Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.     

Deoxyribonuclease I produces staggered cuts in the DNA of chromatin

The relationship of cuts made by deoxyribonuclease I (DNase I, EC. 3.1.4.5) on the two strands of DNA of chromatin has been investigated. DNA was extracted from a DNase I digest of rat liver nuclei and incubated with the large fragment of DNA polyrnerase I. Analysis of the products of this incubation indicates the cuts made by DNase I on opposite strands are staggered with respect to one another. A cut on one strand is about two bases in the 3′ direction or eight bases in the 5′ direction from the position on its own strand which is directly across from the cut on the other strand. A different result is obtained when a DNase I digest of native DNA is analyzed. Current models for the organization of DNA in the nucleosome are discussed with respect to these results.     

Nucleosome segregation in chromatin replicated in the presence of cycloheximide

Ehrlich ascites tumour cells and L cells were grown in the presence of [¹⁴C]thymidine to label DNA replicated under normal conditions and were then cultured in the presence of cycloheximide and [³H]thymidine to label DNA replicated in the absence of histone synthesis, Chromatin from these cells was digested with micrococcal nuclease and with restriction endonuclease BspRI (an isoschizomer of HaeIII). The rates of digestion of the ¹⁴C-labelled and of the ³H-labelled DNA, and the size and buoyant density of the BspRI-generated chromatin fragments showed that: (1) chromatin replicated in the presence of cycloheximide contained half the normal amount of histones; (2) it did not contain long stretches of naked DNA; and (3) it was organized in nucleosomes distributed along DNA in groups of several particles separated by relatively short stretches of histone-free DNA. Control experiments showed that this could not be the result of a long-distance sliding of nucleosomes.These data suggest a bilateral mode of nucleosome segregation during DNA replication.     

A model chromatin assembly system. Factors affecting nucleosome spacing

Poly[d(A-T)].poly[d(A-T)], when reconstituted with chicken erythrocyte core histones and subsequently incubated with sufficient histone H5 in a solution containing polyglutamic acid, forms structures resembling chromatin. H5 induces nucleosome alignment in about two hours at physiological ionic strength and 37 degrees C. The nucleosome spacing and apparent linker heterogeneity in the assembled nucleoprotein are very similar to those in chicken erythrocyte chromatin. Also, condensed chromatin-like fibers on the polynucleotide can be visualized. The binding of one mole of H5 per mole of core octamer is necessary to generate the physiological nucleosome spacing, which remains constant with the addition of more H5. The nucleosome repeat length is not a function of the core histone to poly[d(A-T)] ratio for values lower than the physiological ratio. With increasing ratios, in excess of the physiological value, nucleosome spacing first becomes non-uniform, and then takes on the close packing limit of approximately 165 base-pairs. In addition to eliminating possible base sequence effects on nucleosome positioning, poly[d(A-T)] allows nucleosomes to slide more readily than does DNA, thereby facilitating alignment. Evidence is presented that polyglutamic acid facilitates the nucleosome spacing activity of histone H5, primarily by keeping the nucleoprotein soluble. This model system should be useful for understanding how different repeat lengths arise in chromatin.     

The use of a complementary DNA probe to detect accumulation of mengo RNA in infected cells pretreated with interferon.

Complementary DNA (cDNA) from Mengo virus RNA has been synthesized and used as a probe to measure the synthesis and accumulation of viral RNA in Mengo infected L cell cultures, treated or untreated with interferon. Under experimental conditions used (200 units interferon/ml and 50 virus plaque-forming units/cell) results show that there is some synthesis of Mengo virus RNA in cells treated with interferon. One hour after infection, treated cells contain three times less viral RNA than untreated cells; five hours after infection, this difference has increased to ten fold. As in the control, no fragmented Mengo virus RNA molecules were found in interferon treated cells. The smaller recovery of infectious particles from interferon treated cells as compared to RNA accumulation suggests that not only RNA accumulation is inhibited but also a step posterior in viral maturation.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

Molecular dynamics of calmodulin as monitored by fluorescence anisotropy

Static and dynamic measurements of fluorescence anisotropy have been made for calmodulin, employing both the intrinsic fluorescence of Tyr-99 and Tyr-138 and the fluorescence of dansyl and fluorescein groups attached to Tyr-99, as well as AEDANS groups attached to methionines. All approaches indicate the presence of a significant internal mobility involving the probe for calmodulin in the absence of Ca²⁺. This is diminished in the presence of Ca²⁺.²     

Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions

In the chicken genome there are middle repetitive DNA sequences with a clustered organization. Each cluster is composed of members of different families of repeated DNA sequences and usually contains only one member of each family. Many clusters have the same assortment of repeated sequences but they are in scrambled order from cluster to cluster. These clusters usually exceed 20 × 10³ bases in length and comprise at least 10% of the repeated DNA of the chicken. The repeated sequences that are cluster components are extensively methylated. Methylation was detected by comparing HpaII and MspI digests of total DNA, where the occurrence of the sequence C-m⁵C-G-G is indicated when HpaII (cleaves C-C-G-G) fragments are larger than those generated by MspI (cleaves C-m⁵C-G-G or C-C-G-G). In hybridization experiments with Southern (1975) blots of total DNA digested with either HpaII or MspI, the cloned probes representing clustered repeated sequences showed a dramatic difference in the lengths of restriction fragments detected in the two digests. Many of the sequences that comprise these clusters are methylated in most of their genomic occurrences. There are patterns of methylation that are reproduced faithfully from copy to copy. The overall distribution of methylation within clusters seems to be regional, with long methylated DNA segments interrupted by specific undermethylated regions.     

Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action

Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end has been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Synthesis of specific membrane proteins is a function of DNA replication and phospholipid synthesis in Caulobacter crescentus

Analysis of the effects on membrane function and protein composition of altering phospholipid synthesis in Caulobacter crescentus showed that, like other bacteria, C. crescentus continues to induce a lactose transport system and to synthesize most membrane proteins. However, we show that the incorporation of a set of outer membrane proteins primarily synthesized in stalked cells is dependent on DNA replication which, in turn, is dependent on membrane phospholipid synthesis. Furthermore, the incorporation of another set of membrane proteins, two of which are synthesized primarily in the swarmer cell, appears to be independent of the replication of the chromosome but to be directly dependent on phospholipid synthesis. We have also found that when phospholipid synthesis is blocked, the synthesis of the flagellar proteins is inhibited and that this effect may be mediated by the primary inhibition of DNA replication. Newton has presented evidence that the synthesis of flagellar proteins is dependent on specific execution points in DNA replication and that this connection serves as a temporal regulator of differential protein synthesis (Osley et al., 1977; Sheffery & Newton, 1981). We suggest here that a direct link between the replicating chromosome and the growing membrane might serve, in turn, to dictate the site of membrane assembly of newly synthesized gene products.     

An improved method of reconstitution of adipocyte glucose transport activity

The glucose transport activity of rat epididymal fat cells was reconstituted into egg lecithin liposomes with a high degree of reproducibility. The activity was solubilized with 20 mm sodium cholate in Buffer B (10 mm Tris-HCl, pH 7.5). After elimination of small molecules by gel filtration, the transport activity was incorporated into egg lecithin liposomes (Sigma, Type IX-E, homogeneously dispersed into Buffer B) by sonication (5 s), freezing (?70°C), thawing, and a second sonication (5 s). The sonication was done in a 16.8-mm polystyrene test tube (Sarstedt, 55-468) placed in a cup horn (from Heat Systems Ultrasonics) connected to a Branson's sonicator (W-185) at setting No. 3 (70 W of output). The optimum sample size was 80 μl, and the optimum clearance between the test tube and the sonicator horn was 2–3 mm. The concentration of egg lecithin at the reconstitution step was 25 mg/ml, and that of the microsomal protein was approximately 0.3–0.5 mg/ml. The glucose transport activity of reconstituted liposomes was assayed by incubating the latter with a mixture of d-[³H]glucose and l-[¹⁴C]glucose. The incubation was terminated by the addition of HgCl₂, and the reaction mixture was filtered with a Millipore filter (GSWP). The difference in the rates of uptake of d-glucose and l-glucose was regarded as representing the carrier-mediated glucose transport activity. The results of the assay indicated that the glucose transport activity could be reconstituted in a highly reproducible manner. The reconstituted activity was proportional, within a limit of experimental error, to the amount of protein used for reconstitution and was almost completely blocked by cytochalasin B, phloretin, or HgCl₂. However, a small amount of d-glucose was found to bind with the egg lecithin preparation.     

A specific DNA orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy.

The molecular structure of the single-stranded fd DNA inside its filamentous virion has been stabilized by the photochemical reaction with a psoralen derivative and examined in the electron microscope. The results support the notion that the 6389 nucleotide-long DNA molecule is folded back on itself inside the 1 μm-long protein coat. At one end of the virion, there exists a DNA hairpin region 200±50 base-pairs long. This “end hairpin” is mapped on the fd genome to the site of the replication origin. The most stable in vitro hairpin of fd DNA has been mapped previously to this same site. This unique duplex region of fd DNA may play an important role in the formation of specific protein-DNA complexes which are crucial to stages of the fd life cycle: the adsorption of the phage to the bacteria, the initiation of replication of the single-stranded DNA, and the assembly of newly synthesized DNA strands into the filamentous virions.     

Conservation and DNA sequence arrangement of the DNA polymerase I gene region from Klebsiella aerogenes, Klebsiella pneumoniae and Escherichia coli

Linked multiple mutation is observed after treatment of Escherichia coli with methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and N-ethyl-N-nitro-N-nitrosoguanidine but not ultraviolet light. Induction of linked multiple mutations requires the uvrE⁺ gene product indicating the involvement of the mismatch repair system. The observation of linked multiple mutations is not due to mutagenesis occurring in a subpopulation of cells. Growing point mutagenesis also occurs after treatment with these mutagens but not with ultraviolet light. It is likely that the excess of mutations observed with these mutagens at growing points is at least partly a relative effect, rather than one due to an absolute increase of reactivity at the DNA growing point region. This relative effect may result from the operation of an inducible repair mechanism which removes O⁶-alkylguanine residues from the DNA distal to the bacterial growing point. The adaptive response, first described by Robins &; Cairns (1979) prefers O⁶-methylguanine over O⁶-ethylguanine.     

Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.