EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila.

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms--in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3' untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.     

Trans-inactivation of the Drosophila brown gene: evidence for transcriptional repression and somatic pairing dependence

Position-effect variegation in Drosophila is the variable inactivation of a gene that occurs when it is juxtaposed to heterochromatic regions of chromosomes. The brown gene, required for pteridine pigment in the eye, is unusual in that expression of the unrearranged homolog also is affected. This dominant effect can be very strong, as inactivation is detectable when as many as three trans copies of the gene are present. We show that pteridine reductions coincide with similar reductions in the accumulation of brown mRNA. The dominant effect is suppressed by certain altered structural configurations of the brown region, suggesting that somatic pairing is involved in the phenomenon. We propose that direct transmission of the altered chromatin structure characteristic of position-effect variegation (heterochromatinization) occurs between paired homologs in the region of the brown locus.     

A gene adjacent to satellite DNA in Drosophila melanogaster.

Several copies of a sequence adjacent to 1.688 g/cm3 satellite DNA in the Drosophila melanogaster genome have been isolated by molecular cloning. This sequence, called the Dm142 gene, is homologous to a 1.6-kilobase RNA found in both D. melanogaster embryos and tissue culture cells. One cloned DNA segment includes two copies of the Dm142 gene and 1.688 g/cm3 satellite DNA sequences, which are located between and flanking both gene copies. The Dm142 gene is repeated many times in the D. melanogaster genome, and some copies are not flanked by 1,688 g/cm3 satellite DNA.     

Translation of mRNAs with degenerate initiation triplet AUU displays high initiation factor 2 dependence and is subject to initiation factor 3 repression.

The influence of the rare initiation triplet AUU on mRNA translation was investigated by comparing the activity of two pairs of model mRNAs that differ in the length of Shine-Dalgarno and spacer sequences. Irrespective of the initiation triplet (AUG or AUU), all mRNAs had similar template activity in vitro, but translation of AUU mRNAs depended more on initiation factor (IF) 2 and less on IF3 than that of AUG mRNAs. Increasing the IF3/ribosome ratio from 2 to 10 progressively inhibited the AUU mRNAs and abolished their capacity to compete for translating ribosomes with other mRNAs but did not affect activity of the AUG mRNAs. The effects induced by IF3 are from its different influence on on- and off-rates of the transition 30S preinitiation complex<==>30S initiation complex; depending on the nature of the initiation triplet (AUG or AUU) of the mRNA, IF3 shifts the position of equilibrium toward binding or dissociation of fMet-tRNA, respectively. Stimulation of fMet-tRNA binding and dissociation yields superimposable IF3 titration curves that saturate at an IF3/30S ratio of approximately 1, indicating that the data are from the interaction of one molecule of IF3 with the same 30S binding site. Both effects are either lost or strongly reduced with 30S mutants defective in IF3 binding. Translational repression of AUU mRNAs by IF3 is from the factor-dependent dissociation of fMet-tRNA from 30S subunits, which becomes relevant when excess IF3 interferes with the formation of 70S initiation complex, presumably by interacting with 50S subunit.     

葡萄糖激酶基因启动子G/A变异与空腹高血糖相关

目的　研究葡萄糖激酶 (GCK)基因启动子 - 30G/A变异在中国南方地区汉族人群中的分布并比较其在正常糖耐量 (NGT)者及糖耐量低减 (IGT)者中的差别及其与糖代谢相关指标之间的关系。方法　研究对象为 4 4 4例无糖尿病及亲缘关系的中国南方地区汉族人 ,其中 2 2 2例为NGT ,2 2 2例为IGT。通过PCR RFLP检测GCK启动子 - 30位置的G→A变异所导致BsiHKAⅠ酶切位点的缺失。结果　在IGT及NGT的人群中含有A等位基因的频率分别为 18.2 %及 19.8%。与GG基因型的人群相比 ,带有A等位基因的人群空腹血糖显著增高〔(5 .35± 0 .5 9vs 5 .19± 0 .5 3)mmol/L ,P =0 .0 0 6〕 ,在IGT的人群中 ,带有A等位基因的人群空腹血糖仍显著增高〔(5 .5 4± 0 .5 8vs 5 .35± 0 .5 2 )mmol/L ,P =0 .0 2〕 ,而在NGT的人群中 ,不同GCK基因型者的空腹血糖差异无显著性。结论GCK基因启动子 - 30G/A变异与空腹高血糖水平相关 ,可能对中国人的IGT有一定的影响。     

Immunologic evidence that the gene for L-gulono-gamma-lactone oxidase is not expressed in animals subject to scurvy.

L-Gulono-gamma-lactone oxidase (L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8) is the enzyme that catalyzes the terminal step of L-ascorbic acid biosynthesis in mammalian liver. The absence of the oxidase activity in primates and guinea pigs is the reason why these animals are subject to scurvy, which must be considered an inborn error of metabolism. Attempts were made to determine if a protein immunologically crossreactive with L-gulono-gamma-lactone oxidase is present in these animals. Detergent-solubilized microsomal preparations from guinea pig and African green monkey liver did not precipitate the antisera directed to either rat or goat enzyme, nor did any of the other cell fractions obtained from guinea pig liver react with either antiserum. No crossreactive protein was detectable in guinea pig microsomes even with the sensitive procedure or micro-complement fixation. On the other hand, extracts of all 10 other mammalian (4 orders) liver microsomes tested were shown to contain L-gulono-gamma-lactone oxidase activity that did crossreact with antibodies to the rat and goat enzymes. One explanation of these findings is that, in the guinea pig, and perhaps in primates too, the structural gene for L-gulono-gamma-lactone oxidase is not expressed.     

The slowpoke gene is necessary for rapid ethanol tolerance in Drosophila

BACKGROUND: Ethanol is one of the most commonly used drugs in the world. We are interested in the compensatory mechanisms used by the nervous system to counter the effects of ethanol intoxication. Recently, the slowpoke BK-type calcium-activated potassium channel gene has been shown to be involved in ethanol sensitivity in Caenorhabditis elegans and in rapid tolerance to the anesthetic benzyl alcohol in Drosophila. METHODS: We used Drosophila mutants to investigate the role of slowpoke in rapid tolerance to sedation with ethanol vapor. Rapid tolerance was defined as a reduction in the sedative phase caused by a single previous sedation. The ethanol and water contents of flies were measured to determine if pharmacodynamic changes could account for tolerance. RESULTS: A saturated ethanol air stream caused sedation in <20 min and resulted in rapid tolerance that was apparent 4 hr after sedation. Two independently isolated null mutations in the slowpoke gene eliminated the capacity for tolerance. In addition, a third mutation that blocked expression specifically in the nervous system also blocked rapid tolerance. Water measurements showed that both ethanol and mock sedation caused equivalent dehydration. Furthermore, a single prior exposure to ethanol did not cause a change in the ethanol clearance rate. CONCLUSIONS: Rapid tolerance, measured as a reduction in the duration of sedation, is a pharmacokinetic response to ethanol that does not occur without slowpoke expression in the nervous system in Drosophila. The slowpoke channel must be involved in triggering or producing a homeostatic mechanism that opposes the sedative effects of ethanol.