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The arenavirus nucleoprotein (NP) can suppress induction of type I interferon (IFN). This anti-IFN activity is thought to be shared by all arenaviruses with the exception of Tacaribe virus (TCRV). To identify the TCRV NP amino acid residues that prevent its IFN-countering ability, we created a series of NP chimeras between residues of TCRV NP and Pichinde virus (PICV) NP, an arenavirus NP with potent anti-IFN function. Chimera NP analysis revealed that a minimal four amino acid stretch derived from PICV NP could impart efficient anti-IFN activity to TCRV NP. Strikingly, the TCRV NP gene cloned and sequenced from viral stocks obtained through National Institutes of Health Biodefense and Emerging Infections (BEI) resources deviated from the reference sequence at this particular four-amino acid region, GPPT (GenBank KC329849) versus DLQL (GenBank NC004293), respectively at residues 389-392. When efficiently expressed in cells through codon-optimization, TCRV NP containing the GPPT residues rescued the antagonistic IFN function. Consistent with cell expression results, TCRV infection did not stimulate an IFNβ response early in infection in multiple cells types (e.g. A549, P388D1), and IRF-3 was not translocated to the nucleus in TCRV-infected A549 cells. Collectively, these data suggest that certain TCRV strain variants contain the important NP amino acids necessary for anti-IFN activity.

Transcription of ptsG encoding glucose-specific permease, enzyme IICB(Glc), in Escherichia coli is initiated from two promoters, P1 and P2. ptsG transcription is repressed by Mlc, a glucose-inducible regulator of carbohydrate metabolism. The regulation of ptsG P1 transcription is also under positive control by cyclic AMP receptor protein and cyclic AMP complex (CRP.cAMP) as observed in other Mlc regulon. We report here that Fis, one of the nucleoid-associated proteins, plays a key role in glucose induction of Mlc regulon. ptsG transcription was induced when wild-type cells were grown in the presence of glucose. However, in a fis mutant, the basal level of ptsG transcription was higher but decreased when cells were grown in the presence of glucose, which implies the possibility of regulatory interactions among Fis, Mlc, and CRP.cAMP. Footprinting experiments with various probes and transcription assays revealed that Fis assists both Mlc repression and CRP.cAMP activation of ptsG P1 through the formation of Fis.CRP.Mlc or Fis.CRP nucleoprotein complexes at ptsG P1 promoter depending on the availability of glucose in the growth medium. ptsG P2 transcription was inhibited by Fis and Mlc. Tighter Mlc repression and enhanced CRP.cAMP activation of ptsG P1 by Fis enable cells to regulate Mlc regulon efficiently by selectively controlling the concentration of enzyme IICB(Glc) that modulates Mlc activity.

The possibility that hormone-induced changes in the synthesis of individual proteins may serve as initiating events for the rapidly evolving glucocorticoid-induced metabolic suppressions in rat thymus cells was examined. Synthetic rates of about 2500 individual proteins were screened using giant two-dimensional gel electrophoresis. Dexamethasone induces large, rapid increases in the rates of synthesis of at least three and more subtle increases in another three proteins. Increases in the rates of synthesis of four of these six proteins occur within 15 to 45 min. The time course suggests that changes in the synthesis of some or all of these four proteins may serve to generate the rapidly evolving hormone-induced inhibition of glucose transport. More slowly emerging changes in two other proteins parallel the latter metabolic effects of decreased mitochondrial ATP production and increased nuclear fragility. Cordycepin, an inhibitor of new mRNA processing, prevents the dexamethasone-induced increases in protein labeling. Also, when used alone, cordycepin selectively slows the synthesis of those early proteins that can be induced by dexamethasone, suggesting that the mRNAs coding for the early induced proteins have unusually short half-lives. Short-lived mRNAs for such putative regulatory proteins may allow the cell to respond rapidly to changing needs. Preliminary experiments using giant gel separations of subcellular fractions indicate that two of the induced proteins co-purify with crude plasma and nuclear membrane fractions. None of the induced proteins was detected in gel separations of mitochondria.

When chromatin is extracted from nuclease-digested nuclei by stepwise salt elution, two different classes of newly replicated chromatin can be distinguished. Nascent DNA eluted from nuclei under conditions of low to moderate ionic strength (0.1-0.3 M NaCl) exhibits nucleosomal periodicity and is found in particles which have the same electrophoretic mobility as bona fide H1- or high mobility group protein-containing mononucleosomes. Thus, factors believed to be involved with both the higher order coiling and transcriptionally active state of chromatin are rapidly complexed with newly synthesized DNA and may be retained on parental nucleosomes throughout replication. In contrast, approximately 40% of new DNA is resistant to extraction with solutions of moderate ionic strength. Most of this material is eluted from nuclei by 0.4-0.6 M NaCl. While bulk chromatin that is extracted by 0.4-0.6 M NaCl is organized into nucleosomes, most of the newly replicated "chromatin" from the same fractions lacks subunit structure, as determined by DNA size analyses in polyacrylamide gels, thereby distinguishing this nascent material from newly replicated chromatin eluted at lower ionic strength. Within 15 min all newly synthesized chromatin matures and exhibits the solubility and nucleosomal periodicity characteristics of bulk chromatin. The unusual properties of the "nonnucleosomal" fractions may reflect the structure of newly synthesized DNA prior to its assembly into nucleosomes.

Treatment of rat superior cervical ganglia in culture with nerve growth factor (NGF) increases the amount of radioactive phosphate incorporated into a nuclear protein band. This band migrates coincidentally with H1 histone on 10% sodium dodecyl sulfate/polyacrylamide gels. The increase in phosphate incorporation is at least 70% and occurs only in tissues known to be responsive to NGF. It is not produced by treatment with related peptides, but is observed after the addition of dibutyryl cyclic AMP. An increase in phosphorylation can be detected after 1 h, and can be seen with as little as 10 ng/ml of NGF in the medium. Neither actinomycin D nor cycloheximide inhibits the effect. When the nuclei are extracted with 0.2 M H2SO4 and the extract analyzed on acid-urea/polyacrylamide gels, two NGF-responsive proteins can be detected. One protein again migrates with the H1 histone marker; the other migrates more slowly than H1. These two NGF-responsive proteins have molecular weights of approximately 30,000 and are chromatin-bound. They are not soluble in 5% perchloric acid, but can be extracted from the nuclei with 0.35 M NaCl. No increase in the phosphorylation of these proteins was seen in ganglia from 6-hydroxydopamine-treated rats. The phosphorylation of the proteins in both control and NGF-treated ganglia occurs almost exclusively on serine residues. The amino acid compositions of the two nuclear proteins show that they are different from the H1 histone and different from each other. Both nerve growth factor (NGF) and epidermal growth factor (EGF) increase the incorporation of radioactive phosphate into a specific nuclear protein in cultures of PC12, a clone of rat pheochromocytoma. Purified NGF antibody blocks the effect of NGF, but not that of EGF; EGF antiserum neutralizes the effect of EGF, but not that of NGF. Insulin, glucagon, and dexamethasone are without effect. The increase in phosphorylation due to NGF can be detected within 1 h. Dibutyryl cyclic AMP increases the phosphorylation of this protein, but dibutyryl cyclic GMP does not. Neither the uptake nor the overall incorporation of [32P]orthophosphate is altered by NGF, EGF, or dibutyryl cAMP under the present experimental conditions. The nuclear protein exhibiting increased radioactivity is similar in solubility, size, and amino acid composition to one of the NGF-responsive nuclear proteins from sympathetic ganglia.

Chromatin and nuclear ribonucleoprotein (nRNP) have been prepared from a human carcinoma cell line. Following a 1-hour (3H)uridine pulse, 60 to 70% of the nuclear radioactivity, after removal of nucleoli, was found in the chromatin, the balance in nRNP. This was true whether the chromatin and nRNP were separated by velocity centrifugation or by isopycnic centrifugation on Metrizamide gradients. Radioactivity in chromatin and nRNP was found in high molecular weight RNA, with mean sedimentation coefficients of 20 S and 15 S, respectively, as determined on sodium dodecyl sulfate-sucrose gradients. Experiments on the kinetics of appearance of radioactivity in the RNA of the two fractions suggest that some of the chromatin-associated RNA is precursor to nRNP-RNA. The proteins of nRNP are complex as revealed by sodium dodecyl sulfate gel electrophoresis. The contamination by chromatin protein was estimated to be 5%. Experiments involving short pulses of (3H)tryptophan, and pulse-chase, suggested that the rapidly turning over proteins of nRNP were not complexed with RNA while still associated with chromatin. However, it was also shown that the radioactivity in nRNP following short pulses of (3H)tryptophan did not correspond to the major bands seen on stained sodium dodecyl sulfate gels. It is therefore concluded that the protein of nRNP consists of two classes: species present in large amounts, possibly common to all RNA in nRNP, which are relatively stable and may be complexed to RNA still associated with chromatin; and a large number of rapidly turning over species, each present in small amounts and associated with nRNP only after its release from chromatin.

Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.

Recently, using molecular hybridization techniques with albumin [3H]cDNA, we have determined that in normally fed rats 98% of total liver polyribosomal albumin mRNA sequences are found in membrane-bound polyribosomes (Yap, S. H., Strair, R. K., and Shafritz, D. A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5397-5401). We now observe that a 24- to 30-h withdrawal of food leads to major changes in the amount and subcellular distribution of albumin mRNA molecules. The total amount of cytoplasmic albumin mRNA per liver and concentration of albumin mRNA per unit of membrane-bound polyribosomal RNA are decreased. However, the proportion of albumin mRNA present in the postribosomal supernatant fraction increases dramatically in a short term fast, so that it now represent 60% of total cytoplasmic albumin mRNA sequences. Most of the albumin mRNA sequences in the postribosomal supernatant fraction sediment between 30 S and 50 S. These findings suggest that albumin mRNA is probably stored in the messenger ribonucleoprotein fraction during the fasting state.

The synthesis and phosphorylation of influenza virus nucleoprotein and nonstructural protein were analyzed. The nucleoprotein (NP) was found to be phosphorylated in both infected cells and in isolated virions. The phosphate is in a monoester linkage to a serine residue. Two-dimensional tryptic peptide maps of the 32P-labeled protein, as well as measurements of specific activity, suggests that NP is phosphorylated at one site per molecule. The viral nonstructural (NS 1) protein is also phosphorylated, but on threonine residues. Up to a maximum of two sites per NS 1 molecule could be so modified in infected cells, as demonstrated by two different methods of tryptic peptide analysis and by measurements of the ratio of 32P to 3H-amino-acids incorporated into NS 1 protein species. The NS 1 protein is resolved into four major species of differing isoelectric point in a two-dimensional electrophoretogram. The most acidic species was found to have two phosphorylated sites per molecule, and the next most acidic species contained on the average one phosphate per molecule. Treatment of the phosphorylated species with bacterial alkaline phosphatase demonstrated that the level of phosphorylation is the only identifiable difference between the phosphorylated and unphosphorylated NS 1 species. The distinction between the two unphosphorylated species could not be determined. The distribution of the un-, mono-, and diphosphorylated NS 1 species was characterized at different times after synthesis. These modifications were found to occur very rapidly after translation (30 to 60 s), after transport of the unmodified species from cytoplasm to nucleus of the infected cell. The phosphorylation of NP also takes place rapidly after its synthesis; the site within the cell of the NP phosphorylation has not been unambiguously determined.