Canadian journal of biochemistry最新文献

Mononucleosomes isolated from micrococcal nuclease digests of stationary phase chromatin of the yeast Saccharomyces cerevisiae were compared both compositionally and physiochemically with those from chicken and bovine calf. It was found that while yeast mononucleosomes are similar in composition, their thermal denaturation profiles and circular dichroism spectra indicate a less constrained structure. Furthermore, yeast nucleosomes were discovered to be labile in solutions of low ionic strength and could not be reconstituted by methods applicable to calf and chicken nucleosomes. On the basis of the reconstitution of a hybrid nucleosome containing calf histones H2A, H2B, and H3 and yeast histone H4, it was concluded that variations in the yeast H4 sequence are unlikely to be responsible for the apparent decrease in the stability of yeast nucleosomes. Examinations of histone-histone interactions in free solution revealed a change in the H3-H4 interaction and together with the previously published results of other researchers it was inferred that changes in the H3 sequence might be responsible for this structural variation.

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.

The present results demonstrate that Chinese hamster embryo cell populations in culture can be adapted to grow in the presence of chloramphenicol. It is shown that tryptose phosphate broth and uridine, one of its components, prevent the growth inhibitory effect of the drug. Study of some respiratory parameters (cytochrome c oxidase, cytochrome spectra, oxygen consumption) indicated that neither the broth nor uridine prevented the inhibitory effect of chloramphenicol on mitoribosomal protein synthesis. The cells grew with mitochondria devoid of a functional respiratory chain. Auxotrophy for pyrimidines appeared to result from the absence of dihydroorotate dehydrogenase, a respiratory chain-linked enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis. These and other results suggest that the synthesis of orotic acid may be considered as one of the main contributions of mitochondria to the growth of animal cells in culture.

When pancreatic chromatin fragments were prepared and resolved in the presence of 80 mM NaCl, endogenous poly(ADP-ribose) polymerase activity was found to be maximal in nucleosome periodicities of four to five units and did not respond to any further increases in nucleosomal architecture. Furthermore, in nucleosome complexities spanning 1 through 14 and over unit lengths, polyacrylamide gel electrophoresis on acid-urea and acid-urea-Triton gels has shown pancreatic histone H1 to be the only actively ADP-ribosylated histone species. The extent of ADP-ribosylation of histone H1 was also demonstrated to retard the protein's mobility in acid-urea, acid-urea-Triton, and lithium dodecyl sulfate polyacrylamide gels and to consist of at least 12 distinct ADP-ribosylated species extractable in all nucleosome complexities studied. Finally, extraction and subsequent electrophoresis of total chromosomal proteins in the presence of lithium dodecyl sulfate also evidenced heavy ADP-ribosylation at the level of nonhistone chromosomal proteins of the high mobility group comigrating in the core histone region, as well as in the topmost region of the gels where poly(ADP-ribose) polymerase was found to form a poly(ADP-ribosyl)ated aggregate.

DNA and protein contents of pairs of sister nuclei were determined using a combined Feulgen-dinitrofluorobenzene technique. Sister nuclei were studied in binucleate cells, induced by treatment with 0.1% caffeine, and in sister mononucleate cells of untreated roots. Excised pea roots, grown in culture, were treated with 5-aminouracil to induce mitotic synchrony and with caffeine at the time of peak mitotic index, to provide the maximum number of binucleate cells. The induced binucleate cells form a marked population which was followed through a cell cycle; sister nuclei showed a correlation of volume and protein content, r = 0.79. Protein contents of sister nuclei were rarely identical and at 1 + 2 and 1 + 6 h the difference in protein contents of sister nuclei was significant (p = 0.05). Mean nuclear protein content decreased from 1 + 2 to 1 + 6 h; then, as nuclei entered S phase, their protein content increased. From 1 + 2 to 1 + 14 h the increase in protein content, in absolute amount, was identical in both sister nuclei. This suggests that there was a biphasic pattern of protein uptake; it is differential, in sister nuclei, in the first part of G1 but is identical throughout the rest of interphase. Analysis of sister nuclei from sister mononucleate cells showed a similar pattern of change; this is further evidence, from untreated cells, of a biophasic pattern of protein uptake. Caffeine-treated nuclei had lower protein contents than untreated nuclei, yet they completed a cell cycle and entered mitosis; this suggests that nonessential proteins were no longer present. It is proposed that mitosis is asymmetrical for molecules that regulate rates of macromolecular synthesis, cell growth, and progress through a cell cycle and that once the initial asymmetry has been established, it is maintained throughout interphase, even in binucleate cells in which the two nuclei share a common cytoplasm.

Change in RNA during the juvenile hormone (JH) stimulated synthesis of vitellogenin (Vg) in the fat body of adult female Locusta migratoria have been studied. Total RNA from mature females, but not that from males or prereproductive females, shows a 6300 nucleotide component, which has been isolated by binding to oligo(dT)-cellulose and sucrose gradient centrifugation, and identified as Vg mRNA by translation in Xenopus oocytes. It has been assayed quantitatively by photometric scanning after electrophoresis. During a gonotrophic cycle, Vg mRNA increased rapidly from 0 up to about 1% of the total fat body RNA, or more than 10(6) copies per cell. After destruction of the corpora allata (the source of JH) by treatment with ethoxyprecocene, Vg synthesis was stimulated by injection of 150 micrograms of the JH analog, methoprene. In primary stimulation, Vg mRNA was first detected at 24 h and showed a marked lag in accumulation; in secondary stimulation by methoprene after decay of the primary effect, Vg mRNA was detected after only 12 h and accumulation was much more rapid. Both in the natural cycle and in experimental stimulation, Vg mRNA did not disappear in correlation with declining Vg synthesis, which suggests conservation of mRNA in untranslated form. Total (chiefly ribosomal) RNA showed a different pattern, accumulating markedly during primary and only slightly during secondary stimulation. The data indicate that JH acts selectively (though not necessarily directly) on transcription of the Vg genes.

The heat-shock response has been characterized in cultured fibroblasts of the rainbow trout, Salmo gairdnerii. The response has been elicited by two different stress situations; cells were either subjected to higher temperatures than normal (27 to 29 degrees C as opposed to 22 degrees C) or were incubated in medium containing sodium arsenite (15 to 100 microM final concentration). The response of the cells to these conditions is to synthesize a set of new polypeptides, the "heat-shock polypeptides" (hsps), that are not present or present in extremely low amounts in noninduced cells. Furthermore, during prolonged arsenite induction, the synthesis of normal cellular proteins is repressed. In trout fibroblasts, at least six hsps are detectable. These range from 30 000 to 87 000 in molecular weight and are referred to as hsp30, hsp32, hsp42, hsp62, hsp70, and hsp87. The hsp30 and hsp70 components are the most abundant and can be visualized by Coomassie blue staining of gels after prolonged induction. The heat-shock response is a reversible process in trout cells. Results of in vitro translation of mRNA from induced cells indicate that the control of hsp induction may be at the transcriptional level. Hsp70 from trout comigrates with the major hsp from Drosophila melanogaster on sodium dodecyl sulfate - polyacrylamide gels, suggesting that this protein may be highly conserved in evolution.

Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage. Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 degrees C; alkylated phage was then incubated 24 h at 30 degrees C to induce depurination. These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid. After ejection, the phage preparations were analyzed by electron microscopy. DNA lengths in capsid-DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined. To establish the direction of DNA ejection, E. coli RNA polymerase was bound to capsid-DNA complexes. The results showed that DNA was partially ejected from both alkylated and depurinated phages. In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end. We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end. For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.

Intense gamma-irradiation from a cobalt source differentially affects macromolecular synthesis of cultured mammalian cells. Exposure of monkey BSC-1 or murine fibroblastic L2 cells to 40 or 70 krad (1 rad = 1 x 10(-2) J/kg) abolishes DNA and RNA synthesis almost entirely but reduces the formation of protein much less. A dose-response analysis of irradiation shows that synthesis of total RNA and the messenger component thereof, measured as the poly(A)-containing fraction, are equally diminished. Host cells in which formation of DNA and RNA are minimal can support normal or nearly normal replication and transcription rates of vesicular stomatitis and vaccinia viruses. Therefore, use of pretreatment with gamma-irradiation, as employed here, should prove to be generally useful in examining virus-related transcription under circumstances in which application of drugs affecting gene expression, such as actinomycin D, is deemed undesirable.

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.