(A)GGG(A), (A)CCC(A) and other potential 3' splice signals in primate nuclear pre-mRNA sequences

Several 3' splice signals in nuclear precursor mRNAs have already been known for some time: the AG doublet on the left-hand side of the splice and a run of pyrimidines just upstream of it. More recently it has been noted that the YNYTRAY sequence (where Y is a pyrimidine, R a purine and N any base) is a branching-sequence participating in formation of a lariat structure. Keller and Noon have shown the existence of several putative consensus sequences at this site. In this work, extensive computations of the distributions of 256 quartets in all primate nuclear pre-mRNA intron sequences present in GenBank have been carried out. Several putative signals upstream and downstream of the 3' splice have been detected. These have been compared with the results obtained in analogous computations carried out on all nuclear pre-mRNA introns present in a combined eukaryotic file containing mammal, non-mammalian vertebrate, invertebrate and plant sequences. The distributions of the more interesting oligomers are shown here. Of particular interest are the putative (A)GGG(A) signal 60 nucleotides upstream of the 3' splice site and (A)CCC(A) 3-40 nucleotides downstream of it. A possible splicing model explaining these data and involving formation of alternative hairpin loop structures is proposed.     

Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.)

Gibberellins (GAs) A₁, A₅, and A₂₉ were identified, and also GA₃₂ was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning → paper chromatography → reverse phase C₁₈ high performance liquid chromatography (HPLC) → bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C₁₈ HPLC at Rts where GA_4/7 and GA₈ (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA₃₂ (112 ng of GA₃ equivalents per gram fresh weight), and minor amounts (0.5 ng of GA₃ equivalents) for each of GA₁ and GA₅, respectively, based on the microdrop assay.     

Quantitation of Gibberellins A(1), A(3), A(4), A(9) and a Putative A(9)-Conjugate in Grafts of Sitka Spruce (Picea sitchensis) during the Period of Shoot Elongation

The levels of endogenous gibberellin A₁ (GA₁), GA₃, GA₄, GA₉, and a cellulase hydrolyzable GA₉ conjugate in needles and shoot stems of mature grafts of Sitka spruce (Picea sitchensis [Bong.] Carr.) grown under environmental conditions that were either inductive, hot, and dry, or noninductive, cool, and wet, for flowering, were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated [²H₂]GA₁, GA₃, GA₄, and GA₉ as internal standards. The samples were taken when the shoots had elongated about 30, 70, and 95% of the final shoot length and 17 days after elongation had terminated. The concentration of putative GA₉-conjugate, estimated by GCSIM of GA₉ after cellulase hydrolysis of the highly water soluble fraction, was 33 nanograms per gram fresh weight in the needles of both heat and drought- and cool and wet-treated plants sampled just after bud burst. The concentration gradually decreased to a final value of 13 nanograms per gram fresh weight in the heat and drought-treated grafts and 6 nanograms per gram fresh weight in the cool and wet-treated grafts. The stems contained no detectable putative GA₉ conjugate. Free GA₉ was highest in heat and drought-treated material. For plants subjected to this treatment, GA₉ increased from 22 to 32 nanograms per gram fresh weight in needles and from 1 to 22 nanograms per gram fresh weight in stems during the rapid stem elongation phase. By day 17, after cessation of shoot elongation, GA₉ had decreased to 12 nanograms per gram fresh weight in needles and 9 nanograms per gram fresh weight in the shoot stems. The cool and wet-treated material also showed an increase in GA₉ concentration during shoot elongation. However, the concentration was not as high and was also delayed compared with heat and drought-treated material. By day 17, after cessation of shoot elongation, GA₉ concentration was 9 nanograms per gram fresh weight in needles and 5 nanograms per gram fresh weight in stems for cool and wet treatment plants. The concentration of GA₄ was very low in tissue from both treatments. Fluctuation in concentration of the more polar gibberellins, GA₁ and GA₃, showed the same pattern as fluctuations in the content of GA₉. However, the heat and drought-treated material had lower amounts of GA₁ and GA₃ during the later phases of shoot elongation, than the cool and wet-treated material. These results imply differential metabolism between clones treated with conditions inductive and noninductive for flowering. Higher concentrations of putative GA₉ conjugate and free GA₉ in the hot and dry treatment indicate a higher capacity of synthesizing, for flowering, the physiologically important GA₄ in the heat and drought-treated material. This synthesis does not, however, result in a buildup of the GA₄ pool, probably because of a high turnover rate of GA₄. The cool and wet-treated material had higher amounts of GA₁ and GA₃, indicating that the differentiation was preferentially directed toward vegetative growth.     

Synthesis and coding properties of poly(c 1 A), poly(c 3 A), poly(c 7 A) and poly(h 6 A)

The homopolynucleotides poly(c¹A), poly(c³A), poly(c⁷A) and poly(h⁶A)⁺⁺ were synthesized from their corresponding nucleoside diphosphates using polynucleotide phosphorylase. With the exception of poly(h⁶A), which displayed no hypochromicity, the homopolynucleotides showed melting profiles similar to poly(A). All these polynucleotides, poly(h⁶A), poly(c⁷A), poly(c³A) and poly(c¹A) stimulated the binding of Lys-tRNA to ribosomes; the coding activity of poly(c¹A), however, was very low. Poly(h⁶A) was found to be less specific for Lys-tRNA than poly(A). The data supports the exclusive formation of Watson-Crick type base pairs and contradicts Hoogsteen base pairing in codon-anticodon recognition. Since, however, poly(h⁶A), which can form only one hydrogen bridge per base pair, stimulated the binding of Lys-tRNA comparably to poly(A), the coding activity of the homopolynucleotides tested is discussed in respect to their secondary structure as well as to the pK-values of their 6-amino groups.     

Inhibitors from Carob (Ceratonia siliqua L.): II. Effect on Growth Induced by Indoleacetic Acid or Gibberellins A(1), A(4), A(5), and A(7)

Two inhibitory fractions (B₁ and C) from extracts of immature fruit of carob were tested for their ability to inhibit the action of indoleacetic acid (IAA) in three bioassays. There was no reduction of IAA-induced reactions in the Avena curvature test, abscission of debladed coleus petioles, or growth of cucumber hypocotyls. The highest ratio of inhibitor to IAA was 10,000 times greater than the ratio necessary to inhibit by 50% the growth caused by an equivalent amount of gibberellin A₃ in pea seedlings. At the highest concentration used, fraction C alone caused curvature of Avena coleoptiles. The inhibitory fractions appeared to enhance the effect of IAA in the cucumber test.     

Phospholipase A(2)

Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.     

Chimpanzee lipoprotein(A): Relationship between apolipoprotein(A) isoform size and the density profile of lipoprotein(A) in animals with different heterozygous apo(A) phenotypes

In a previous study [C. Doucet et al., J. Lipid Res 35:263–270, 1994], we have shown that plasma lipoprotein (a) [Lp(a)] levels were significantly elevated in a population of unrelated chimpanzees as compared to those in normolipidemic human subjects. Nonetheless, the inverse correlation between Lp(a) levels and apolipoprotein (a) [apo(a)] isoforms typical of man was maintained in the chimpanzee. In the present study, we describe the density profiles of apo B- and apo A1-containing lipoproteins and of Lp(a) in chimpanzee plasmas heterozygous for apo(a) isoforms after fractionation by single spin ultracentrifugation in an isopycnic gradient. The distribution of apo(a) isoforms in the density gradient was also examined by SDS-agarose gel electrophoresis and immunoblotting using chemiluminescence detection. In all double-band phenotypes examined, the smallest isoform was present along the entire length of the density gradient. The density distribution of the second isoform varied according to the size difference between the respective isoforms. Two isoforms close in size (difference in apparent molecular mass ? 60 kDa) were present together in every gradient subfraction. On the contrary, when the two isoforms displayed distinct molecular mass (maximal difference in apparent molecular mass = 340 kDa), then the largest was principally present in the densest fractions of the gradient (d > 1.1 mg/ml). These observations suggest that Lp(a) particles with small apo(a) isoforms are more susceptible to interact with other lipoproteins than are Lp(a) particles with large isoforms.     

PHYLOGENETIC POSITION OF SYMBIODINIUM (DINOPHYCEAE) ISOLATES FROM TRIDACNIDS (BIVALVIA), CARDIIDS (BIVALVIA), A SPONGE (PORIFERA), A SOFT CORAL (ANTHOZOA), AND A FREE-LIVING STRAIN

The genus Symbiodinium is the commonly observed symbiotic dinoflagellate (zooxanthellae) that forms mutual associations with various marine invertebrates. Numerous studies have revealed that the genus is comprised of a group of diverse taxa, and information on the phylogenetic relationships among the genus’ members is increasing. In this study, small subunit (SSU) ribosomal RNA (ssrRNA) gene sequences were determined for 15 more Symbiodinium strains from 12 relatively unstudied host taxa (Indo-Pacific tridacnids, cardiids, sponge, and soft coral), 1 hitherto unreported free-living Symbiodinium strain, and 4 other Symbiodinium strains from four other host taxa (Indo-Pacific zoanthid, foraminifer, jellyfish, and mid-Pacific hard coral). Their respective phylogenetic positions were inferred, and strains that are either closely related to or distinct from previously reported Symbiodinium taxa were revealed. The cultured Symbiodinium strains isolated from individuals of six species of tridacnids and three species of cardiids all had identical ssrRNA gene sequences, are closely related to S. microadriaticum Freudenthal, and are indistinguishable from the RFLP Type A strain previously reported. However, the ssrRNA gene sequences of clam symbionts that were obtained via gene cloning were different from those of the cultured isolates and represent strains that are close to the RFLP Type C strains. The Symbiodinium-like dinoflagellate from the Indo-Pacific sponge Haliclona koremella De Laubenfels is distinct from any of the Symbiodinium taxa studied and may be similar to the symbiont previously isolated from the stony coral Montipora patula Quelch. The isolates from the soft coral Sarcophyton glaucum Quoy et Gaimard and from the zoanthid Zoanthus sp. are both very closely related to S. pilosum Trench et Blank. The free-living Symbiodinium isolate is very closely related to the symbiont isolated from the Indo-Pacific foraminifer Amphisorus hemprichii Ehrenberg, which in turn is distinct from the Red Sea strain isolated from a similar host. Theisolate from Cassiopeia sp. is different from S. microadriaticum F., the type species harbored by Cassiopeia xamachana Bigelow, and is instead very closely related to S. pulchrorum Trench isolated from a sea anemone. The symbiont from the stony coral M. verrucosa Lamarck is a sister taxon to the symbionts isolated from the foraminifera Marginopora kudakajimensis Gudmundsson and Sorites orbiculus Forskål. These data suggest that polymorphic symbioses extend from cnidarians to some bivalve, foraminifer, and jellyfish host species.     

Poly(A) polymerase from Vigna unguiculata seedlings. A bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63,000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity.     

A Co(III) derivative of concanavalin A

Co(III) has been stoichiometrically incorporated into jack bean concanavalin A. The Co(III) protein still possesses a binding site for an additional divalent transition metal ion which together with Ca(II) can induce the sugar binding ability. No H₂O₂ oxidation of Co(II) occurs with demetallized concanavalin A activated with Ca(II) and Co(II) unless Co(II) is present in a stoichiometric excess. Evidence is presented to indicate that kinetically stable Co(III) is bound to a completely different location than the thermodynamically stable Co(II) protein site.     

The discovery and synthesis of novel adenosine receptor (A(2A)) antagonists

In high throughput screening of our file compounds, a novel structure 1 was identified as a potent A(2A) receptor antagonist with no selectivity over the A1 adenosine receptor. The structure-activity relationship investigation using 1 as a template lead to identification of a novel class of compounds as potent and selective antagonists of A(2A) adenosine receptor. Compound 26 was identified to be the most potent A(2A) receptor antagonist (Ki = 0.8 nM) with 100-fold selectivity over the A1 adenosine receptor.     

A novel nuclear human poly(A) polymerase (PAP), PAP gamma.

Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. Here we show that the 90-kDa isoform is the product of the gene PAPOLG, which is distinct from the previously identified genes for poly(A) polymerases. The 90-kDa isoform is referred to as human PAP gamma (hsPAP gamma). hsPAP gamma shares 60% identity to human PAPII (hsPAPII) at the amino acid level. hsPAP gamma exhibits fundamental properties of a bona fide poly(A) polymerase, specificity for ATP, and cleavage and polyadenylation specificity factor/hexanucleotide-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of hsPAPII. Mutational analysis and sequence comparison revealed that hsPAP gamma and hsPAPII have similar organization of structural and functional domains. hsPAP gamma contains a U1A protein-interacting region in its C terminus, and PAP gamma activity can be inhibited, as hsPAPII, by the U1A protein. hsPAPgamma is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on this and previous studies, it is obvious that multiple isoforms of PAP are generated by three distinct mechanisms: gene duplication, alternative RNA processing, and post-translational modification. The exclusive nuclear localization of hsPAP gamma establishes that multiple forms of PAP are unevenly distributed in the cell, implying specialized roles for the various isoforms.     

Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L

[17-¹³C,³H]-Labeled gibberellin A₂₀ (GA₂₀), GA₅, and GA₁ were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). ¹³C-Labeled GA₂₉, GA₈, GA₅, GA₁, and 3-epi-GA₁, as well as unmetabolized [¹³C]GA₂₀, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-¹³C, ³H]GA₂₀ to all three genotypes. ¹³C-Labeled GA₈ and 3-epi-G₁, as well as unmetabolized [¹³C]GA₁, were identified by GC-SIM from feeds of [17-¹³C, ³H]GA₁ to all three genotypes. From feeds of [17-¹³C, ³H]GA₅, ¹³C-labeled GA₃ and the GA₃-isolactone, as well as unmetabolized [¹³C]GA₅, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-¹³C, ³H]GA₅ to [¹³C]GA₁, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA₂₀, as follows: (a) GA₂₀ → GA₁ → GA₈; (b) GA₂₀ → GA₅ → GA₃; and (c) GA₂₀ → GA₂₉. The in vivo biogenesis of GA₃ from GA₅, as well as the origin of GA₅ from GA₂₀, are conclusively established for the first time in a higher plant (maize shoots).     

Poly(A): A new evolutionary probe

The poly(A) segment increases in length as the complexity of the organism increases. The shortest poly(A) size (10–15 AMP units) exist on mRNA from bacteria. The largest poly(A) segments (180–200 AMP units) exist in highly differentiated tissue. The natural log of average poly(A) size has evolved in a linear manner with the time of evolution of different organisms. This relationship places the origin of mRNA, and possibly life, at 3·85 ± 0.2 billion years ago. The poly(A) length of mitochondrial mRNA places the origin of mitochondria within eucaryotes at 2· 1 billion years ago.     

Expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in HL-60 cells: dependence on cell cycle phases

The present studies investigated changes in expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in samples of HL-60 promyelocytic cells differing in the actual presence of cells in various phases of the cell cycle induced by the double thymidine block method. Real-time PCR technique was used for obtaining data on mRNA expression. Statistical analysis of the data revealed that the mRNA expression of adenosine A(1), A(2a), and A(3) receptors is dependent on the cell cycle phase. G(0)/G(1) and G(2)/M phases were characterized by a higher mRNA expression of adenosine A(1) receptors and a lower one of adenosine A(2a) and A(3) receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A(2b) receptors was independent on the cell cycle phase. The results indicate the plasticity of mRNA expression of adenosine receptors in the investigated promyelocytic cells and its interaction with physiological mechanisms of the cell cycle.     

(A1-D-alanine) insulin (author's transl)]

Starting from porcine insulin, A1-glycine was substituted by D-alanine and by L-alanine for comparison. Replacement of A1-glycine by L-alanine revealed the known decrease in the biological activity. [A1-D-Alanine]insulin, however, has the same blood sugar lowering activity as insulin and is slightly more active in its influence on the glucose uptake into the rat diaphragm. The specific binding to insulin receptors of rat liver is decreased as, compared to insulin, but increased as compared to the L-alanine analogue.