Histone synthesis and deposition in the G1 and S phases of hepatoma tissue culture cells

Hepatoma tissue culture cells were synchronized in G1 and in S phase in order to examine the level of synthesis of different histone types and to determine the rate, timing, and location of their deposition onto DNA. We observe a basal level of synthesis in G1 (5% of that seen in S phase) for H2A.1, H2A.2, H3.2, H2B, and H4. The minor histone variants X and Z are synthesized at 30% of the rate observed in S cells. The rate of synthesis of the ubiquinated histones uH2A.1,2 is not as depressed in G1 cells as seen for H2A.1 and H2A.2. Histones synthesized in G1 are not deposited on the DNA of these cells at equivalent rates. Thus, histones H3.2 and H4 are not deposited significantly until S phase begins, at which time deposition occurs selectively on newly synthesized DNA. The deposition of H2A.1, H2A.2, H2B, X, and Z proceeds in G1; however, it occurs to a 2-4-fold lower extent than seen for the deposition of H1, HMG 14, and HMG 17. The deposition of all histones synthesized in S phase occurs rapidly, but there are variations in the sites of deposition. Thus, newly synthesized H3.1, H3.2, and H4 deposit primarily on newly replicated DNA whereas H2A.1, H2A.2, uH2A.1, 2, and H2B deposit only partially on new DNA (30%) and mostly on old. H1, HMG 14, and HMG 17 are deposited in an apparently fully random manner over the chromatin. To interpret these observations, we propose a model which includes a measure of histone exchange on the chromatin fiber. The model emphasizes the dynamics of histone-histone and histone-DNA interactions in regions of active genes and at replication forks.     

Modification of histones immediately following synthesis

The acetylation of newly synthesized histones has been studied by exploiting the ability of sodium butyrate to inhibit deacetylation. Upon arrival in the nucleus a significant fraction of histones H2a, H2b, and H3 is in the parental unmodified form. In contrast histone H4 first appears primarily in the diacetylated form. During an ensuing 135 min the diacetylated H4 is slowly further modified.     

Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system.

6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.     

Histone segregation on replicating chromatin

We have reinvestigated the mode of segregation of preexisting histones onto replicating chromosomes. Since our previous data have indicated that only histones H3 and H4 do not appear to move from their association with the DNA strand with which they are bound until the next round of replication, we have concentrated our attention on these two histones. The strategy we have employed involved density labeling of DNA and radiolabeling of the histones of interest. Subsequently, we followed the association of histones and DNA during further rounds of DNA replication. One can make predictions concerning the nature of the association between specific histones and particular DNA strands depending on the mode of deposition. The results have confirmed our previous findings that histones segregate randomly. The possibility that such a result is a consequence of turnover of radiolabel in non-histone proteins and subsequent reutilization for histone synthesis has been tested directly. This process appears to be occurring to only a very limited extent. The implications of these conclusions for chromatin structure and gene control are discussed.     

Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

Only a small fraction of avian erythrocyte histone is involved in ongoing acetylation.

We have studied histone acetylation in chicken erythrocytes. We find that about 30% of the histone in these cells is acetylated, however the majority of these histones are not in a dynamic steady state typical of other chicken cells and of mammalian cells, but rather are frozen in this state of modification. A very small fraction of erythrocyte histones are being modified normally but cannot be detected as shifting to higher levels of acetylation upon treatment with butyrate because the amount of histone so modified is small. Nonetheless, chicken erythrocytes incorporate 3H-acetate into histones about 40% as well as seen in the dynamically active HTC cells. This is most likely due to the formation of very high specific activity Acetyl CoA pools in erythrocytes which have very low levels of coenzyme A. We conclude that these genetically inactive cells are involved in only a minor way with histone acetylation.     

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.     

The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.