Xenopus laevis serum albumins are encoded in two closely related genes.

cDNA clones containing sequences complementary to Xenopus laevis albumin mRNA have been identified in a collection of cDNA clones made from poly(A)+ RNA prepared from male Xenopus laevis liver. Although all the albumin cDNA clones crosshybridise, restriction enzyme and heteroduplex analysis show that there are 2 closely related albumin mRNA sequences. The 2 albumin mRNAs are only mismatched by 8% but could be isolated by positive selection using stringent hybridization conditions. Oocytes injected with the 2 purified mRNAs, secreted either the 68,000 or 74,000 dalton albumin into the culture medium showing that the 2 albumins of X. laevis serum are encoded in the 2 closely related mRNAs. Measurements of the abundance of albumin mRNA show that the 2 albumin mRNAs together account for about 9% of total poly(A)+ RNA in male Xenopus laevis liver but the mRNA coding for the 74,000 dalton mRNA is about twice as abundant as that coding for the 68,000 dalton mRNA.     

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20-80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.     

Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.     

Nature of albumin exported from the frog oocyte following microinjection of mouse liver mRNA

Microinjections of mouse liver mRNA into Xenopus laevis oocytes induced efficient export of a polypeptide with an apparent Mr or 68,000 which was immunoprecipitable with anti-mouse albumin antibody. Analysis of the anti-albumin precipitate of the exported protein by two-dimensional gel electrophoresis showed that the electrophoretic behavior precisely coincided with that of authentic mouse serum albumin. This result indicates that the Xenopus oocyte may perform secretory processes similar or identical to those occurring in liver cells with respect to the processing of albumin.     

Estrogen dramatically decreases albumin mRNA levels and albumin synthesis in Xenopus laevis liver

The levels of albumin mRNA in Xenopus laevis liver were measured at various times after injection of estradiol using two different methods involving hybridization of cloned albumin cDNA to total liver RNA. The absolute levels of albumin mRNA fell by more than 95% during the first 4 days following estrogen treatment, then slowly returned to normal levels over the following 12 days. Albumin synthesis paralleled the albumin mRNA levels during the first 8 days after injection; but, 16 and 32 days after injection, albumin synthesis again decreased while albumin mRNA remained at normal levels. The time courses of the effects of estrogen on albumin and vitellogenin mRNA levels were different. Whereas albumin mRNA levels were minimal 4 days after estradiol injection, vitellogenin mRNA levels were maximal 8 days after injection.     

Divergence of the Two Albumins of X. laevis

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis , differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Divergence of the two albumins of X. laevis. Evidence for the glycosylation of the major 74K albumin

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis, differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

Expression of functional thrombin receptors in xenopus oocytes injected with human endothelial cell mRNA.

Human endothelial cell thrombin receptors were functionally expressed in Xenopus laevis oocytes by injection of RNA extracted from human umbilical vein endothelial cells. Oocytes injected with endothelial cell RNA responded to thrombin with a Ca2(+)-dependent depolarizing current whose size depended on the amount of RNA injected. In oocytes expressing thrombin receptors, thrombin caused homologous but not heterologous desensitization. Both the catalytic and anion-binding exosites of thrombin were necessary to elicit depolarizing currents. Thus, Xenopus laevis oocytes injected with mRNA from human endothelial cells express Ca2(+)-dependent thrombin receptors which share many common features with thrombin receptors on intact endothelial cells. Xenopus oocytes may, therefore, be used as a screening system in the expression cloning of the endothelial cell thrombin receptor.     

Phorbol ester increases the level of interleukin 2 mRNA in mitogen-stimulated human lymphocytes

TPA alone did not induce the production of IL 2 in human tonsillar lymphocytes but enhanced the PHA-induced IL 2 production by seven-fold. That the effect of TPA was due to an increase in IL 2 mRNA was demonstrated by examining the amount of IL 2 mRNA translatable in Xenopus laevis oocytes, and by Northern blotting analysis using IL 2 cDNA as a probe. In these ways, it was shown that TPA alone did not induce any significant IL 2 mRNA synthesis, but when added together with PHA it increased the level of IL 2 mRNA by at least 10-fold, as compared with that induced by PHA alone.     

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.     

Ribosomal-protein synthesis is not autogenously regulated at the translational level in Xenopus laevis

Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied. For this purpose ribosomal (r) proteins were prepared from X. laevis ribosomal subunits and group fractionated by ion-exchange chromatography. They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins. The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls. Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with [35S]methionine for different times. In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.     

Changes in the Level of Histone H4 mRNA in Xenopus leavies Embryogenesis.

In Xenopus laevis , the change in the amount of histone H4 mRNA per embryo measured by Northern blotting methods follows a unique change during early embryogenesis: It starts to increase first at the blastula stage, doubles by the gastrula stage then decreases considerably at the neurula stage, and then increases again from the tailbud stage on. The present paper establishes these developmental changes, and furthermore, provides evidence that the synthesis of H4 mRNA starts or at least increases to a detectable level at the midblastula stage as shown by S–1 protection analysis of the expression of paternal histone H4 genes in X. borealis (♀) and X. laevis (♂) hybrid embryos.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.     

LiCl disrupts axial development in mouse but does not act through the beta-catenin/Lef-1 pathway

Chimera and cell marking studies suggest that axial determination in mouse embryos occurs at postimplantation stages. In contrast, Xenopus laevis axes are determined early due to the asymmetric distribution of maternally derived factors in the one-cell zygote. In our earlier study we used lithium chloride (LiCl) to perturb development of mouse axes. Here we investigate whether the lithium induced axial defects in mouse are being mediated by the beta-catenin/Lef-1 pathway as in Xenopus laevis. In lithium treated embryos we did not observe any changes in the amount or localization of beta-catenin protein. Furthermore, the lack of Lef-1 mRNA in treated and untreated embryos indicates the LiCl induced axial defects in the mouse are not mediated by the beta-catenin/Lef-1 pathway.