vitro labeling of DNA molecules using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair has been examined as an alternative to existing methods of replacement synthesis labeling. It is shown that exonuclease III is able to act in a common restriction enzyme buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 50 mM NaCl] to produce a population of base-paired primer:template molecules which decrease uniformly in single-strand length with time. After heat inactivation of the exonuclease III and in the presence of radiolabeled deoxynucleotides the polymerase I reaction faithfully resynthesizes full-length molecules, asymmetrically labeled to high specific activity.

Base Composition↗
PubMed1983

Chromatin assembled in the presence of cytosine arabinoside has a short nucleosome repeat.

Incubation of MSB cells with cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) inhibits 3H-thymidine incorporation into nascent DNA while nucleosome core histone synthesis proceeds in molar stoichiometry at about 20% of control rates. The excess nascent histone is incorporated into chromatin and nucleosome cores are assembled normally on the small amount of DNA which is synthesized at submaximal levels of ara-C. This DNA becomes packaged into a shortened nucleosome repeat, however. These results indicate that the nucleosome core is a strongly conserved unit of chromatin replication and suggest that the stoichiometry of nascent histone to DNA may be one factor influencing the establishment of the nucleosome repeat length. It cannot be the only factor, however, since the closely packed nucleosomes made in the presence of ara-C begin to return to their normal spacing within six hours after reversal.

Cell Line↗
PubMed1983

Stability of the conservative mode of nucleosome assembly.

The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.

Animals↗
PubMed1983

Decreased protein staining after chemical crosslinking.

Reaction of several proteins with the homobifunctional amino group-directed reagents dithiobissuccinimidyl propionate or methylmercaptobutyrimidate is shown to decrease the subsequent Coomassie blue or silver staining of these proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decreased dye response is not due to the proximity of the crosslinked proteins since the monomers generated after bisection of the crosslinkers with beta-mercaptoethanol show reduced staining. Quantitation of dye absorbance relative to protein content (assayed by radioactivity) indicates that the decreased staining effect is variable, depending on the particular stain, crosslinker, and protein.

Chemical Phenomena↗
PubMed1981

Sequence sensitivity of histone binding.

The nucleotide sequence selectivity of histone binding has been measured by thermal denaturation of reconstituted nucleoproteins. When DNAs of different average base compositions competed for the binding of purified histone fractions during in vitro reconstitutions in the presence of salt and urea, a decreasing (A + T)-binding preference was observed following the order H1 greater than H2B greater than H5 greater than H2A greater than [H2A + H2B] greater than [H2A + H2B + H3 + H4], [H1 + (H2A + H2B + H3 + H4)2]. Nucleoprotein complexes formed under conditions shown to yield more physiologically comparable nucleosome structures revealed a minimal (A + T)-binding preference. These results suggest that homotypic and heterotypic histone interactions decreased the nucleotide sequence selectivity of nucleosome binding.

Base Sequence↗
PubMed1977

Conservative assembly and segregation of nucleosomal histones.

The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3-4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2-3 generations.

Animals↗
PubMed1975

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.

Animals↗
PubMedDate not supplied

Conservative segregation of nucleosome core histones.

Density labelling studies have shown that nascent histones are not mixed with parental histones during the assembly of nucleosome cores. However, experiments in other laboratories, examining histone deposition with respect to newly synthesized DNA, have been interpreted as suggesting that a substantial proportion of core histones (greater than 15%) are randomized at each chromatin replication. The data presented here support our previous results in showing that conservatively assembled nucleosome core histone octamers are conservatively segregated over successive cell generations. It is also shown that the nucleosome cores assembled during 1-beta-D-arabinofuranosylcytosine inhibition of DNA synthesis are conservatively segregated for a minimum of five or six cell generations. These results suggest that the nonrandom assembly of nucleosome cores is not merely a coincidence of the mechanism of histone transport into the nucleus and that the conservative mode of nucleosome segregation is a fundamental feature of chromatin replication, one which is stable to modulations in chromatin packaging.

Animals↗