Histone Messenger Research Articles

Size and sequence homology of masked maternal and embryonic histone messenger RNAs

The messenger RNAs for five classes of histone proteins are shown by competitive RNA-DNA hybridization to be stored in the unfertilized egg of the sea urchin, Lytechinus pictus. The masked mRNAs for f2b, f2a2, f3 and f2al histones migrate in polyacrylamide slab gels with the same mobility as the histone mRNAs that are synthesized after fertilization and are found engaged in protein synthesis on polysomes. The masked maternal and embryonic mRNAs for histone f2a1 are identical in mobility when analyzed in a gel system capable of resolving differences estimated as small as 4–5 nucleotides in length. We conclude that these histone mRNAs synthesized during oogenesis and inactive prior to fertilization are not activated during embryogeny by alteration in their molecular size.

Interspecific "common" repetitive DNA sequences in salamanders of the genus Plethodon.

Intermediate repetitive sequences of Plethodon cinereus which comprised about 30% of the genomic DNA were isolated and iodinated with 125I. About 5% of the 125I-repetitive fraction hybridized with a large excess of DNA from P. dunni at Cot 20. About half of the 125I-DNA in the hybrids was resistant to extensive digestion with S-1 nuclease. The average molecular size of the S-1 nuclease-resistant fraction was about 100 nucleotide pairs. The melting temperature of the S-1 nuclease-resistant fraction was about 2 degrees lower than that of the corresponding fraction made with P. cinereus DNA. These results are taken to indicate the presence in the genomes of P. cinereus and P. dunni of evolutionarily stable "common" repetitive sequences. The average frequency of repetition of the common repetitive sequences is about 6,000 X in both species. The common repetitive fraction is also present in the genomes of other species of Plethodon, although the general populations of intermediate repetitive sequences are markedly different from one species to another. The cinereus--dunni common repetitive sequences could not be detected in plethodontids belonging to different tribes, nor in more distantly related amphibians. The profiles of binding of the common repetitive sequences to CsCl or CS2SO4-Ag+ density gradient fractions of P. dunni DNA suggested that these sequences consisted of heterogeneous components with respect to base compositions, and that they did not include large amounts of the genes for ribosomal RNA, 5S RNA, 4S RNA, or histone messenger RNA. In situ hybridization of the 3H-labelled intermediate repetitive sequences of P. cinereus to male meiotic chromosomes of the same species gave autoradiographs after an exposure of seven days showing all 14 chromosomes labelled. The pattern of labelling appeared not to be random, but was impossible to analyse on account of the irregular shapes and different degrees of stretching of diplotene and prometaphase chromosomes. In situ hybridization of the same sequences to meiotic chromosomes from P. dunni gave autoradiographs after 60 d exposure in which all chromosomes were labelled. These heterologous in situ hybrids can only have involved the "common" repetitive sequences.

In vitro synthesis of yeast ribosomal proteins

Most yeast messenger RNAs contain a polyA fragment of about 50 nucleotides [l-4]. However, since a substantial amount of the pulse-labelled RNA from yeast polysomes lacks polyA [2,4] the presence of a polyA tract is not a property common to all yeast mRNAs. It is not known whether lack of polyA is limited to mRNA species coding for specific classes of protein or whether it is a reflection of the physiological state (e.g. age) of mRNA species in general. The only eukaryotic messenger RNA known to be devoid of polyA is histone messenger [5] . If indeed only specific mRNA species lack polyA, then messenger RNA for ribosomal protein, because of the physiological analogy between histones and ribosomal protein, would be a likely constituent of the mRNA fraction lacking polyA. Just as histones, ribosomal proteins are synthesized in the cytoplasm [6-81 and then transported into the nucleus [9]. The complex set of nucleocytoplasmic interactions required in each case might be influenced by the absence of polyA from the mRNA involved. If ribosomal protein messengers could be shown to occur in the fraction lacking polyA, isolation of these messengers and study of the genetic organization of the genes coding for ribosomal protein would be enormously facilitated. We separated polyA-containing mRNA from mRNA lacking polyA by chromatography on oligodTcellulose columns and translated both fractions in vitro using a wheat germ system. The products were analyzed using the method recently developed by us for identification of yeast ribosomal protein [lo]. The results presented in this paper demonstrate

Regulation of cell cycle stage-specific transcription of histone genes from chromatin by non-histone chromosomal proteins.

RNA transcripts from chromatin of S phase but not G1 cells contain histone-specific sequences. Chromatin reconstituted with S phase non-histone chromosomal proteins transcribes histone messenger RNA sequences whereas chromatin reconstituted with G1 non-histone proteins does not. These results suggest that transcription of histone genes is regulated during the cell cycle and that non-histone proteins have a key role in this regulation.

Hybridization analysis of histone messenger RNA: association with polyribosomes during the cell cycle.

Hybridization of cell cycle stage-specific polyribosomal RNA's to histone complementary DNA indicates that histone messenger RNA sequences are present on polyribosomes of Hela S3 cells only during the period of DNA replication.

Cell cycle stage-specific transcription of histone genes.

DNA complementary to histone messenger RNAs was utilized to assay the in vitro transcripts from chromatin of G1 and S phase HeLa S3 cells for the presence of histone specific sequences. The complementary DNA was prepared by transcribing, with an RNA-dependent DNA polymerase, histone messenger RNAs isolated from the polyribosomes of S phase HeLa S3 cells to which adenylic acid residues had been enzymatically added. While the RNA transcripts of S phase chromatin contained histone specific sequences, such sequences were not detected in the RNA transcripts from G1 chromatin. These results suggest that transcription of the genes for histone polypeptides is restricted to the S phase of the cell cycle.

Individual histone messenger RNAs: Identification by template activity

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that coelectrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein synthesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.

Further evidence of transcriptional and translational control of histone messenger RNA during the HeLa S3 cycle

Using a translational assay and new analytical procedures we have found that: • —Histone mRNA can be detected both associated with polyribosomes and in the postribosomal supernatant of S phase HeLa S3 cells. • —Inhibition of DNA replication by cytosine arabinoside treatment causes histone mRNA to completely disappear from polyribosomes, and little histone mRNA can be detected in the postribosomal supernatant of inhibited cells. These data indicate that histone mRNA does not accumulate in the cytoplasm after the inhibition of DNA replication. • —Histone mRNA species cannot be detected in the postribosomal supernatant of G1 cells synchronized by selective detachment. This observation, together with the previous finding that histone mRNA is not present on G1 polyribosomes, is consistent with the idea of a transcriptional block in histone mRNA production and transport to the cytoplasm during G1.

Invitro synthesis of DNA complementary to polyadenylated histone messenger RNA

Summary The messenger RNA's for histone polypeptides were isolated from S phase HeLa S3 cells and adenylic acid residues were enzymatically added to the 3′-OH end of the molecules with a eukaryotic ATP: polynucleotidylexotransferase. The polyadenylated histone messenger RNA's were then used as templates for the synthesis of complementary DNA's by RNA-dependent DNA polymerase from Rous sarcoma virus.

Reiteration frequency of histone coding sequences in man

A “10S”-RNA fraction with the characteristics of histone messenger was isolated from pulse labeled synchronized HeLa cells. Two properties indicate that this fraction consists of histone messenger sequences. First, its appearance in the cell cytoplasm is dependent upon continued DNA replication; and second, it is capable of cross hybridization with isolated histone DNA of sea urchin. The rate of hybridization of this RNA with an excess of human DNA suggests that there are 10–20 copies of each histone coding sequence in the human haploid genome.

Translation of histone messenger RNA by homologous cell-free systems from synchronized HeLa cells.

Cell-free systems have been prepared from HeLa cells synchronized in S phase and from the same cells treated with cytosine arabinoside to inhibit DNA synthesis. Translation of HeLa cell histone mRNA by these cell-free systems is equally efficient. The cell-free systems have been characterized for K+ and Mg2+ optima which are similar to those of the mouse ascites cell-free system. One major difference between these two cell-free systems is the reduced efficiency of initiation of the HeLa system. This can be overcome by the addition of rabbit reticulocyte initiation factors, which stimulate 4–5-fold translation of histone mRNA. The possibility that a translational inhibitor of histone synthesis is lost during the preincubation of the HeLa extracts has been tested by using cell-free systems prepared with non-preincubated extracts. In addition, it has been established that nuclear HeLa cell histones inhibit protein synthesis in a non-specific way; when added to an ascites or HeLa cell-free system they do not inhibit specifically the translation of histone mRNA and thus cannot act as specific translational repressors.

Effects of cordycepin, hydroxyurea and cycloheximide on histone mRNA synthesis in synchronized HeLa cells.

The effects of cordycepin, hydroxyurea and cycloheximide on the synthesis of histone mRNA in synchronized HeLa cells were studied by quantitating the RNA, using translation in a rabbit reticulocyte cell-free system. It was found that biologically active histone mRNA is synthesized in the presence of cordycepin. This result strengthens the findings by others that histone mRNA does not contain poly(A). Hydroxyurea and cycloheximide, when used at concentrations that inhibit cellular DNA synthesis, had different effects on histone messenger activity. While polyribosomal messenger activity rapidly declined after addition of hydroxyurea it was not impaired by cycloheximide. These results might help to elucidate regulatory mechanisms involved in the coupled synthesis of DNA and histones in HeLa cells.

Messenger RNA turnover in mouse L cells

The turnover of polyadenylic acid-containing messenger RNA and histone messenger RNA, which lacks poly(A), was studied in exponentially growing mouse L cells by measuring the kinetics of approach to steady-state uridine labeling. Constant specific activity of precursor pools was verified by showing that the data for stable RNA components, like ribosomal RNA and transfer RNA, follow theoretically predictable curves. In agreement with a previous report by Greenberg (1972), the data for poly(A)-containing mRNA (poly(A)(+)mRNA) follow theoretical curves for a class of molecules turning over with first-order (stochastic) kinetics. Cells growing with doubling times of 13·5 hours at 37 °C and 41 hours at 30 °C exhibited mean lifetimes for their poly(A)(+)mRNA of 15 hours and 42 hours, respectively, suggesting a parallelism between growth and turnover rates. The kinetic data for histone mRNA are not indicative of a stochastic process. Rather, they suggest an age-dependent decay or a zero-order (ordered) turnover with a mean lifetime of about six hours. One model, which gave a good fit to the data, considers that the histone messages persist for a fixed duration of the cell cycle, e.g. the DNA synthetic phase, and are then destroyed in a “sensitive period” after this phase. These results are discussed with regard to the possible implications of the poly(A) sequences in messenger RNA aging.

Studies on the translational control of histone synthesis: I. Translation of histone messenger RNA by heterologous cell-free systems prepared from cells inactive in DNA synthesis

The translation of histone mRNA has been studied in a cell-free system prepared from mouse ascites cells treated with cytosine arabinoside to inhibit DNA synthesis. No difference in the efficiency of translation was observed with respect to an extract of control untreated cells. Moreover, a rabbit reticulocyte cell-free system can translate histone mRNA; thus, cells inactive in DNA synthesis can translate this mRNA. A search for an inhibitor of translation of histone mRNA was carried out by adding postribosomal supernatant from S-phase-synchronized HeLa cells treated with cytosine arabinoside, to the ascites cell-free system. No effect on the translation of histone mRNA was detected. Even when the cell-free system was reconstituted with ascites ribosomes and postribosomal supernatant from S-phase HeLa cells blocked in DNA synthesis, no effect on translation of histone mRNA was observed. These results suggest that the regulatory mechanism which couples histone to DNA synthesis does not involve synthesis of a factor specifically necessary for the translation of histone mRNA, nor the formation of a stable inhibitor. We cannot exclude, however, that an inhibitor of translation may be formed in amounts corresponding exactly to those of the histone mRNA present at the time of the block in DNA synthesis and that for this reason we have failed to find it in the postribosomal supernatant.

Identification of presumptive histone messenger RNA in rapidly labelled polysomal RNA of mouse myelomas

Polysomal RNA was isolated from subcutaneous myeloma tumors in BALB/c mice after injection of (5- 3H)uridine directly into the tumors. Analysis of the polysomal RNA by polyacrylamide gel electrophoresis revealed one major peak of radioactive RNA in the region between 5-S and 18-S RNA with properties consistent with its identification as a messenger RNA. Three pieces of evidence indicated that the species of RNA was probably not myeloma messenger RNA but was histone messenger RNA: 1. 1. The amount of radioactivity in this peak was about the same for two myeloma cell lines that produce widely different amounts of light chains. 2. 2. A peak of radioactive RNA was observed in the same position on the acrylamide gel if polysomal RNA from HeLa cells was isolated and analyzed with methods similar to those used with the myeloma cells. 3. 3. Treatment of tumor-bearing mice with 100 μg/g of cytosine arabinoside inhibited incorporation of [5- 3H]uridine into this peak of RNA by more than 75 %.

Distribution of histone messenger RNA among free and membrane-associated polyribosomes of a mouse myeloma cell line

The distribution of histone messenger RNA among free polyribosomes and two classes of membrane-associated polyribosomes was studied in an immunoglobulin-secreting mouse myeloma cell line. The two classes of membrane polyribosomes were distinguished on the basis of their sensitivity to dissociation from membranes upon exposure to high concentrations of salt or during repeated centrifugation through sucrose. Histone messenger RNA is specifically excluded from that class of polyribosomes most tightly associated with cellular membranes.

Histone mRNA in eggs and embryos of [formula omitted][formula omitted

Histone messenger RNA is detectable in both the maternal RNA which is stored in the unfertilized sea urchin egg and in the RNA species which are synthesized de novo after fertilization. Hybridization competition experiments show that sequences similar to pulse-labeled 912S RNA from morulae are present in total RNA from unfertilized eggs as well as that from later stages. The proportion of histone mRNA in cellular RNA increases after fertilization, reaching a maximum at the morula stage. Although these messengers are still present in hatched blastulae and gastrulae, they represent a smaller proportion of total RNA compared with earlier stages.

Minimum estimate of the lifetime of histone messenger RNA of HeLa cells

The lifetime of histone mRNA of HeLa cells has been studied by its kinetic of approach to steady-state labeling. Cells preincubated with low concentrations of actinomycin D to inhibit rRNA synthesis, were incubated with ((3)H)uridine. Linear incorporation of uridine was observed for only two hours under the conditions chosen. Polyribosomes were isolated from cells incubated overnight with trace amounts of ((14)C)uridine and for 30 to 150 min with ((3)H)uridine. RNA was extracted from polyribosomes and fractionated by polyacrylamide gel electrophoresis. Histone mRNA was identified as a peak migrating in a characteristic position, which was absent in gels of RNA obtained from cells treated with the inhibitor of DNA synthesis cytosine arabinoside. The kinetic of labeling of histone mRNA was linear up to 150 min, which represents a minimum estimate of the lifetime of this mRNA.

Identification of histone messenger RNA from HeLa cells. Appearance of histone mRNA in the cytoplasm and its translation in a rabbit-reticulocyte cell-free system.

Histone mRNA from HeLa cells synchronized by a double thymidine block has been identified and characterized. Its synthesis starts immediately after the cells are released from the thymidine block.Pulse labelling for 60 min with [5‐3H]uridine at different times of the S‐phase showed that radioactively labelled histone mRNA enters polyribosomes with a maximum during the first hour of S‐phase, i.e., before the maximum of DNA synthesis.Histone mRNA sediments on sucrose gradients in the 8–10‐S region and separates on polyacrylamide gels into three RNA species with molecular weights of about 1.5 × 105, 1.8 × 106, and 2.1 × 105, respectively.Polyribosomal 8–10‐S RNA from synchronized cells in S‐phase directs the synthesis of histones in a rabbit reticulocyte cell‐free system. Histones synthesized in vitro were identified by polyacrylamide gel electrophoresis and by high‐voltage paper electrophoresis of tryptic peptides. Polyribosomal 8–10‐S RNA from cells in which DNA synthesis was blocked by thymidine or hydroxyurea had only a marginal histone mRNA activity.

Evidence that all messenger RNA molecules (except histone messenger RNA) contain poly(A) sequences and that the poly(A) has a nuclear function

The appearance of newly formed messenger RNA in polyribosomes of HeLa cells Is inhibited by over 85% by 3′deoxyadenosine (Penman, Rosbash & Penman, 1970) probably due to the failure of normal attachment of poly(A) to heterogeneous nuclear RNA in the presence of this drug (Darnell, Philipson, Wall & Adesnik, 1971). Results presented here show that the labeled RNA which does reach polysomes in the presence of 3′deoxyadenosine can be characterized as messenger RNA which contains smaller poly(A) segments than normal messenger RNA. The results of the present experiments suggest that all, or almost all, HeLa cell messenger RNA molecules (except for histone messenger RNA) are derived from nuclear RNA molecules which contain poly (A). The 3′deoxyadenosine-sensitive step, presumably the addition of poly(A) to heterogeneous nuclear RNA, occurs in the cell nucleus a few minutes after the DNA-dependent synthesis of the RNA molecule destined to form messenger RNA.