Messenger RNA regulation in human diploid fibroblasts

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.     

RNA containing poly-A sequence in Pharbitis nil cotyledons and its template activity in vitro

RNA containing poly-A sequence was separated from the totalnucleic acid of morning glory cotyledons by cellulose columnchromatography. The RNA was preferentially retained on poly-Uimmobilized filter and had high template activity in a bacterialcell-free protein synthesizing system. Electrophoretic analysissuggested that this poly-A RNA is 10 S RNA and the size of thepoly-A sequence is about 3.5 S. Sub-cellular distribution ofthe poly-A RNA was also investigated. (Received December 7, 1973; )     

Analysis of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells

The molecular size and poly-A content of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells were measured. KCl was found to be essential for synthesis of high molecular weight RNA: when 0.4 M KCl was added to the reaction mixture, the average molecular size of the RNA formed was 14S; without KCl the average molecular size was 5S. A significant amount of poly-A sequences was found in RNA synthesized in the presence of alpha-amanitin, suggesting that RNA polymerase I and/or III may synthesized some RNA containing poly-A in isolated nuclei.     

Interrelationship between protein synthesis and mRNA metabolism in rat liver cells]

It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.     

Effect of hydrocortisone on the properties of rat liver polyribosomes, metabolism, and template activity of polysomal poly-A-containing RNA

The effect of hydrocortisone on the amount of newly synthesized polyribosomal poly-A+-RNA and its translation activity and the distribution of polyribosomes in the induction dynamics according to their size were studied. It was shown that 3-5 hours after intraperitoneal injection of hydrocortisone the incorporation of labelled precursors into polyribosomal poly-A+-mRNA is increased, which is accompanied by rapid accumulation of mRNA in the polyribosomes. Under prolonged induction those parameters come down to the initial level. 4-7 hours after the injection of the hormone the relative amount of heavy polyribosomes (350-412S) in liver cells is increased. It was found that hydrocortisone significantly changes the specific translation activity of polysomal poly-A+-mRNA: it shows an increase 2-4 hours after the hormone injection and returns to the initial level 12 hours after the injection.     

Poly adenylic acid sequences in mitochondrial RNA

RNA isolated from a mammalian mitochondria was investigated for Poly-A sequences. 34–39% of mitochondrial RNA was found to contain Poly-A as tested by two different techniques. The Poly-A sequences are 150–180 nucleotides (4–5S) long. The possibility of poly-A containing RNA being a cytoplasmic contaminant was excluded by treating mitochondria with RNAse and digitonin.     

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.     

Induction of cell cycle progression by adenovirus E1A gene 13S- and 12S-mRNA products in quiescent rat cells.

Rat 3Y1 cell lines that express either adenovirus type 12 E1A 13S mRNA or 12S mRNA in response to dexamethasone treatment were established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines were growth arrested, and the induction of cell cycle progression was analyzed by flow cytometry after switch on of the cDNA by the addition of dexamethasone. The results indicate that the 13S- or 12S-mRNA product alone has the ability to cause progression of the cell cycle at a similar rate. The simultaneous addition of epidermal growth factor accelerated the rate of cell cycle progression in the transition from the G0/G1 phase to the S phase.     

Isolation and nucleotide sequence of a full-length cDNA coding for aldolase B from human liver.

Two recombinant clones, pA2 and pA3, containing cDNA sequences for human aldolase B have been isolated from a full length human liver cDNA library. The larger one, pA3, has been subcloned in M13 phage and completely sequenced with the chain terminator method. The sequence covers 1,600 nucleotides including the whole coding region (1,050 nucleotides), 67 nucleotides from the 5' non-coding region and the whole 3' non-coding region, 440 nucleotides long, down to the poly-A tail. Comparison with rabbit aldolase A and with a partial sequence of rat aldolase B, shows a homology of about 76% for aldolase A and of about 94% for aldolase B, which indicates that the sequenced cDNA codes for the liver isoenzyme. This is the first complete sequence reported for human aldolase B. The pA3 clone strongly hybridizes to 18S mRNA from human adult liver as expected from the size of the isolated cDNA.     

Tetrahymena tubulins and in vitro translation of Tetrahymena RNA.

Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymena alpha tubulin did not comigrate with either brain or flagellar alpha tubulins, although brain, flagellar, and ciliary beta tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W, S, HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A+ RNA had 2 prominent protein bands which comigrated with alpha and beta tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products of poly-A- RNA.     

Purification and properties of biologically active rainbow trout testis protamine mRNA.

At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.     

核仁含磷蛋白B_(23)mRNA的部分提纯

用蔗糖梯度离心法对Novikoff肝癌细胞的多聚(A)~+mRNA进行了链长分部。沉降在13S和15S的组分6和组分7富集了B_(23)mRNA,其体外转译产物可特异地被抗蛋白B_(23)的抗体免疫吸附,被吸附的蛋白在SDS-PAGE中迁移到大约37,000道尔顿的区带。另外,用多聚核糖体免疫吸附技术提纯了少量B_(23)mRNA,它在体外转译系统中也指导合成了分子量约为37,000道尔顿的蛋白质。     

Chicken histone H5: selection of a cDNA recombinant using an extended synthetic primer

We describe the use of a synthetic primer to select a cDNA recombinant clone containing H5 coding sequences. The strategy used was as follows: 1. Prepare oligo(dT) cellulose-bound mRNA from chicken reticulocytes and select 11S-18S material from sucrose gradients. 2. Use this RNA fraction both to prepare a cDNA library and as a template for H5-specific cDNA synthesis using a synthetic primer. 3. Screen out most globin cDNA recombinants with oligo(dT)-primed globin cDNA. 4. Search for H5 recombinants using H5 specific cDNA and verify the identity by DNA sequencing. Our screening suggests an H5 mRNA abundance of about two parts per thousand in chicken reticulocyte poly(A)-containing RNA. The isolation of an H5 cDNA recombinant clone is an initial step in the study of H5 genes and their relationship to H1 and core histone genes.     

A sensitive in vitro protein synthesizing system from Ehrlich ascites mitochondria

The S-25 fraction prepared from digitonin washed mitochondria is highly active in poly(U) directed phenylanine incorporation when supplemented with t-RNA. Ribosomes prepared from the S-25 fraction contain 58S monomeric ribosomes and 40S and 29S subunits. Further, these ribosomes contain 21S and 13S rRNA. No detectable cytoplasmic specific ribosomal particles and also rRNA was detected in the mitochondrial S-25 preparation. Ribosomes from mitochondrial S-25 have specific requirement for mitochondrial specific supernatant factors for complete activity.     

Design and evaluation of an oligonucleotide-microarray for the detection of different species of the genus Kitasatospora

An oligonucleotide-microarray method was developed for the detection of Kitasatospora species in soil samples. The 16S-23S rDNA internal transcribed spacer (ITS) sequence of these antibiotics-producing actinomycetes was applied to design short oligonucleotide probes. Two different 26-mers were synthesized, specific to each species used. Additionally, four oligonucleotide probes were designed to evaluate the system. The oligonucleotides were spotted onto slides of the ArrayTube microarray system and examined with a new silver-labeling detection technique. Prior to hybridization analysis, the 16S-23S rDNA were amplified by polymerase chain reaction both from bacterial cells and environmental samples using two actinomycetes specific primers containing a 5' biotin labeling. The type strains of eight Kitasatospora species included in this study were K. phosalacinea DSM 43860, K. setae DSM 43861, K. cochleata DSM 41652, K. cystarginea DSM 41680, K. azatica DSM 41650, K. mediocidica DSM 43929, K. paracochleata DSM 41656, and K. griseola DSM 43859. The actinomycetes-specific primers were shown to amplify the entire 16S-23S rDNA ITS region from all tested strains. More importantly, the described technique allows the detection of Kitasatospora strains from soil samples by extracting metagenomic DNA followed by a PCR amplification step. This indicates that the oligonucleotide-microarray method developed in this study is a reliable tool for the detection of Kitasatospora species in environmental samples.     

Functional heterogeneity of the 30S ribosomal subunit of E. coli

Summary When 30S ribosomal subunits from E. coli are incubated with poly U, two separable components are recovered by zonal centrifugation of the incubation mixture. The faster sedimenting component is an aggregate of 30S subunits and poly U, while the slower one corresponds to the 30S ribosomal subunit. One ribosomal protein, protein 30S-1 is predominantly present in the faster sedimenting aggregate. The amount of poly U-30S subunit complex formed in the incubation mixture is limited by the amount of protein 30S-1 present. Consequently the number of ribosomal binding sites available for Phe-tRNA is limited in a similar fashion by the presence of protein 30S-1. When 30S ribosomal subunits are reconstituted in the absence of protein 30S-1, very little poly U or Phe-tRNA binding capacity is manifest under our assay conditions. We conclude that protein 30S-1 is required for maximum capacity of ribosomes to bind mRNA. Since this protein is present only on a fraction of the ribosome at any one time, it must exchange from one ribosome to another during protein synthesis.Abbreviations Poly U (polyuridylic acid) - t-RNA (transfer ribonucleic acid) - mRNA (messenger ribonucleic acid) - Phe (phenylanine) - A₂₆₀ unit (unit of material which gives an optical density of 1.0 at 260 nm in a one centimeter optical path)