The polyadenylate polymerases from yeast.

Poly(A) polymerase activity was first detected in yeast extracts, primarily in association with the ribosomal fraction, by Twu and Bretthauer in 1971 (Twu, J. S., and Bretthauer, RK. (1971) Biochemistry 10, 1576-1582). This activity has now been separated into three distinct enzymes by chromatography on DEAE-cellulose. Each of the three enzymes can catalyze the incorporation of adenylate residues from ATP into a polyadenylate (poly(A)) tract at the 3' terminus of a primer RNA. Enzyme I elutes at 0.07 M ammonium sulfate from the DEAE-cellulose column, utilizes the mixed polynucleotide poly(A,G,C,U) or ribosomal RNA most efficiently in vitro, and may be responsible in vivo for the initiation of the poly(A) tracts found on yeast messenger RNA. Enzyme II elutes from the column at 0.20 M ammonium sulfate, requires poly(A) itself or an RNA primer containing a 3'-oligo(A) tract, and may be responsible in the nucleus for the elongation of tracts initiated by enzyme I. Enzyme III elutes from the column at 0.56 M ammonium sulfate and is present in low amounts in nuclear extracts. It may be involved in adding poly(A) tracts to messenger RNA in mitochondria. These enzymes also have the intrinsic capacity for the incorporation of cytidylate residues from CTP, which correlates with the finding of cytidylate residues in the poly(A) tracts present in the yeast RNA which is rapidly labeled in vivo. About 75% of the total poly(A) polymerase activity of yeast is enzyme I, most of which is present in the soluble protein fraction of the whole yeast extract. About 20% of the total poly(A) polymerase is enzyme II, and 1 to 5% is enzyme III. All three of the yeast poly(A) polymerases require an RNA primer with a free 3'-hydroxyl group, show no requirement for a DNA template, require Mn-2+ for optimal activity, have pH optima of 8.5, and are inhibited by GTP, CTP, UTP, and native yeast DNA. Polymerases I and II have similar molecular weights by gel filtration.     

The presence of polyadenylate sequences in the ribonucleic acid of a higher plant

Rice callus tissue in suspension culture was labeled $\text{in}$$\text{vivo}$ with ³²PO₄^?3. Approximately 4% of the total RNA extracted from the tissue could be bound to nitrocellulose filters in the presence of 0.5 M KCl. The base composition of this material was: A, 30.6; U, 32.7; G, 18.0; and C, 18.6%. If the RNA was digested with both T₁ and pancreatic ribonucleases prior to binding, the base composition of the bound material was: A, 97.8; U, 0.0; G, 1.6; and C, 0.5%. About 14% of the RNA isolated from purified polyribosomes could be bound to filters in 0.5 M KCl. It is concluded that higher plant RNA contains polyadenylate sequences.     

Size heterogeneity of polyadenylate sequences in mouse globin messenger RNA

Heterogeneity in the length of the poly(A) region has been demonstrated in mouse α and β-globin messenger RNAs. This finding is based on the initial observation that only 30% of the globin mRNA purified by oligo(dT)-cellulose affinity chromatography binds to Millipore filters under conditions where other poly(A)-containing mRNAs have been shown to bind, and the subsequent finding that the bound and non-bound fractions contain different size classes of poly(A). The poly(A) size was determined by polyacrylamide gel electrophoresis of the T₁ and pancreatic RNAase-resistant fragments. The unbound mRNA fraction gives a fragment 35 to 45 adenine nucleotides long, while the bound mRNA contains two fragments with average lengths of 55 to 65 and 75 to 120 nucleotides.The heterogeneity of the poly(A) region is present in both α and β-globin mRNAs as both Millipore-bound and unbound RNA fractions directed the synthesis of comparable amounts of mouse α and β-globin chains.Change in the distribution of the various size classes of poly(A) was analyzed by Millipore binding assays after various times of labeling in vivo. The percentage of labeled mRNA bound to Millipore filters decreased with time, suggesting either a shortening of the poly(A) region or differential synthesis of mRNAs containing shorter poly(A) at earlier stages in erythropoeisis.     

The occurrence of gas-vacuolate bacteria in lakes

In order to assess the importance of gas vacuoles in planktonic bacteria we carried out a survey of 35 lakes of different types. At least 39 morphologically distinct species of gas-vacuolate bacteria were encountered. All of the freshwater lakes, and one of two seawater-flooded quarries, which became thermally stratified in summer, contained gas-vacuolate bacteria in their anaerobic hypolimnia; however, with one exception, none was found in isothermally mixed lakes. This pattern of distribution supports the idea that gas vacuoles are important in providing buoyancy, a function which is relevant only in non-turbulent aquatic systems.     

Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA.

Ribonucleoprotein particles containing heterogeneous nuclear RNA (Pederson, 1974) were isolated from HeLa cells and digested with ribonucleases A and T₁ at high ionic strength. The nuclease-resistant material, comprising 9.4% of the initial acid-insoluble [³H]adenosine radioactivity, was further fractionated by poly(U)-Sepharose chromatography. The bound fraction eluted from the column with 50% formamide and banded in cesium sulfate gradients (without aldehyde fixation) at a buoyant density characteristic of ribonucleoprotein (1.45 g/cm³). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this material revealed two Coomassie blue-stained bands. The major polypeptide had a molecular weight of 74,000 a less prominent band had a molecular weight of 86,000. The RNA components contained 74.4 mol % AMP and 17.7 mol % UMP. Polyacrylamide gel electrophoresis of the RNA, labeled with [³H]adenosine, demonstrated the presence of molecules 150 to 200 nucleotides in length (poly(A)), as well as molecules 20 to 30 nucleotides long (oligo(A)). Both poly(A) and oligo(A) sequences have previously been identified in HeLa heterogeneous nuclear RNA. These data demonstrate that both the poly (A) and oligo(A) sequences in HeLa heterogeneous nuclear RNA exist in vivo tightly complexed with specific proteins.     

The occurrence of Ds-like sequences in cereal genomes

Summary The occurrence of DNA sequences similar to the Ds-element of sh-m5933 maize (Ds-like sequences) was studied in other representatives of the Gramineae. The approximate number of copies of such sequences found under gentle and stringent conditions of washing was determined by dot-hybridization. It was shown that in the maize genome the number of copies of Ds-like sequences exceeds about ten-fold the content of such sequences found in wheat, rye and barley genomes. Quantitative differences in Ds-like sequences between wheat species with various genomes and ploidies (when estimated per genome) as well as between different H. vulgare varieties was not determined. The various melting points (T_m) of DNA-duplexes formed when the Ds-element is hybridized with wheat, rye and barley DNA respectively do not show significant differences and are essentially lower than the T_m of the Ds-element (by 8°–9°C). Thus, these duplexes have 9–11% of nucleotide substitutions in comparison to Ds sh-m5933. The data obtained permit one to suppose the presence of a series of Ds-like sequences heterogenous for the length and degree of homology to the Ds-element isolated from the shrunken locus (sh-m5933) of maize DNA.     

The occurrence of the Entner-Doudoroff pathway in bacteria

The occurrence of the two key enzymes of the Entner-Doudoroff pathway, gluconate-6-phosphate dehydrase and 2-keto-3-deoxygluconate-6-phosphate aldolase, was determined in approximately 150 strains belonging to 37 different bacterial genera. The following results were obtained:

1. 24 out of 37 genera have at least one representative with the Entner-Doudoroff mechanism. It is thus more widespread than previously thought.
2. The Entner-Doudoroff mechanism occurs mainly in gram-negative bacteria with a DNA base composition in the range 52–70% GC. Eighty-five per cent of these organisms contain the system, while only 20% (6 strains) of the gram-negative organisms with less than 52% GC possess both enzymes.
3. This pathway is absent in all gram-positive organisms investigated except in 5 out of 12Nocardia strains.
4. Erwinia and some strains of theAchromobacter-Alcaligenes group are exceptional, since they possess only 2-keto-3-deoxygluconate-6-phosphate aldolase.

     

The occurrence of hopanoid lipids in Bradyrhizobium bacteria

Abstract Lipid extraction procedures followed by GLC and GLC-MS analysis were used to investigate the triterpenoid content in Bradyrhizobium and Rhizobium bacteria. Unlike the tested strains of Rhizobium bacteria, a range of triterpenoids e.g., squalene and different classes of hopanoid derivatives were detected in bacteria from all Bradyrhizobium strains investigated (different strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii as well as Bradyrhizobium sp.). Furthermore, related compounds were identified from some hopanoid lipids (e.g., diplopterol) that carried an additional methyl group in their molecular structure. The hopanoid content was high in some strains and accounted for more than 40% of the total lipid fraction (e.g., in strains Bradyrhizobium japonicum USDA 110 and USDA 31), while other strains contained only about a tenth of that amount (e.g., Bradyrhizobium japonicum ATCC 10324 and Bradyrhizobium sp. ( Lupinus ) ATCC 10319).     

The occurrence of denitrification in extremely halophilic bacteria

Abstract A total of 97 aerobic and facultatively anaerobic bacteria, and 3 Candida albicans , were tested for their ability to inhibit the growth of Haemophilus influenzae . Only strains of Pseudomonas aeruginosa showed any inhibitory effect and all 5 strains tested clearly inhibited the growth of all 10 strains of H. influenzae . The inhibition of H. influenzae . by Ps. aeruginosa may play a role in the establishment of chronic Ps. aeruginosa infections in the respiratory tracts of patients with bronchiectasis and cystic fibrosis (CF).