Precursors of U4 small nuclear RNA.

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.

Nucleolar ultrastructure and transcription of preribosomal RNA in Rous sarcoma virus-transformed chick embryo fibroblasts treated with alpha-amanitin.

Uninfected and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts (CEF) were exposed to various concentrations of alpha-amanitin for different lengths of time. At a concentration of 4 micrograms alpha-amanitin/ml, RSV-transformed cells were shown to maintain a normal rate of transcription of all classes of RNA, whereas in uninfected cells transcription was reduced to a very low level. These observations cannot be accounted for by a difference in the penetration rate of alpha-amanitin through the plasma membrane. Investigation of the ultrastructure showed that the degree of nucleolar fragmentation induced by alpha-amanitin was comparable in both types of cells. Persistence of transcription with concomitant nucleolar fragmentation in alpha-amanitin-treated RSV-transformed CEF is not in accord with the hypothesis that nucleolar integrity is required for preribosomal RNA transcription.

Silver staining of nucleolar granules in tumor cells.

With the aid of a simple silver-staining procedure, large numbers and unusual arrays of nucleolar argyrophilic granules were found in Novikoff hepatoma, KB, and HeLa cells. Some of these arrays consisted of linearly arranged discrete granules, and others were in two to three rows each containing three to five granules. Corresponding formations were not found in either the normal or regenerating liver nucleoli which contained an argyrophilic network in which the dark granules were apparently associated with the less dark argyrophilic fibrils of a reticulum. The nucleolar argyrophilic granules were readily identifiable in the separated daughter nuclei of the tumor cells in telophase, suggesting that the increased nucleolar activity of the G1 phase begins in these cells even before cell division has been completed.

Regulation of DNA chain elongation in SV3T3 cells in relation to growth rate.

Regulation of DNA synthesis was investigated in SV40 transformed 3T3 cells exhibiting variable growth rates and residence times in S phase when cultured in the presence of different serum concentrations. Pulse-labeled DNA was chased into large molecular weight material in vivo much more slowly in slowly growing cells than in cells growing at the normal rate. Consistent with this, the joining of short (less than 10 S) chains to form long (greater than 10 S) chains by whole cell lysate system in vitro was greatly impaired in slowly growing cells compared to controls. Thus the lengthening of S phase in SV3T3 cells growing slowly in low serum is reflected in a reduced rate of DNA chain elongation. The presence of cycloheximide during chase in vivo reduced the rate of conversion of pulse-labeled molecules into large molecular weight DNA in both slowly growing and normally growing cells.

Maturation of replicating simian virus 40 DNA molecules in isolated nuclei by continued bidirectional replication to the normal termination region.

Mature SV40 DNA synthesized for different periods of time either in isolated nuclei or in intact cells was highly purified and then digested with restriction endonucleases in order to relate the time of synthesis of newly replicated viral DNA to its location in the genome. Replication in nuclei supplemented with a cytosol fraction from uninfected cells was a faithful continuation of the bidirectional process observed in intact cells, but did not exhibit significant initiation of new replicons. SV40 DNA replication in cells at 37 degrees C proceeded at about 145 nucleotides/min per replication fork. In the absence of cytosol, when DNA synthesis was limited and joining of Okazaki fragments was retarded, bidirectional SV40 DNA replication continued into the normal region where separation yeilded circular duplex DNA molecules containing one or more interruptions in the nascent DNA strands. In the presence of cytosol, this type of viral DNA was shown to be a precursor of covalently closed, superhelical SV40 DNA, the mature from of viral DNA.

Isolation of the transfer RNA genes of bacteriophage T4 and transfer RNA synthesis in vitro.

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not know.

The structural organization of nuclear pre-mRNA. II. Very long double-stranded structures in nuclear pre-mRNA.

High molecular weight nuclear pre-messenger RNA (pre-mRNA or hnRNA) isolated from Ehrlich ascites carcinoma cells contains besides moderately long (100--200 base pairs) snap-back double-stranded structures, also longer double-stranded structure containing at least 300--800 base pairs. Their double-stranded nature was proved by Cs2SO4 gradient centrifugation. Very long double-stranded sequences are not able to snap-back after RNA melting. While the moderately long double-stranded RNA (dsRNA) is renatured at C0t1/2 approximately equal to 5-10(-4), the very long dsRNA shows a higher complexity (C0t1/2 approximately equal to 2-10(-2). They also hybridize to less reiterated class of DNA than moderately long dsRNA. Two classes of dsRNA are represented by different sequences as followed from cross-renaturation experiments. Very long dsRNA forms stable hybrids with 20% of total poly(A)+mRNA of cytoplasm. The properties of different classes of ds structures present in nuclear pre-mRNA are compared and their possible nature is discussed. The presence of very long dsRNA may reflect either the symmetric transcription of structural genes, or the transcription from those DNA sequences which are complementary to each other but located in different parts of the genome.

The effects of protein synthesis inhibition, and of mutations rna1.1 and rna82.1, on the synthesis of small RNAs in yeast.

Strains of Saccharomyces cerevisiae have been constructed that possess temperature-sensitive defects in tRNA precursor (pre-tRNA) splicing and which also lack the processing endonuclease that acts at the 3'-terminus of 5 S rRNA and 35 S rRNA precursors (pre-rRNAs). The unspliced pre-tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre-tRNA splicing-defective strains with a functional pre-5 S RNA processing enzyme. The pre-RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse-labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild-type strains, an RNA 145 +/- 2 nucleotides in length.

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.

Acceleration of ribosome formation in rat liver in response to hydrocortisone.

The amount of nuclear pre-rRNA (precursor to rRNA) and rRNA species and their labelling in vivo with [14C]orotate were determined in hormone-deficient and treated rats. On the basis of the data obtained, the rates of synthesis and processing of pre-rRNA in the two groups of animals were compared. It is shown that the rate of nucleolar RNA synthesis is increased twice, its half-life is reduced about 1.4 times and the endonuclease processing of primary pre-rRNA is channeled through the shortest maturation pathway. Thus, an accelerated ribosome formation takes place in response to hydrocortisone. The overproduced ribosomes are not retained in the nucleoplasm and are immediately transferred to the cytoplasm.

Metabolism of caffeine to nucleic acid precursors in mammalian cells.

Caffeine is rapidly metabolized in human and mouse cells in culture by de-methylation: within 1-3 h of exposure to millimolar concentrations of labeled caffeine, more than 90% of the pool consists of labeled products of metabolism and less than 10% is still caffeine. The methyl groups seem to be transferred and used in the de novo synthesis of thymine, guanine, and adenine to nucleic acids. Normal fibroblasts, Lesch-Nyhan fibroblasts, xeroderma pigmentosum fibroblasts, HeLa cells, wild type mouse cells, and adenine phosphoribosyl-transferase-deficient mouse cells all seem to metabolize caffeine similarly.

[Intensity of the synthesis of RNA precursors in blood lymphocytes of cattle infected with leukemia virus]. / Intensivnost' sinteza bmRNK v limfotsitakh krovi krupnogo rogatogo skota, infitsirovannogo virusom leikoza.

The peripheral blood lymphocytes of the cattle with lymphatic leukemia experimentally induced by BLV and of the animals from the same group with persistent lymphocytosis show the lower level of the pre-RNA-synthetic activity per cell when compared with the lymphocytes of healthy cattle (as evaluated by autoradiography with 3H-uridine as a precursor). At the same time the RNA-synthetic activity in the population as a whole increases as a result of growth of the RNA synthesizing cells (a labelling index), which is confirmed by enhancement of DNA-dependent RNA-polymerase activities and 3H-uridine in vitro incorporation into lymphocytes revealed by the liquid scintillation counting method.

[Characteristics of the organization and expression of individual genes in eukaryotic cells]. / Osobennosti organizatsii i ékspressii individual'nykh genov v kletkakh éukariotov.

The modern views on the organization and expression of single genes in eukaryotic cells, RNA structure and biosynthesis are reviewed. The problems of unstable gene localization ("mobile dispersed genetic elements") of some structural genes of cellular and viral origin are discussed. Peculiarities of transcription and processing of mRNA, pre-mRNA splicing are described. The information on the mechanisms of pre-mRNA splicing in normal and virus-infected eukaryotic cells, the role of low molecular nuclear RNA in the RNA splicing as signals for less than recognition greater than by specific nucleases and RNA synthetases are also regarded.

[Kinetic regularities of RNA synthesis. VII. Accumulation of RNA precursors and the inverse problem]. / Kineticheskie zakonomernosti sinteza RNK. VII. Nakoplenie predshestvennikov RNK i obratnaia zadacha

Applicability limits are specified for earlier obtained solutions of particular inverse problems of accumulation kinetics of newly synthesized RNA using isotope label for immitating unstationary state in stationary cell cultures.

[Reutilization of labelled DNA precursors by the iris cells during the regeneration of the lens and retina in newts]. / Reutilizatsiia mechenykh predshestvennikov DNK kletkami raduzhiny pri regeneratsii linzy i setchatki u tritonov

In the adult crested newts, the proliferating cells of the proximal and distal iris zones reutilize the labelled DNA precursors 4 days after triple injections of 3H-thymidine. The density of radioautographs (intensity of labelling per nucleus) under reutilization is 2.5--3 times weaker than under 3H-thymidine incorporation.