Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase.

Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide. Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract. The in vitro synthesized polypeptides were identified by tryptic peptide analysis. Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657). Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed. When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.     

Adenovirus Type 2-Simian Virus 40 Hybrid Population: Evidence for a Hybrid Deoxyribonucleic Acid Molecule and the Absence of Adenovirus-Encapsidated Circular Simian Virus 40 Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) from the adenovirus-encapsidated particles of the adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid population plaque variant (Ad2(++) HEY), known to yield SV40 virus with high efficiency, was studied by equilibrium density centrifugation followed by ribonucleic acid-DNA hybridization employing virus-specific complementary ribonucleic acids synthesized in vitro. These techniques establish linkage between the Ad2 and SV40 components in the adenovirus-encapsidated particles of this population. The linkage is alkali-resistant and presumably covalent; thus, the Ad2 DNA and SV40 DNA are present in a hybrid molecule. Velocity centrifugation studies in alkaline sucrose gradients eliminated the possibility that supercoiled circular SV40 DNA is present in the adenovirus capsids. The DNA obtained from the adenovirus-encapsidated particles of the Ad2(++) HEY population appears to consist of nonhybrid Ad2 DNA and Ad2-SV40 hybrid DNA molecules.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses II. Relationship of Adenovirus 2 Deoxyribonucleic Acid and Simian Virus 40 Deoxyribonucleic Acid in the Ad2+ND1 Genome

A nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), has been plaque-isolated from an Ad2-SV40 hybrid population. This virus, unlike the defective Ad-SV40 hybrid populations previously described, replicates without the aid of nonhybrid adenovirus helper. Consequently, the hybrid virus deoxyribonucleic acid (DNA) can be obtained free of nonhybrid adenovirus DNA. The DNA of the Ad2(+)ND(1) virus was shown by ribonucleic acid (RNA)-DNA hybridization to consist of nucleotide sequences complementary to Ad2- and SV40-specific RNA. Techniques of equilibrium density and rate zonal centrifugation were employed to demonstrate that these Ad2 and SV40 nucleotide sequences were linked together in the same DNA molecules by alkali-resistant bonds. Calibration curves were established relating the amount of tritium-labeled SV40-specific RNA (prepared in vitro or in vivo) bound to given amounts of SV40 DNA in a hybridization reaction, and these curves were employed to determine the equivalent amount of SV40 DNA in the Ad2(+)ND(1) molecule. From the results obtained, it was estimated that 1% of the Ad2(+)ND(1) DNA consists of SV40 nucleotide sequences.     

A vertical submarine electrophoresis apparatus for polyacrylamide minigels

A vertical submarine electrophoresis apparatus for use with minislab polyacrylamide gels is described. The design allows polyacrylamide gels to be run with the same ease and convenience that agarose gels are run with horizontal submarine apparatuses. The vertical submarine features a single buffer chamber with a restriction between the upper and the lower portions of the chamber. Acrylamide gels, cast between 9 X 10-cm glass slides, are inserted into the restriction and are completely immersed in buffer. Thus, current flows primarily through the gel itself, but some current flows through the buffer in the restriction surrounding the gel. Because water-tight separation of buffer chambers is not necessary, time-consuming and/or expensive procedures such as sealing with agarose or using fragile notched glass plates are eliminated. The apparatus can be set up to run a gel in less than 30 s. It is versatile in that gels of varying thickness (0.5, 0.8, 1.5, and 3 mm) can be run on a single apparatus. The apparatus has been used for sodium dodecyl sulfate gels, low ionic strength native gels for nucleoprotein complexes, and composite acrylamide-agarose gels.     

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.     

Electrophoretic Analysis of Ribosomal and Viral Ribonucleic Acids with a Simple Technique for Slicing Low-Concentration Polyacrylamide Gels

The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.     

Stimulation of RNA synthesis in cowpea (Vigna sinensis) seedlings by gibberellic acid and adenosine 3′,5′-cyclic monophosphate

There is about 50% stimulation in the incorporation of [³H]uridine into total RNA of cowpea following the application of gibberellic acid (GA₃) and adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Cyclic AMP is very specific in its action. Co-fractionation of ³H- and ¹⁴C-labelled RNA on acrylamide-agarose gels reveal a control by GA₃ and cyclic AMP predominantly on its polydisperse fraction. Both GA₃ and cyclic AMP appear to act through a similar mechanism.     

Nature of Ribonucleic Acid Synthesis During Early Sporulation in Saccharomyces cerevisiae

Phosphate uptake in sporulating cultures of Saccharomyces cerevisiae has been found to occur approximately 2 h after the transfer to sporulation medium. Early ribonucleic acid synthesis begins at approximately 4 h and continues to 8 h. Incorporation of phosphate into acid-extractable precursor pools parallels phosphate uptake. In triple-labeling experiments it was observed that the breakdown of vegetatively synthesized ribonucleic acid is not a significant source of precursors for ribonucleic acid synthesis during sporulation. The majority of the ribonucleic acid made in a 10-min period during sporulation does not migrate on gels with precursor or mature ribosomal ribonucleic acid.     

Specialized lambda transducing bacteriophage which carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase.

A number of specialized lambda transducing bacteriophages which carry the Escherichia coli gene guaB were isolated from E. coli. One of these bacteriophages, lambda cI857 Sam7 d guaB-2, also carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase (EC 6.1.1.21). Histidyl-transfer ribonucleic acid synthetase activities in induced and uninduced lysogens carrying lambda d guaB-2 indicate that the phage carries the entire structural gene and that the gene is under the control of an E. coli promoter. These conclusions were confirmed by the in vivo production of a protein encoded by the phage which comigrates with authentic histidyl-transfer ribonucleic acid synthetase on two-dimensional polyacrylamide gels.     

Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. Quantitative analysis of their interaction in a fractionated reticulocyte cell-free system.

The polyadenylic acid-containing messenger ribonucleic acids of eukaryotic cells are rapidly isolated and deproteinized in a simple and gentle one-step procedure. The polyribosome fraction, dissolved in 0.5 M NaCl/0.5 percent sodium dodecyl sulfate, is passed through an oligo(dT)-cellulose column which is then washed with the solvent until proteins and contaminating ribonucleic acids are fully removed. Deproteinized messenger ribonucleic acid is then eluted by lowering the ionic strength. This method gives highly purified and active messenger ribonucleic acids from all tissues tested. The yield is approximately 1.5 to 2 percent of the polyribosomal ribonucleic acid. Messenger ribonucleic acids are assayed in a rabbit reticulocyte-derived, messenger-dependent, cell-free protein-synthesizing system modified from Crystal et al. (Crystal, R. G., Nienhuis, A. W., Elson, N. A., and Anderson, W.F. (1972) J. Biol. Chem. 247, 5357-5368). This system synthesizes proteins at an almost linear rate for at least 2 hours. During this period, each globin messenger ribonucleic acid directs the synthesis of several globin molecules. Each active ribosome synthesizes a globin molecule every 6 to 7 min, but only a small fraction of the ribosomes or messengers are active at any instant. Translation occurs mainly on di- and monoribosomes although larger sized polysomes also occur. Several lines of evidence suggest that globin messenger ribonucleic acid requires "activation" before it can be utilized and that a messenger activation step of protein synthesis initiation is rate-limiting in this cell-free system.     

Nucleotide sequence of phenylalanine transfer ribonucleic acid from pea (Pisum sativum, Alaska).

Phenylalanine transfer ribonucleic acid from peas (Pisum sativum, Alaska) was completely digested with beef pancreatic ribonuclease (RNase I) and with ribonuclease T1. The resulting oligonucleotides were compared with those from the corresponding hydrolyses of phenylalanine transfer ribonucleic acid from wheat germ. The structures of both ribonucleic acids appeared to be identical. This report is the first to show that identical structures for the same specific acceptor transfer ribonucleic acid are present in two different plant species.     

Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A.

Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Specific small nuclear RNAs are associated with yeast spliceosomes

Two different methods have been devised for the analysis and purification of spliceosomes formed in a yeast in vitro splicing system. The first method relies on the electrophoretic separation of ribonucleoprotein particles in composite acrylamide-agarose gels. A large fraction of added substrate is located in spliceosomes, the formation of which can be shown to be dependent on the presence of both a yeast 5' splice junction and a TACTAAC box on the RNA substrate. The second method relies on oligo(dT)-cellulose chromatography of spliceosomes formed with a polyadenylated substrate. Purification of spliceosomes by either method indicates that at least three small nuclear RNAs, approximately 160, 185, and 215 nucleotides in length, are specifically associated with yeast spliceosomes.     

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.     

Regional distribution of polyadenylated mRNA in Xenopus laevis embryos

Xenopus laevis embryos were dissected into dorsal and ventral regions in post-gastrula stages. Polyadenylated and nonpolyadenylated ribonucleic acids were separated on oligo (dT) cellulose and translated in vitro. The radioactivity incorporated into the translation products directed by polyadenylated and nonpolyadenylated messenger ribonucleic acids shows that in the dorsal region most proteins are synthesized on polyadenylated messenger ribonucleic acid templates in all the stages, while in the ventral region the major templates seem to be, until the neural fold stage, nonpolyadenylated messenger ribonucleic acids. Later the polyadenylated messenger ribonucleic acid activity there too increases.     

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Soybean Hypocotyl Cells: Their Relation to Messenger Ribonucleic Acid

Plant ribonucleic acids which have high adenosine monophosphate concentrations were studied. Purified deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid fractions both contained poly-adenosine monophosphate sequences (those from the latter being longer than those from the former); without these poly-adenosine monophosphate sequences their base compositions were the same. The average poly-adenosine monophosphate sequence from purified tenaciously bound ribonucleic acid was 160 residues long, as measured by gel electrophoresis. However, base hydrolysis and chromatography indicated one 3′-nucleoside (adenosine) per 71 nucleotides, giving a chain length of 72 residues. The dominant species in the cytoplasm, as measured by radioactive precursor incorporation, was tenaciously bound ribonucleic acid, whereas deoxyribonucleic acid-like ribonucleic acid was present in greater amounts in the nucleus. This work provides evidence that deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid represent forms of messenger ribonucleic acid in soybean, with deoxyribonucleic acid-like ribonucleic acid residing in the nucleus, perhaps as the messenger ribonucleic acid precursor, and tenaciously bound ribonucleic acid residing, as the active messenger ribonucleic acid, in the cytoplasm.     

Adenylate-Rich Sequences in Vesicular Stomatitis Virus Messenger Ribonucleic Acid

Vesicular stomatitis virus (VSV)-specific messenger ribonucleic acid (mRNA) species contain sequences of adenylate-rich RNA which are more heterogeneous in their migration through sodium dodecyl sulfate-polyacrylamide gels than the corresponding fractions from HeLa cell mRNA. VSV virion RNA contains no adenylaterich sequences. The possible role of such sequences in the mRNA species of a cytoplasmically replicating virus is discussed.     

Cytokinins and Transfer Ribonucleic Acids. I. Presence of Cytokinins in Various Amino-acid Specific Transfer Ribonucleic Acids of Baker's Yeast

Soluble ribonucleic acid of baker's yeast was fractionated by countercurrent distribution and assayed for both cytokinin activity, using tobacco pith callus tissue, and for certain specific amino-acid acceptor activities. Two groups of fractions showed cytokinin activity, one of which corresponds to serine and tyrosine transfer ribonucleic acids which are known to contain isopentenyl adenine, while the other corresponds lo undetermined species of ribonucleic acids.