Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos (S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, $\text{dP1}\text{dt}\text{= M·f·n·}\text{A}\text{t}$, where $\text{dP1}\text{dt}$ = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and $\text{A}\text{t}$ = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ($\text{dP1}\text{dt}$). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo^?1 hr^?1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo^?1 hr^?1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (f^zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f^zygotic and $\text{dP1}\text{dt}$, the rate of elongation of nascent histone polypeptide chains ($\text{A}\text{t}$) during the first 6 hr of development was estimated; $\text{A}\text{t}$ remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.     

Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system

A small quantity of unsaturated diacylglycerol (DG) sharply decreased the Ca²⁺ and phospholipid concentrations needed for full activation of a Ca²⁺-activated, phospholipid-dependent multifunctional protein kinase described earlier (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979)$\text{J.}\text{Biol.}\text{Chem.}\text{254}$. 3692–3695). In the presence of unsaturated DG and micromolar order of Ca²⁺, phosphatidylserine (PS) was most relevant with the capacity to activate the enzyme, whereas phosphatidylethanolamine and phosphatidylinositol (PI) were far less effective. Phosphatidylcholine was practically inactive. It is possible, therefore, that unsaturated DG, which may be derived from PI turnover provoked by various extracellular stimulators, acts as a messenger for activating the enzyme, and that Ca²⁺ and various phospholipids such as PI and PS seem to play a role cooperatively in this unique receptor mechanism.     

Calcium stimualtion of protein kinase C in the absence of added phospholipids

The activity of protein kinase C as isolated and described by Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. $\text{252}$, 7610–7616, can be markedly stimulated by Ca²⁺ in the presence of 4 mM Mg²⁺. This Ca²⁺ dependency does not require the presence of phospholipids or exogenous calmodulin. The increase in activity in the presence of Ca²⁺ is blocked by fluphenazine in the presence of 30 mM 2-mercaptoethanol. These results suggest that a calmodulin-like moiety may be a subunit of prokinase C.     

Anuran erythrocytes and liver both contain satellite histone Hls

In addition to the usual major Hl sub-fractions, a “satellite” histone Hl^s, similar in amino acid composition and molecular weight to mammalian Hl^o, is present in both liver and erythrocyte nuclei of $\text{Xenopus laevis}$ and $\text{Rana catesbeiana}$ (as in freshwater turtles), but no erythrocyte-specific histone resembling the H5 of birds and some fish was found. Such lysine-rich satellite histones may share functional homologies while displaying tissue-specific variations in composition.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Differential phosphorylation of histone H1 subfractions in vivo.

A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles ($\text{Rana}\text{catesbeiana}$). Two H1 subfractions were obtained which differed in terms of net incorporation of [³²P]phosphate $\text{in}\text{vivo}$. Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [³²P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [³²P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues.     

Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts

We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈10–15}\text{min}$ for monoacetylated histone H4) compared to the slower rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈140–200}\text{min}$ for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.     

Rapid modulation by progesterone and tamoxifen of estradiol effects on nuclear histone acetylation in the uterus of the fetal guinea pig

The effect of estradiol, progesterone, tamoxifen, estradiol + progesterone or estradiol + tamoxifen on the [³H]acetylation of histones in the fetal uterus of guinea pig was studied. The fetuses were injected subcutaneously ‘in situ’ with the hormones or $\text{tamoxifen}\text{+ [}{}^3\text{H}\text{]}\text{acetate}$ alone. In 10 min, estradiol stimulated the acetylation of histone 10–12-times with respect to the control animals. Progesterone and tamoxifen blocked this effect. It is suggested that histone acetylation is an early step induced by estrogen action during intrauterine life and that progesterone and tamoxifen suppress this mechanism very effectively.     

A rapid procedure to detect in situ DNA/RNA hybrids

We developed a new method for detecting DNA/RNA hybrids formed $\text{in}\text{situ}$ using $\text{anti-}\text{DNA}\text{RNA}$ antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesised $\text{in}\text{vitro}$ from cloned $\text{Drosophila}$ histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.     

Postnatal increase in histone H1a in the rat pancreas

Adult rat pancreas contains an unusually high amount of histone H1a; $\text{H1a}\text{H1}$ ratio = 0.27. Histone H1a is not detectable in embryonic or newborn rat pancreas. This histone begins to appear at about 1 week postnatally and reaches the adult level about 5 weeks after birth. There is an inverse relationship between the appearance of histone H1a and DNA synthesis, as indicated by the nuclear labeling index after [³H]thymidine autoradiography.     

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

Do copper ions influence the reduction of ferricytochrome C by O−2?

Recently, it was suggested that the measured rate of reduction of ferricyto chrome $\text{C}$ by O^?₂ below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157–168).The high values were attributed to the presence of impurities of copper, which compete for O^?₂. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome $\text{C}$ in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome $\text{C}$.It was previously shown by us and by others that the reduction yield of ferricytochrome $\text{C}$ by O^?₂ is 100%. We measured the rate of reduction of ferricytochrome $\text{C}$ by Cu(I), and found that this reaction is slow: $\text{k = (1.5±0.5) · 10}{}^3\text{M}{}^{\mathrm{?1}}\text{) ·}\text{s}{}^{\mathrm{?1}}$.Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome $\text{C}$ by O^?₂.     

Evidence of the presence of a latent active site in the large subunit of rat kidney gamma-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. $\text{73}$, 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. $\text{74}$, 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-¹⁴C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the ¹⁴C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Activation of poly(adenosine diphosphate ribose) polymerase with UV irradiated and UV endonuclease treated SV 40 minichromosome

SV 40 minichromosomes were used as a molecular model of eukaryotic chromatin to probe the nature of the lesion responsible for UV stimulation of poly (ADPR) polymerase. UV irradiation of the minichromosomes with doses between 50 and 1000 J/m² did not increase their ability to stimulate the activity of purified poly(ADPR) polymerase. In contrast, when the minichromosomes were UV irradiated and then treated with $\text{M.}\text{luteus}$ UV endonuclease, there was a marked increase in their ability to stimulate poly(ADPR) polymerase. This stimulation was completely suppressed when histone Hl was added to the poly(ADPR) polymerase assay. These studies demonstrate in a purified $\text{in}\text{vitro}$ system that damage caused by UV irradiation alone is not sufficient to stimulate poly(ADPR) polymerase activity. Only when DNA is nicked at the site of UV damage by UV endonuclease is there stimulation of poly(ADPR) polymerase.     

Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Characterization of L5178Y leukemic cells which rapidly develop and lose implantation ability.

Two populations of L5178Y murine leukemic cells, maintained by different methods, were studied for their implantation ability in BDF₁ mice. Implantation ability was measured by number of tumor nodules formed, liver weight, and day of death of the animal. 1) Cells from a population grown for 10 years $\text{in vitro}$ had no implantation ability; i.e., no tumor nodules were formed when injected into the tail vein. After 30 days of growth in the peritoneal cavity of BDF₁ mice, these same cells were injected into the tail vein and 10 days later had produced over 200 liver tumor nodules. When cells taken from these tumors were recultured for 60 days $\text{in vitro}$, they lost the acquired implantation ability, but regained it after another single peritoneal passage. 2) L5178Y murine leukemic cells grown for six years in ascites tumor cells were extremely tumorigenic; over 200 tumor nodules appeared in the liver after tail vein injection. These cells were not rendered less tumorigenic and did not lose their implantation ability by $\text{in vitro}$ culturing for 60 days. The results suggest that implantation ability is a property of the cell's growth environment; furthermore, they have strong implications for the $\text{in vivo}$ and $\text{in vitro}$ manipulation of this property.