Reassessment of histone gene expression during cell cycle in human cells by using homologous H4 histone cDNA.

The representation of H4 histone mRNA sequences in RNAs isolated from G1 and S phase HeLa cells was assessed by use of a homologous H4 histone cDNA. S phase cells were obtained by double thymidine block, and G1 cells were obtained by double thymidine block or mitotic selective detachment. Nuclear and cytoplasmic RNAs from S phase cells hybridized with H4 histone cDNA as did nuclear and cytoplasmic RNAs from G1 cells synchronized by double thymidine block. In contrast, significant levels of hybridization were not observed between H4 histone cDNA and nuclear, polysomal, or postpolysomal cytoplasmic RNAs of G1 cells synchronized by mitotic selective detachment. Double thymidine block yields a G1 cell population containing 20-25% S phase cells whereas the G1 population obtained by mitotic detachment contains less than 0.1% S phase cells. The ability of H4 histone cDNA to hybridize with the RNAs from G1 cells obtained after release from double thymidine block can therefore be explained by the presence of S phase cells in such a G1 population--an artifact of the synchronization procedure. We interpret these results to be consistent with the presence of H4 histone mRNA sequences during the S but not G1 phase of the cell cycle in continuously dividing HeLa S3 cells.     

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.     

Quantitative and qualitative changes in histone gene expression during early mouse embryo development.

There are large amounts of histone mRNA present in mouse eggs. These RNAs are rapidly degraded, as are other mRNAs, after fertilization and prior to the second cleavage. During cleavage, the histone mRNA accumulates as the embryo divides. The same sets of histone genes are expressed in eggs and embryos, although there are large qualitative differences in the amounts of particular histone mRNAs. The function of the egg histone mRNA is unknown. The amount of histone mRNA in cleaving and blastocyst embryos is probably sufficient to code for the blastocyst histone proteins.     

Cell cycle-dependent regulation of thymidine kinase activity introduced into mouse LMTK- cells by DNA and chromatin-mediated gene transfer.

We report on the expression of thymidine kinase (tk) activity in synchronized populations of mouse cells that have been transformed to the tk+ phenotype with purified DNA from various sources or with metaphase chromosomes from human cells. The viral (herpes) tk gene is constitutively expressed but, in all other cases examined, the activity is regulated as in normal tk+ mouse cells: There is a dramatic increase at the beginning of the S-phase. This regulation is observed whether the transgenome is stably integrated into the host genome or whether it is still in an unstable nonintegrated state.     

The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein.

The retinoblastoma gene product (Rb) has been established as a tumor suppressor and cell cycle regulator, although its mechanism of action remains obscure. The observations that several Rb-binding viral oncoproteins all associate with the nuclear matrix suggest that these interactions may occur on this structure. To determine whether Rb itself is a component of the matrix, we extracted synchronized cultured cells to isolate matrix proteins while preserving nuclear architecture. Immunoblot and immunolabeling data show that a significant portion of hypophosphorylated Rb associates with the matrix only during early G1. Mutant Rb in tumor cells did not associate with the matrix, whereas Rb-reconstituted cells contained abundant matrix-bound Rb. Rb is distributed widely throughout the matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants. Core filaments of the matrix contained no detectable Rb. Our screening of expression libraries for potential Rb-associated proteins has identified several that are part of the matrix. Specifically, the peripheral matrix proteins lamin A and C bound Rb in vitro. We therefore suggest that Rb interactions with the nuclear matrix may be important for its ability to regulate cell cycle progression.     

Cell cycle-dependent induction of mutations along a yeast chromosome.

The relation between DNA replication and the action of the mutagen N-methyl-N'-nitro-N-nitroso-guanidine has been studied in Saccharomyces cerevisiae. The frequenceis of reversion to prototrophy of six auxotrophic markers located along one arm of chromosome VII were examined as a function of the vegetative cell cycle. Exponentially growing cells were treated with nitrosoguanidine and then separated by zonal rotor centrifugation into fractions equivalent to stages in the cell cycle. The frequency of reversion for five of the six markers is greatest during the period of DNA replication. Each marker has a single point of maximum reversion, approximately 10-fold greater than the frequency observed at other points in the cell cycle. For any one marker the effect of nitrosoguanidine is restricted to an interval shorter than the period of DNA replication. The two markers most distant from each other, ade5 and leul, both have their highest reversion frequency early during DNA replication. The peak reversion frequency for lys5 is somewhat later, while the peaks for tyr3 and trp5 occur near the end of DNA replication. The results indicate that nitrosoguanidine acts primarily during DNA replication and that different markers appear to be affected at different intervals during the DNA biosynthetic period. If nitrosoguanidine does act at the growing point of DNA replication, these observations indicate that the initiation of DNA replication occurs at specific times during the period of DNA synthesis and at specific initiation sites. Further, there must be more than one point of initiation of DNA replication on one arm of chromosome VII.     

Regulated expression of an extrachromosomal human beta-interferon gene in mouse cells.

Beta(fibroblast)-interferon mRNA and protein are induced by the synthetic double-stranded RNA poly(I) X poly(C) in cultured human fibroblasts. To study the mechanism of this induction, we have isolated a human beta-interferon gene and inserted it in a vector plasmid containing DNA of the bovine papilloma virus. After removal of bacterial plasmid sequences, the bovine papilloma virus-beta-interferon recombinant was used to morphologically transform mouse fibroblasts. Analysis of DNA from the transformed cell lines indicated that this recombinant is propagated as a stable multicopy extrachromosomal element. Human beta-interferon mRNA and protein are inducible by poly(I) X poly(C) in all of these cell lines, and the mRNA is indistinguishable from beta-interferon mRNA synthesized by induced human cells.     

Cell cycle-dependent uptake of putrescine and its importance in regulating cell cycle phase transition in cultured adult mouse hepatocytes

Previous studies in which investigators have induced the rate of polyamine uptake in vitro have used either inhibitors of polyamine biosynthesis or growth factors that induce cell proliferation. Recently, however, we have described the induction of putrescine uptake in cultured adult mouse hepatocytes and have shown that uptake is independent of both intracellular polyamine levels and proliferation. Although proliferation was not apparent in those studies, data suggested that, after isolation, the cells entered G1 of the cell cycle. In this study, we have examined whether the induction of putrescine uptake is a function of entry into the cell cycle and whether uptake activity is essential for optimal progression into the S phase. Using ribonuclease reductase subunit M1 as a marker of entry into the cell cycle, we have shown that hepatocytes enter G1 during the first 4 hr of culture. Both putrescine uptake and ornithine decarboxylase activity increased as the cells entered G1. Treatment of the cells with retinoic acid (10 to 33 mumol/L) prevented them from entering G1 and also inhibited the induction of the putrescine transporter by up to 90%. In contrast, initiation of G1 to S phase transition markedly down-regulated the activity of the transporter. Thus induction of the putrescine transporter in isolated hepatocytes appears to be a G1-specific event. Culturing the hepatocytes in the presence of 1,1'-bis[3-(1'-methyl-[4,4'-bipyridinium]-1-yl)-propyl]- 4,4'-bipyridinium, a potent competitive inhibitor of putrescine uptake, resulted in a 47% decrease in intracellular putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line.

We have introduced a functionally rearranged murine kappa light chain immunoglobulin (Ig) gene into an Abelson murine leukemia virus-transformed lymphoid cell line. Plasmid pSV2gpt-kappa 41, containing the kappa light chain gene from the myeloma MOPC41 and the selectable marker gene gpt, was introduced into 81A-2 cells by the calcium phosphate coprecipitation technique. Cells expressing the gpt gene were selected by growth in medium containing mycophenolic acid. One transfected cell line, kappa-2, was shown to make kappa mRNA and polypeptide chains and to assemble the kappa chain product with gamma 2b heavy chains to form an apparently complete IgG2b. When bacterial lipopolysaccharide was added to the growth medium, levels of kappa mRNA and polypeptide increased, showing regulated expression of the introduced kappa gene.     

Cell cycle regulation of human histone H1 mRNA.

A cloned genomic DNA fragment containing a human histone H1 gene has been used to analyze histone H1 gene expression in two human cell lines (HeLa S3 and WI-38). The cellular abundance of histone H1 mRNA was compared with that of core (H2A, H2B, H3, and H4) histone mRNAs as a function of the cell cycle: core and H1 histone mRNA levels are related both to each other and to the apparent rate of DNA synthesis and are rapidly destabilized after DNA synthesis inhibition. The use of three synchronization protocols, and of transformed and normal diploid cells in culture, suggests that the detected core and H1 histone mRNA levels are regulated by similar mechanisms in continuously dividing human cell lines and nondividing cells stimulated to proliferate.     

Cell cycle-dependent modulation of telomerase activity in tumor cells.

Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. We tested this hypothesis by arresting human tumor cell lines at different stages of the cell cycle. Induction of quiescence by serum deprivation did not affect telomerase activity. Cells arrested at the G1/S phase of the cell cycle showed similar levels of telomerase to asynchronous cultures; progression through the S phase was associated with increased telomerase activity. The highest level of telomerase activity was detected in S-phase cells. In contrast, cells arrested at G2/M phase of the cell cycle were almost devoid of telomerase activity. Diverse cell cycle blockers, including transforming growth factor beta1 and cytotoxic agents, also caused inhibition of telomerase activity. These results establish a direct link between telomerase activity and progression through the cell cycle.