ISSN:0192-253X    

DOI:10.1002/dvg.1020140602

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe developmentally regulated inhibitor of eIF‐4 function found in unfertilized sea urchin eggs has been partially purified and its mechanism of action studied in vitro using purified recombinant eIF‐4α and cell‐free translation systems. The results demonstrate that although the phosphorylation of eIF‐4α is necessary to promote protein synthesis, it is not sufficient to maintain all aspects of eIF‐4 function. The egg inhibitor does not change eIF‐4α phosphorylation state. During the blockage of initiation caused by the egg inhibitor, eIF‐4α remains phosphorylated but accumulates in a 48S initiation intermediate. This suggests that the egg inhibitor functions by preventing the release of eIF‐4α from the small ribosomal subunit. The characteristics of the inhibitor in a reticulocyte translation system demonstrate that eIF‐4 activity is inhibited within 3–6 min. However, the inhibitor's characteristics in a mRNA‐dependent translation system contrast with this. Preincubation with the inhibitor for 5–25 min prior to the addition of mRNA does not prevent endogenous eIF‐4 from participating in translation but diminishes its ability to be reutilized, consistent with the accumulation of eIF‐4α on the small ribosomal subunit. The ribosomal localization of the inhibitor suggests that it could prevent eIF‐4α release by direct binding. The gradual inactivation of the inhibitor following fertilization indicates that it represents a component of a novel regulatory cascade that modulates eI

ISSN:0192-253X    

DOI:10.1002/dvg.1020140603

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe stimulation of translation in starfish oocytes by the maturation hormone, 1‐methyladenine (1‐MA), requires the activation or mobilization of both initiation factors and mRNAs [Xu and Hille, Cell Regul. 1:1057, 1990]. We identify here the translational initiation complex, eIF‐4F, and the guanine nucleotide exchange factor for eIF‐2, eIF‐2B, as the rate controlling components of protein synthesis in immature oocytes of the starfish,Pisaster orchraceus.Increased phosphorylation of eIF‐4E, the cap binding subunit of the eIF‐4F complex, is coincident with the initial increase in translational activity during maturation of these oocytes. Significantly, protein kinase C activity increased during oocyte maturation in parallel with the increase in eIF‐4E phosphorylation and protein synthesis. An increase in the activities of cdc2 kinase and mitogen‐activated myelin basic protein kinase (MBP kinase) similarly coincide with the increase in eIF‐4E phosphorylation. However, neither cdc2 kinase nor MBP kinase phosphorylates eIF‐4E in vitro. Casein kinase II activity does not change during oocyte maturation, and therefore, cannot be responsible for the activation of translation. Treatment of oocytes with phorbol 12‐myristate 13‐acetate, an activator of protein kinase C, for 30 min prior to the addition of 1‐MA resulted in the inhibition of 1‐MA‐induced phosphorylation of eIF‐4E, translational activation, and germinal vesicle breakdown. Therefore, protein kinase C may phosphorylate eIF‐4E, after very early events of maturation. Another possibility is that eIF‐4E is phosphorylated by an unknown kinase that is activated by the cascade of reactions stimulated by 1‐MA. In conclusion, our results suggest a role for the phosphorylation of eIF‐4E in the activation of translation during maturation, similar to translational regulation during the stimulation of growth i

ISSN:0192-253X    

DOI:10.1002/dvg.1020140604

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIn the amphibianXenopus laevis, the elongation factor 1α proteins (EF‐1α) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF‐1α gene (EF‐1αS) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF‐1α (conversion of EF‐1α‐GDP to EF‐1α‐GTP) is assured by the EF‐1βγ complex. We show here that inXenopus laevisembryos, contrary to the situation for EF‐1α, EF‐1β, and EF‐1γ mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF‐1β and EF‐1γ genes from the zygotic gencme occurs several hours after that of the somatic EF‐1αS gene. Therefore, during earlyXenopusdevelopment the expression of these three elongation factors is not co‐ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the earlyXenop

ISSN:0192-253X    

DOI:10.1002/dvg.1020140605

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have previously identified a 12 nucleotide long sequence element, the TCE, that was demonstrated to be necessary for translational control of expression in the male germ line ofDrosophila melanogaster(Schäferet al., 1990). It is conserved among all seven members of theMst(3)CGPgene family, that encode structural proteins of the sperm tail. The TCE is invariably located in the 5′ untranslated region (UTR) at position + 28 relative to the transcription start site. In this paper we analyse the mode of action of this element. We show that protein binding occurs at the TCE after incubation with lestis protein extracts fromDrosophila melanogaster.While several proteins are associated with the translational control element in the RNA, only one of these proteins directly crosslinks to the sequence element. The binding activity is exclusively observed with testis protein extracts but can be demonstrated with testis extracts from otherDrosophilaspecies as well, indicating that regulatory proteins involved in translational regulation in the male germ line are conserved. Although binding to the TCE can occur independent of its position relative to the transcription start site of the in vitro transcripts, its function in vivo is not exerted when shifted further downstream within the 5′ UTR of a fusion gene. In addition to being a translational control element the TCE also functions as a transcriptional regulator. Consequently, a DNA‐protein complex is also formed at the TCE. In contrast to the RNA‐protein complexes we find DNA‐protein complexes with protein extracts of several tissues ofDrosophila melanogaster.© 1993 Wile

ISSN:0192-253X    

DOI:10.1002/dvg.1020140606

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractInChlamydomonas rein‐hardtii, flagellar amputation stimulates an induction in the synthesis of flagellar proteins which allows the cells to rapidly regenerate their flagella. The induction involves the coordinate accumulation and rapid degradation of a large number mRNAs, including those encoding the tubulins. The post‐induction degradation of induced tubulin mRNAs has been shown to differ from the consti‐tutive turnover pathway in two ways: (1) the rate of degradation is accelerated, and (2) degradation is prevented by inhibition of protein synthesis. In this report, it is shown that the post‐induction degradation of all deflagellation‐induced mRNAs examined is prevented by cycloheximide (CX), suggesting they all may be degraded via the same pathway. A cell‐free decay system has been developed to investigate the degradation pathway. At least two characteristics of tubulir mRNA degradation are reproducible in these extracts: (1) endogenous α‐tubulin mRNA is less stable than constitutive mRNAs in the same extract and (2) α‐tubulin mRNA in extracts prepared from CX‐treated cells (CX ex‐tracts) is significantly more stable than it is in extracts from untreated cells (control extracts). This indicates that the mechanism by which CX blocks rapid degradation of tubulin mRNA in vivo is not simply by preventing its translation and suggests the involvement of an alteredtrans‐factor. The difference in tubulin mRNA stability in the two extracts is maintained when the extracts are prepared under conditions that dissociate ribosomes from mRNPs, indicating intact polysome structure is not necessary. Tubulin mRNA‐containing polysomes isolated from control and CX extracts are equally stable when assayed alone. However, the poly‐somes from control extracts are more sensitive to exogenous RNAse treatment than are those from CX extracts, indicating a structural difference. There are no detectable differences in soluble factors that influence tubulin mRNA degradation rate between control and CX extracts; addition of excess soluble factors to either control or CX extracts does not alter the tubulin mRNA degradation in the extract, nor does a simple one‐to‐one combination of the two extracts result in stabilization or destabilization of the whole population of tubulin mRNAs in the mixture. The deflagellation‐induced mRNAs, as a group, are shown to be particularly susceptible to a nuclease activity in extracts, inhibitable by vanadyl ribonucleoside complexes, which does not appear to attack constitutive mRNAs. It is proposed that a structural difference in the tubulin mRNPs produced in the presence and absence of CX underlies their differences in stabilities, and that a common nuclease targets the induced flagellar prot

ISSN:0192-253X    

DOI:10.1002/dvg.1020140607

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTheCaenorhobditis elegansXX animal possesses a hermaphrodite germ line, producing first sperm, then oocytes. In this paper, we report the genetic identification of five genes,mog‐2, mog‐3, mog‐4, mog‐5, andmog‐6, that influence the hermaphrodite switch from sper‐matogenesis to oogenesis. Inmcg‐2‐mog‐6mutants, spermatogenesis continues past the time at which hermaphrodites normally switch into oogenesis and no oocytes are observed. Therefore, in these mutants, germ cells are transformed from a female fate (oocyte) to a male fate (sperm). Thefem‐3gene is one of five genes that acts at the end of the germline sex determination pathway to direct spermatogenesis. Analyses ofmog;fem‐3double mutants suggest that themog‐2‐mog‐6genes act beforefem‐3;thus these genes may be in a position to negatively regulatefem‐3or one of the other terminal regulators of germline sex determination. Double mutants offem‐3and any one of themogmutations make oocytes. Using these double mutants, we show that oocytes from anymog;fem‐3double mutant are defective in their ability to support embryogenesis. This maternal effect lethality indicates that each of themoggenes is required for embryogenesis. The two defects inmog‐2‐mog‐6mutants are similar to those ofmog‐1: all sixmoggenes eliminate the sperm/oocyte switch in hermaphrodites and cause maternal effect lethality. We propose that themog‐2‐mog‐6mutations identify genes that act withmog‐1to effect the sperm/oocyte switch. We further speculate that themog‐1‐mog‐6mutations all interfere with translational control

ISSN:0192-253X    

DOI:10.1002/dvg.1020140608

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractFertilization ofUrechis coupooocytes stimulates dramatic changes in the pattern of protein synthesis. This shift is brought about entirely through selective translation of the large pool of maternal mRNAs synthesized and stored during oogenesis. My laboratory has identified cDNA clones to more than 20 differentUrechismaternal mRNAs. These have been used to determine whether the complementary mRNAs are translated in oocytes or embryos, and to analyze the polyad‐enylation status of the mRNAs at different stages. For 14 of the mRNAs, multiple, overlapping cDNA clones were isolated, and the complete sequence of the mRNA molecule was determined. Of these 14 mRNAs, half are from the subset that is translated in growing and full‐grown oocytes, but not in embryos. These 7 mRNAs have poly(A) tails before fertilization. The other 7 are from the subset that is not translated at any time before fertilization, and has very short poly(A) tails in oocytes. After fertilization these mRNAs are recruited onto polysomes and extensively polyadenylated. The sequence data from the two classes of maternal mRNAs was compared in an attempt to identify consensus sequences that could regulate translation directly, or indirectly, by controlling polyadenylation or secondary structure formation. Two features of the sequences correlate very well with the translation and polyadenylation of the different mRNAs‐the identity of the base immediately preceding the AUG start codon, and the presence of the sequences UUUUA and UUUUUA in the 3′ untranslated region. © 1993 Wiley

ISSN:0192-253X    

DOI:10.1002/dvg.1020140609

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractDuring meiotic maturation ofSpisulaoocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA‐specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle‐otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence‐specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu‐cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis‐acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993

ISSN:0192-253X    

DOI:10.1002/dvg.1020140610

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWnt‐1andWnt‐3aproto‐on‐cogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showingWnt‐1and‐3aexpression in developing cranial and spinal cord regions and from studies of mutant mice whoseWnt‐1genes have undergone targeted disruption by homologous recombination.Wnt‐1null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of theWnt‐1deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation ofWnt‐1expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid‐ and hindbrain abnormalities as those seen in theWnt‐1null mutant mice. Attentuation ofWnt‐1expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset ofWnt‐1expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout ofWnt‐3a, a gene structurely related toWnt‐1, targeted the forebrain and midbrain region, which were hy‐poplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts ofWnt‐1andWnt‐3atargeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20‐25 nu‐cleotides) make this antisense oligonucleotide/ whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embr

ISSN:0192-253X    

DOI:10.1002/dvg.1020140611

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY