The synthesis and isolation of DNA complementary to histone mRNA from mouse embryos

A 9S poly(A)-RNA preparation isolated from mouse embryos was shown to stimulate the synthesis of histones in an ascites cell free extract. This RNA preparation was used for the synthesis of a highly labelled cDNA probe complementary to histone mRNA. Hybridisation of this cDNA probe to rRNA showed that 52% of the cDNA consisted of sequences complementary to rRNA. The histone mRNA specific cDNA was purified by hybridising the impure cDNA to rRNA followed by removal of the single-stranded histone cDNA by hydroxylapatite chromatography.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.     

Organization, nucleotide sequence, and chromosomal mapping of a tandemly repeated unit containing the four core histone genes and a 5S rRNA gene in an isopod crustacean species.

A tandemly repeated unit of 6553 bp containing a copy of the four core histone genes H2B, H2A, H3, and H4, and also a 5S rRNA gene, was amplified by PCR from genomic DNA of the isopod crustacean Asellus aquaticus. The linkage between 5S rRNA genes and histone genes has been so far observed in only one other organism, the anostrac crustacean Artemia salina. The gene cluster was cloned and sequenced. The histone genes, in their 3' flanking region, have the interesting feature of possessing two different mRNA termination signals, the stem-loop structure and the AATAAA sequence. A part of the PCR product was used as a probe in FISH experiments to locate the gene cluster on an inter-individually variable number of chromosomes from 6 to 12 per diploid cell, always in a terminal position and never associated with the heterochromatic areas. Fluorescence in situ hybridization (FISH) was also performed on preparations of released chromatin and the reiteration level of the gene cluster was determined as approximately 200-300 copies per haploid genome.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.     

Coordinate control and selective expression of the full complement of replication-dependent histone H4 genes in normal and cancer cells

The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described. We have determined, by real-time quantitative PCR and RNase protection, that the human histone H4 genes are not equally expressed and that a subset contributes disproportionately to the total pool of H4 mRNA. Differences in histone H4 gene expression can be attributed to observed unequal activities of the H4 gene promoters, which exhibit variations in gene regulatory elements. The overall expression pattern of the histone H4 gene complement is similar in normal and cancer cells. However, H4 genes that are moderately expressed in normal cells are sporadically silenced in tumor cells with compensation of expression by other H4 gene copies. Chromatin immunoprecipitation analyses and in vitro DNA binding assays indicated that 11 of the 15 histone H4 genes interact with the cell cycle regulatory histone nuclear factor P, which forms a complex with the cyclin E/CDK2-responsive co-regulator p220(NPAT). These 11 H4 genes account for 95% of the histone H4 mRNA pool. We conclude that the cyclin E/CDK2/p220(NPAT)/histone nuclear factor P signaling pathway is the principal regulator of histone H4 biosynthesis.     

Non-Coordinated Synthesis of RNA's in Pre-Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre- and post-gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 10⁴ molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.     

Ribosomal RNA genes from Prevotella bryantii: organization and heterogeneity

FiveP. bryantii B₁4 16S rRNA gene copies and their flanking regions were cloned and analyzed. A genomic library was constructed and screened with oligonucleotide DNA probe specific for 16S rRNA gene ofP. bryantii. Five out of six different copies of 16S RNA gene were recovered and sequenced. Only minor differences (0.3–1.2%) between copies were detected within the 1541 bp long sequence. The impact of the sequence variability of 16S rRNA gene copies on phylogenetic positioning ofP. bryantii was determined. All five sequences from clonedP. bryantii B₁4 16S rRNA genes were placed in the same operational taxonomy unit. Control regions of all five analyzed rRNA operatons were almost identical and three candidate for promoter sequences were identified by Neutral Network Promoter Prediction. Spacer regions between 16S-rRNA and 23S rRNA genes in all five cloned copies were 543 bp long and genes for tRNA^lle and tRNA^Ala were identified inside this regions.     

DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips

Histone H2A mRNA is selectively expressed in scattered subpopulations of cells in the pea (Pisum sativum) root apical meristem. To study whether this specific expression was associated with the cell cycle, a double-labeling technique was used to identify cells replicating DNA during S phase and those expressing H2A mRNA. Cells in S phase were detected by [3H]thymidine incorporation and autoradiography, whereas cells containing H2A mRNA were identified by in situ hybridization using digoxigenin-labeled probes. Approximately 92% of the [3H]thymidine-labeled S-phase cells expressed H2A mRNA and 85% of cells that expressed H2A mRNA were in S phase. In root tissue located basal to the promeristem, synchronous co-located expression was observed in scattered packets of proliferating cells. Furthermore, neither H2A mRNA nor S-phase cells could be detected within the quiescent center or mature root cap. When DNA synthesis was inhibited with hydroxyurea, a commensurate and specific decrease in steady-state levels of H2A mRNA was found. We conclude that cell-specific expression of pea histone H2A mRNA is replication dependent and that H2A mRNA is transiently accumulated during a period of the cell cycle that mostly overlaps the S phase. We propose that the overlap between H2A expression and S phase could occur if H2A mRNA accumulation began in late G1 and abated in late S.     

The normalization of gene expression data in melanoma: investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR

We examined a panel of 26 melanoma and fibroblast samples (tissues and cultured cells) to evaluate the suitability of two commonly used housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA (rRNA), for quantitative real-time PCR. Both genes showed significant variations within the individual cell line and tissue groups. Although no overall trends were observed in the expression of the 18S rRNA, GAPDH was up-regulated in melanoma tissue and cultured cells compared with the corresponding normal samples. In melanoma and fibroblast cell lines and tissues, absolute quantification appears to be more appropriate than normalizing messenger RNA (mRNA) expression via GAPDH or 18S rRNA housekeeping genes.     

The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines.

We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma SV-Py 3T3), hamster lung fibrobalsts (v79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species. The ratio of mRNA to rRNA varied form 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in seescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.     

RD 114 Virus-Specific Sequences in Feline Cellular RNA: Detection and Characterization

RNA extracted from cat cells contains sequences homologous to RD-114 viral RNA. The sequences are measured by molecular hybridization with a single-stranded DNA probe synthesized by the virion polymerase using the endogenous viral RNA as template. Viral-specific RNA has been detected in all cells of cat origin tested thus far, but not in cells of other animals, except for the virus-producing human rhabdomyosarcoma cell, RD-114. The extent of hybridization of the DNA probe to cellular RNA was equivalent to that obtained with viral 70S RNA indicating that an equal extent of viral specific sequences is present in all cat cells as well as in RD-114 cells. The amounts of this viral RNA reach approximately 100 copies per cell in cat cells, while virus-producing RD-114 cells contain about 1,000 copies per cell. The viral RNA is present in cat cells in two distinct sizes of about 35S and 18S, whereas in RD-114 cells virus-specific RNA is quite heterogeneous in size.     

A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures.

A technique is described to identify the rare sequences within an RNA molecule that are available for efficient interaction with complementary DNA probes: the target RNA is digested by RNase H in the presence of a random pool of complementary DNA fragments generated from the same DNA preparation that was used for target RNA synthesis. The DNA region was amplified by PCR, partially digested with DNase and denatured prior to RNA binding. In the presence of single-stranded DNA fragments the RNA was digested with RNase H such that, on average, each molecule was cut once. Cleavage sites were detected by gel electrophoresis either directly with end-labeled RNA or by primer extension. The pattern of accessible sites on c- raf mRNA was determined and compared with the known profile of activity of oligonucleotides found in cells, showing the merit of the method for predicting oligonucleotides which are efficient for in vivo antisense targeting. New susceptible sites in the 3'-untranslated region of c- raf mRNA were identified. Also, four RNAs were probed to ascertain to what extent structure predicts accessibility: the P4-P6 domain of the Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a part of rat 18S rRNA.     

Expression of histone genes in a G1-specific temperature-sensitive mutant of the cell cycle

The expression of genes coding for the four core histones (H2A, H2B, H3, and H4) was studied in tsAF8 cells. These baby hamster kidney-derived cells are a temperature-sensitive (ts) mutant of the cell cycle that arrest in G1 at the restrictive temperature. When serum-deprived tsAF8 cells are stimulated with serum, they enter the S phase at the permissive temperature of 34 degrees C, but are blocked in G1 at the nonpermissive temperature of 39.6 degrees C. Northern blot analysis using cloned human histone DNA probes detected only very low levels of histone RNA either in quiescent tsAF8 cells or in cells serum stimulated at the nonpermissive temperature for 24 h. Cellular levels of histone RNA were markedly increased in cells serum stimulated at 34 degrees C for 24 h. Temperature shift-up experiments after serum stimulation of quiescent populations showed that the amount of histone RNA was related to the number of cells that entered the S phase. Those cells that synthesized histone RNA and entered the S phase were capable of dividing. This is the first demonstration in a mammalian G1-specific ts mutant that the expression of H2A, H2B, H3, and H4 histone genes depends on the entry of cells into the S phase of the cell cycle.     

Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells.

The production of cytoplasmic and nucleolar rRNA species was examined in HeLa cells infected with high multiplicities of adenovirus type 5. Both 28S and 18S rRNA newly synthesized in infected cells ceased to enter the cytoplasm as reported previously (N. Ledinko, Virology 49: 79-89, 1972; H. J. Raskas, D. C. Thomas, and M. Green, Virology 40: 893-902, 1970). However, the effects on 28S cytoplasmic rRNA were observed considerably earlier in the infectious cycle than those on 18S rRNA. The inhibition of cellular protein synthesis and of the appearance in the cytoplasm of labeled cellular mRNA sequences (G. A. Beltz and S. J. Flint, J. Mol. Biol. 131: 353-373, 1979) were also monitored in infected cultures. During the later periods of an infectious cycle, from 18 h after infection, nucleolar rRNA synthesis and processing and exit of 18S rRNA from the nucleus were inhibited, probably reflecting the failure of infected cells to synthesize normal quantities of ribosomal proteins. The earliest responses of cellular RNA metabolism to adenovirus infection were, however, the rapid and apparently coordinate reductions in the levels of newly synthesized 28S rRNA and cellular mRNA sequences entering the cytoplasm.