The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3HJuridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.The biosynthesis of histones is generally thought to occur periodically in the cell cycle and to be tightly coupled to DNA replication. Early studies utilizing synchronized HeLa cells showed that newly synthesized histones could be found associated with chromatin only in S-phase cells (2,14,27). These cells contained an abundance of small polyribosomes with nascent polypeptides that comigrated with histones in gel electrophoresis; these small polysomes were not apparent in G1 cells (2). When cells undergoing DNA replication are treated with inhibitors of DNA synthesis there is a rapid loss of histone production (2, 4). Gallwitz and Mueller (8) showed that histone proteins were not synthesized in vitro on microsomes isolated from such cells, and Borun et al. (1) confirmed these results by a direct mRNA translation assay.Despite extensive investigations into the mechanisms responsible for the temporal pattern of histone synthesis in the cell cycle, there is still considerable disagreement concerning the level(s) at which histone protein synthesis is regulated. A number of studies support the view ...

Insulin activates a tyrosine-specific cAMP-independent protein kinase when added directly to detergent extracts of differentiated 3T3-L1 adipocytes and humal placental membranes. The kdnase is also activated by antibody to the insulin receptor and, to a lesser extent, by proinsulin. It catalyzes the phosphorylation of the 92,000-dalton component of the insulin receptor, histone, and casein; in each case, tyrosine is the principal amino acid modified. Under the conditions used to activate the kinase, insulin does not affect the rate of dephosphorylation ofthe receptor or of histone. The insulin-activated kinase is copurified with the human placental insulin receptor until the final elution from insulin-Sepharose. It remains to be established whether the ldnase and the insulin receptor are separate molecules.The initial step in insulin action is the interaction of insulin with its high-affinity cell surface receptor. The transduction of the signal from the insulin-receptor complex ultimately involves phosphorylation and dephosphorylation ofspecific cellular proteins. In 3T3-L1 and other cells, insulin stimulates the phosphorylation of both ribosomal protein S6 (1-3) and the enzyme, ATP citrate (pro-3S)-lyase (4-9). In each case, phosphorylation occurs on serine residues and is cAMP independent. Insulin also stimulates the cAMP-dependent phosphorylation ofa cyclic nucleotide phosphodiesterase resident in rat liver plasma membranes (10). Recently, Kasuga et al. (11) reported that the addition of insulin to intact lymphocytes and hepatoma cells brought about the incorporation of 32P into the 94,000-dalton subunit of the insulin receptor. Kasuga et aL. also have shown that a detergent-solubilized extract of rat liver plasma membranes phosphorylates tyrosine residues on the insulin receptor (12).We present evidence for the insulin-activated phosphorylation of the insulin receptor in extracts of insulin-sensitive 3T3-Li adipocytes and in partially purified preparations of the human placental insulin receptor. The kinase from both sources phosphorylates tyrosine residues in both the receptor and, in exogenous substrates, is activated specifically by molecules that interact with the insulin receptor and is cAMP independent. MATERIALS AND METHODS[y-32P]ATP (3,000 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) was from Amersham. Purified cAMP-dependent protein kinase inhibitor was a gift from J. Demaille; human antibody to the human insulin receptor (B-9) was provided by C. R. Kahn, and rabbit antibody to rat liver membrane insulin receptor was from S. Jacobs. Insulin and proinsulin were from Eli Lilly, and epidermal growth factor (EGF) was from Collaborative Research (Waltham, MA). Histone H2B was from Worthington, and casein a-S was provided by E. Bingham. Agarose-bound wheat germ agglutinin and formalin-fixed protein A-bearing Staphylococcus aureus (Pansorbin) were from Vector and Calbiochem, respectively. Bovine brain calmodulin (13) and rat liver ATP citrate lyase (9) were prepared as described, and ribosomal 40S sub...

We have earlier reported that anti-TAR PNA conjugated with the membrane-transducing peptide transportan inhibits transactivation of the HIV-1 LTR resulting in decreased production of HIV-1 virions by chronically infected H9 cells (N., Kaushik, A., Basu, P., Palumbo, R.L., Myers, V.N., Pandey, 2002. Anti-TAR polyamide nucleotide analog conjugated with a membrane permeating peptide inhibits HIV-1 production. J. Virol. 76, 3881-3891). In this study, we have found that the PNA(TAR)-transportan conjugate is efficiently internalized by cells and kinetics analysis reveals a sigmoidal curve with a cooperativity index of 6, indicating very rapid cellular uptake. Additionally, analysis of uptake at varying temperatures or in the presence of phenylarsine oxide revealed that the mechanism of uptake is neither receptor-dependent nor occurs via endocytosis. We also found that the PNA(TAR)-transportan conjugate exhibits potent virucidal activity as HIV-1 virions pretreated with the conjugate were rendered noninfectious, suggesting that the conjugate may also permeate the virus envelope. The anti-HIV-1 virucidal activity of the conjugate may be useful either in topical formulations designed to block HIV-1 infection or as a prophylactic agent for inactivation of HIV-1 in the circulating plasma prior to attachment and entry.

We have studied 31 male germ cell tumors (GCTs) for probable mutations in codons 12, 13, and 61 of HRAS, KRAS, and NRAS oncogenes using the polymerase chain reaction. Twenty of the thirty-one tumors exhibited NRAS gene mutations, 14 in codon 61, and six in codon 12, whereas no mutations were detected in HRAS and KRAS genes. The NRAS mutations were equally prevalent in seminomatous and nonseminomatous GCTs. Thus 13 of 22 seminomas, six of seven embryonal carcinomas, and one of two mixed tumors exhibited mutations. Two non-seminomatous tumors (an embryonal carcinoma and a yolk sac/teratoma) had mutations in both codons 12 and 61. The high frequency of NRAS mutations observed in the present study suggests that NRAS gene products may play an important role in growth regulatory functions of premalignant and malignant germ cells.