R. David Cole

Isolation of mitotic apparatus containing vesicles with calcium sequestration activity

We present the first report of isolate mitotic apparatus with vesicular calcium sequestration. Phase-contrast, differential interference contrast and polarized light microscopy as well as transmission and scanning electron microscopic examinations revealed structures comparable to mitotic apparatus in vivo. Numerous membrane-bound vesicles which retained their osmotic activity were present throughout. Microtubules, yolk, ribosomes and condensed chromatin were also present. The protein composition of mitotic apparatus was not dramatically altered by treatment with 0.5% Triton X-100, even though vesicles were destroyed and yolk was extracted. Calcium sequestration was demonstrated with ATP-dependent accumulation of 45Ca by mitotic apparatus whose vesicles were left intact. Compared with controls for which no nucleotide was added, accumulation by mitotic apparatus with intact vesicles was enhanced to 184% when it was present. When ATP was supplemented with the divalent ionophore A23187, the calcium retention level was comparable to that of the control to which no nucleotide was added. Finally, the calcium accumulation by mitotic apparatus treated with either of the nonhydrolyzable ATP analogs AMPPCP or AMPPNP resulted in calcium retention levels similar to those of controls. The solubilization of vesicles with Triton X-100 abolished calcium accumulation in the presence or absence of any of the above additives. Resolution of vesicles on sucrose step gradients after 45Ca-oxalate loading with ATP or AMPPCP indicates that a specific vesicular fraction sequesters 45Ca.

A minireview of microheterogeneity in H1 histone and its possible significance

Subtypes of H1 histone vary in primary structure, and the higher organisms that have been studied each seem to have about a half-dozen subtypes. The proportions of these subtypes vary with the progress of differentiation as seen in embryonic development, hormonally induced changes, spermatogenesis, and terminal differentiation. The H1 subtypes differ among themselves in their ability to condense DNA and small chromatin fragments. They have the potential, therefore, of causing different parts of the chromatin to be condensed to different degrees.

Membrane phospholipids associated with nuclei and chromatin: melting profile, template activity and stability of chromatin.

Abstract A substantial amount of phospholipid, the distribution pattern of which resembled that found in microsomal membranes, was recovered in chromatin prepared from whole rat liver (“straight” chromatin). The amount of phospholipid, which is taken to indicate the presence of membrane fragments, was reduced in chromatin prepared from purified nuclei (control nuclear chromatin) and was extremely reduced if obtained from nuclei pretreated with Triton X100 (Triton nuclear chromatin). Electron microscopy showed that detergent treatment removed the outer nuclear membrane and the resulting chromatin sometimes appeared partially dehydrated. Labelling of phospholipid with 32 P, [ 3 H]choline and [ 14 C]acetate showed that the over-all rate of turnover of total phospholipid in outer nuclear membrane approximated that of microsomal membranes, but those of inner nuclear membrane were about four times more stable. This difference was reflected in labelling kinetics in chromatin preparations which indicated that phospholipids or membrane fragments in chromatin from whole tissue or nuclei untreated with detergent were derived from the endoplasmic reticulum and the outer nuclear envelope and those in Triton nuclear chromatin from only the inner nuclear membrane. The presence of varying amounts of phospholipids was of little consequence to the melting profile or template activity of the three types of fresh chromatin preparations. But storage at 2 °C (with 10 −3 m -NaN 3 ) had a drastic effect on the stability of straight chromatin and control nuclear chromatin, which resulted in lower protein/DNA ratios, progressive drop in T m and an enhancement of template activity. Triton-treated nuclear chromatin was stable in this respect for over one month, thus suggesting that proteases associated with the endoplasmic reticulum or the outer nuclear envelope contaminate chromatin prepared without detergent treatment of the tissue or nuclei.

H1 histones from mammalian testes: The widespread occurrence of H1t☆

Abstract H1t is a testis-specific H1 histone variant recently isolated from the rat [13]. We have now identified H1t in testicular extracts from mice, rabbits, hamsters, bulls, and boars, thus establishing its widespread presence in animals. H1t from all species could be differentiated from standard somatic H1 forms by a variety of criteria. It was poorly extracted by 5% trichloroacetic acid (TCA), migrated more rapidly than standard H1 forms during electrophoresis in the presence of sodium dodecyl sulfate (SDS), and eluted more slowly than standard H1 species during chromatography over cross-linked polyacrylamide beads (Bio-Gel P100). Using an improved purification procedure, H1t was isolated from two new species, mouse and rabbit. In these species as with the rat, H1t is readily identified by its low lysine (ca 19 mol%) and high arginine (6–7 mol%) content as compared with that of standard somatic H1 forms (ca 25% lysine and 3% arginine).

Hormonal effects on amino acid incorporation into lysine-rich histones in the mouse mammary gland

Abstract Mammary tissue explanted from mice was cultured in chemically defined media containing radioactive amino acids and either insulin or a combination of insulin, cortisol and prolactin. The incorporation of lysine was determined in five chromatographically resolved peaks which were identified as lysine-rich histones by Chromatographic and electrophoretic behavior, by solubility in trichloroacetic acid, by ratio of lysine: arginine incorporation, and by lack of tryptophan incorporation. Cortisol and prolactin, which are lactogenic hormones in mice, depress the incorporation of lysine in one of the chromatographic components and later elevate incorporation in another. The incorporation pattern of explants cultured in insulin matched that of in vivo incorporation pattern for mammary tissue in late pregnancy while the incorporation pattern for explants cultured in insulin, cortisol, and prolactin matched that of in vivo incorporation in lactating mice. The in vitro effects develop between 16 and 32 hours of culture during a wave of DNA synthesis and mitosis in the epithelial cells. While the mechanism of these effects is not yet established, these observations indicate that the multiplicity of lysine-rich histones is physiologically important.

Purification and characterization of bovine liver β-glucuronidase

Abstract Cellulose ion-exchange chromatography was used to purify bovine liver β-glucuronidase by an improved method. An 18% yield of enzyme having a specific activity of approximately 2 × 10 5 Units per milligram was obtained. The purified enzvme appeared homogeneous by ion-exchange chromatography on several adsorbents, gel filtration, electrophoresis in starch gel and polyacrylamide gel, and ultracentrifugation. From sedimentation-equilibrium, a molecular weight of about 280,000 was calculated. With dry weight as a measure of protein, the E 1 cm 1% at 280 mμ was 17. The amino acid composition was determined. These enzyme preparations contained 3–6% carbohydrate, and glucosamine was detected in acid hydrolyzates by ion-exchange chromatography. The purified enzyme contained less than one mole of phosphate per mole of enzyme. The K m with phenolphthalein glucuronide as a substrate was about 0.05 m m . A broad pH-activity curve with a single optimum at pH 4.8 was observed, and the enzyme was most stable between pH 4 and 7. The enzyme could be chromatographed on diethylaminoethyl-cellulose in the presence of 7.7 m urea, with full recovery of activity after dilution of the urea. It was also found to be markedly stable to autolysis in liver homogenates at 37 °.

Degradation of histones during the manipulation of isolated nuclei and deoxyribonucleoprotein

Abstract 1. 1. Incubation of isolated nuclei or deoxyribonucleoprotein for an hour at 37° in a neutral medium generated new components at the expense of the authentic histones. The amino acid composition of these artifacts indicated that they were derived from authentic histone, and their relative electrophoretic mobilities in polyacrylamide gels showed that they were somewhat smaller than the authentic histone. N-Terminal analysis confirmed that proteolysis occurred during the incubation which produced the artifacts. 2. 2. Many degradation products could be resolved from authentic histones electrophoretically or chromatographically, but all the resolving systems tried (perhaps except one) failed in one case or another to resolve some artifacts. The chromatographic behavior of the degradation products which were resolved resembled components normally found in histone preparations derived from rapid, strong-acid extraction. 3. 3. It was concluded that many degradation products could easily escape detection, and that those which were resolved could easily be mistaken for endogenous histones since they fit the general definition of histone very well.

Further studies on the biosynthesis of very lysine-rich histones in isolated nuclei

Abstract 1. 1. The characteristics of the in vitro amino acid incorporation into histones and other proteins of isolated calf thymus nuclei have been extensively investigated. The kinetics of incorporation into very lysine-rich histones, other histones, and soluble nucleoplasmic proteins have been studied. The quantitatively different susceptibilities to puromycin inhibition of these nuclear proteins were further studied over a range of puromycin levels. Electron-microscopic analysis of the whole cell content of various thymus nuclear preparations, prepared by Ficoll barrier sedimentation, was correlated with amino acid incorporation into histones, demonstrating that histones are indeed synthesized within the nucleus. An unexpected inverse relationship between the specific activities of the nuclear proteins and the concentration of nuclei during incubation was observed. This inverse relationship was found to be quantitatively different for different classes of nuclear proteins and has been attributed to dilution of the radioactive amino acids by unlabelled amino acids released from the nuclei during incubation. 2. 2. By the use of an improved fractionation procedure the very lysine-rich histone preparation was resolved into 4 undegraded components. Determination of specific activities of these 4 very lysine-rich histones showed that their rates of synthesis did not differ significantly one from another.

Shell formation by capsid protein of f2 bacteriophage

Abstract The solution conditions necessary for the aggregation of the coat protein of f2 bacteriophage into virus sized shells in the absence of RNA have been investigated. There is a broad pH optimum between 5.5 and 7.0, and as expected the aggregation is strongly concentration dependent. The shells form only below 25 °C, but do so rapidly at these temperatures. Ionic strength is important in the formation of shells, and the species of ions are crucial. The presence of RNA promotes shell formation under conditions where shells normally will not form spontaneously. The stability of shells once formed is quite great, and in fact may be equivalent to the stability of whole phage. Using these data and other information, a structure for shells and phage has been proposed.

The applicability of extraction by trichloroacetic acid to the preparation of very lysine rich histones from the mammary gland

Abstract The technique of De Nooij and Westenbrink (1) for extraction of very lysine-rich histones from the calf thymus was found applicable to the rabbit mammary gland. In order to increase yields and minimize heating, the tissue was pulverized with solid C0 2 prior to blending. The concentration of the TCA 4 by which histones were extracted was not critical over at least the range 2.5 to 4.5%. The final yield was about 14 mg per 100 gm of wet tissue. Only a little contamination was detected in these preparations by amino acid analyses, electrophoresis on polyacrylamide gels, and ion exchange chromatography. A systematic study of the protein extracted by a series of TCA concentrations revealed that lower (less than 2.5%) TCA concentrations extracted, together with the very lysine-rich histones, significant amounts of contamination. Chromatographically this contamination was well resolved from four peaks of very lysine-rich histone. The absolute amounts and relative proportions of the latter four components were independent of the TCA concentration used for extraction.