Trans splicing of mRNA precursors in vitro

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.     

Potent inhibition of mammalian progesterone synthesis by 2 alpha-cyanoprogesterone.

2 alpha-Cyanoprogesterone potently inhibits the conversion of [3H]pregnenolone into progesterone catalysed by bovine corpora lutea, bovine adrenal cortex and human term placenta microsomes (microsomal fractions), yielding IC50 (concentration causing 50% inhibition) values of 66 nM, 120 nM and 700 nM respectively. By contrast, it is an exceedingly poor inhibitor of the isomerization of pregn-5-ene-3,20-dione, yielding IC50 values between 50 and 70 microM. On this basis, 2 alpha-cyanoprogesterone would appear to be an extraordinarily selective inhibitor of the 3 beta-hydroxysteroid dehydrogenase. Dixon plots indicate that it is a very-tight-binding competitive inhibitor of the corpus-luteum enzyme, yielding a Ki of 15 nM. In the bovine adrenal cortex and human placenta the steroid is less potent and inhibits the dehydrogenase non-competitively with Ki values of 150 nM and 1.0 microM respectively. Thus 2 alpha-cyanoprogesterone inhibits the corpus-luteum dehydrogenase with substantial selectivity. Because of its high affinity for the ovarian enzyme, the presence of low-micromolar concentrations of 2 alpha-cyanoprogesterone can promote a complete cessation of progesterone synthesis in corpora-lutea microsomes for several hours. Since this effect is observed in the presence of saturating concentrations of pregnenolone (50 microM), it is predicted that this inhibitor may be even more potent in vivo. 2 alpha-Cyanoprogesterone displays very low affinity for the human progesterone receptor, yielding a Kd of 600 nM as against a Kd of 1.6 nM for progesterone. It is suggested that 2 alpha-cyanoprogesterone may be a selective inhibitor of ovarian progesterone synthesis and may act as an effective anti-gestational agent in vivo.     

Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

Aspects of the neuroendocrine control of ovulation and broodiness in the domestic hen

The neuroendocrine control of ovulation and broodiness in the domestic hen involves complex interactions between hypothalamic neuropeptides, neurotransmitters, and ovarian steroids which regulate the secretion of luteinizing hormone (LH) and prolactin. Nuclear progesterone receptor is localized in many neurons throughout the hypothalamus but is absent from LHRH neurons. Hence, the positive feedback action of progesterone on LH release is not mediated by a genomic mechanism within the LHRH neuron. Precursors of 5-hydroxytryptamine (5HT) and dopamine (DA) inhibit the preovulatory release of LH, while the turnover rates of these neurotransmitters in the anterior hypothalamus decrease when preovulatory levels of LH are at their highest. Further, a population of receptors for 5HT which occurs in the anterior hypothalamus in laying birds is absent in nonlaying, incubating hens. Taken together, these observations suggest that the preovulatory surge of LH is mediated by a transitory decrease in the inhibitory action of 5HT and possibly DA, on the secretion of LHRH. Neurons containing 5HT may play a role in the regulation of prolactin release and, more specifically, in the control of broodiness. Drugs which enhance the function of 5HT neurons stimulate prolactin release while increased prolactin secretion in incubating hens is associated with an increase in the turnover of 5HT in the anterior hypothalamus. No receptors for 5HT were demonstrable in the anterior pituitary gland, showing that the prolactin-releasing activity of 5HT must be mediated by a prolactin-releasing factor (PRF). A candidate for a physiological PRF is vasoactive intestinal polypeptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs

Splicing complexes were analyzed by electrophoresis on a native low-percentage polyacrylamide gel. Two distinct heparin-resistant complexes, A and B, are assembled specifically on an RNA precursor containing authentic 5' and 3' splice sites. This assembly is ATP-dependent. Kinetic experiments suggest that complex A is converted with time to a larger, slower migrating complex B. Complexes A and B detected by gel electrophoresis correspond to material sedimenting at 25S and 35S, respectively. Substrate RNA containing only the 3' splice site is capable of forming the smaller complex A but not complex B. Complex A protects sequences upstream of the 3' splice site, encompassing the branch site and polypyrimidine tract from digestion by RNAase T1. U2 snRNA, but not U1 snRNA was detected in both complexes A and B by Northern hybridization analysis. Interestingly, an endogenous large complex containing U2 snRNP could be detected in nuclear extracts.     

Detection of a homologous series of C26-C38 polyenoic fatty acids in the brain of patients without peroxisomes (Zellweger''s syndrome).

The brains of patients with inherited abnormalities in peroxisomal structure and function contain greatly increased proportions of a homologous series of unique polyenoic fatty acids with carbon chain lengths ranging from 26 to 38. Based on evidence by chemical ionization and electron impact mass spectrometry before and after catalytic hydrogenation, and argentation t.l.c., these lipids have been tentatively identified as 26:5, 28:5, 30:5, 30:6, 30:7, 32:5, 32:6, 32:7, 34:5 and 34:6 fatty acids. A further two fatty acids eluting at very high temperatures from gas chromatography columns have been tentatively identified on the basis of their chemical ionization mass spectra as 36:6 and 38:6 fatty acids.     

The occurrence of polyenoic fatty acids with greater than 22 carbon atoms in mammalian spermatozoa.

Fatty acids with carbon chain lengths greater than 22 (VLCFA) have been detected in boar, ram, bull and human spermatozoa. Saturated and mono-unsaturated fatty acids were present in all spermatozoa but, except for human spermatozoa, polyenoic fatty acids were quantitatively the most important components. Marked differences in polyenoic fatty acid composition were observed. Whereas human spermatozoa contain predominantly di-, tri- and tetraenoic fatty acids with up to 32 carbon atoms, boar, ram and bull spermatozoa also contain pentaenoic and/or hexaenoic acids with up to 34 carbon atoms. Human and boar spermatozoa differ markedly from those of the ram and bull in that only n-6 series acids are present.     

Endogenous opioid peptides: do they mediate the acute antihypertensive action of clonidine in humans?

The involvement of endogenous opioid peptides in the antihypertensive action of acutely administered clonidine, a centrally acting adrenergic agonist, was studied in humans. Eight hypertensive subjects received clonidine 0.2 mg orally, naloxone 8 mg i.v. followed by a 0.13 mg/min infusion, and both drugs together on separate days. Clonidine resulted in a significant decrease in mean blood pressure, which was not affected by concomitant treatment with naloxone. Naloxone alone or with clonidine caused significant elevations in plasma aldosterone, not mediated by increased plasma renin activity. Plasma beta-endorphin was not increased after clonidine administration. In humans, the antihypertensive effects of acute clonidine administration do not appear to be mediated by the release or action of endogenous opioids.     

Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes.

Codon usage data has been compiled for 110 yeast genes. Cluster analysis on relative synonymous codon usage revealed two distinct groups of genes. One group corresponds to highly expressed genes, and has much more extreme synonymous codon preference. The pattern of codon usage observed is consistent with that expected if a need to match abundant tRNAs, and intermediacy of tRNA-mRNA interaction energies are important selective constraints. Thus codon usage in the highly expressed group shows a higher correlation with tRNA abundance, a greater degree of third base pyrimidine bias, and a lesser tendency to the A+T richness which is characteristic of the yeast genome. The cluster analysis can be used to predict the likely level of gene expression of any gene, and identifies the pattern of codon usage likely to yield optimal gene expression in yeast.     

Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.