Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA.

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 mM cordycepin for 12 h, concentration of 3'-dATP in cytosol of oocytes increased to 0.7 mM, while that of ATP remained constant at around 1.2 mM. Incorporation of [32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6-8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.     

Primary structure and differential expression of beta-amylase in normal and mutant barleys

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Ris? mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.     

Functional expression of urea channels in amphibian oocytes injected with frog urinary bladder mRNA.

In amphibian urinary bladder epithelium, vasopressin increases passive urea permeability, concomitant with the appearance of a facilitated urea transport. Amphibian oocytes from Xenopus laevis and Rana esculenta were microinjected with total or fractionated poly(A+) RNA isolated from frog urinary bladder epithelial cells. After several (3-5) days at 18 degrees C, the urea flux was assayed by measuring the uptake and efflux of [14C]urea in water-injected and mRNA-injected oocytes. A 2 to 3-fold increase of urea transport was detected in oocytes injected either with total mRNA or with a 6-10 kilobase mRNA fraction, when compared with water-injected oocytes. This expression of urea channels was inhibited by 0.1 mM phloretin (50% inhibition) and 0.1 mM nitrophenylthiourea (up to 70% inhibition). On the contrary, no expression was detected in brain mRNA-injected oocytes. These results show the specific functional expression of the phloretin- and NPTU-sensitive urea channel (or carrier) from frog urinary bladder epithelial cells, providing an approach for the expression cloning of these urea channels.     

Structure of adenosine deaminase mRNAs from normal and adenosine deaminase-deficient human cell lines.

The structure of human adenosine deaminase mRNA from normal and mutant lymphoblasts was examined by sequence analysis of a cDNA for normal mRNA and electrophoretic analyses of DNA fragments generated by S1 endonuclease cleavage of mRNA-cDNA hybrids. The 1,533-base sequence of the cloned cDNA represents the complete mRNA sequence with the possible exception of some of the 5' untranslated region. S1 nuclease analyses of hybrids between cloned cDNA and normal adenosine deaminase mRNA confirmed that a 76-base sequence in a previously examined adenosine deaminase cDNA is an intron. S1 nuclease analyses of mRNAs from seven mutant cell lines demonstrated that four of the mutants, those in the GM-2471, GM-2756, GM-4258, and GM-2606 cells, contain small defects, such as single-base changes, that are not detectable by the S1 nuclease technique. Three of the mRNAs, those in GM-3043, GM-2294, and GM-2825A cells, do contain defects detectable with S1 nuclease. These defects differ from each other and have been mapped to specific regions of the mRNA. Some or all of these defective mRNAs are postulated to result from anomalous RNA processing.     

A method for [3H]mannose labeling of Asn-linked oligosaccharides on recombinant glycoproteins synthesized in Xenopus oocytes.

We have developed an efficient method for labeling the Asn-linked oligosaccharides of recombinant glycoproteins synthesized in Xenopus laevis oocytes. By coinjecting GDP-[3,4-(3)H]mannose with mRNA for human cathepsin D, it was possible to incorporate as much as 1800 cpm per oocyte into each of the two Asn-linked oligosaccharides of this glycoprotein. Overall, about 50% of the microinjected GDP-[3,4-(3)H]mannose was incorporated into Asn-linked oligosaccharides, a 10-fold greater value than that obtained when [2-(3)H]mannose was microinjected. Less than 10% of the injected GDP-[3,4-(3)H]mannose was metabolized to water or converted to amino acids. This technique should facilitate studies of Asn-linked oligosaccharide biosynthesis, processing, and structure in recombinant proteins synthesized in Xenopus oocytes.     

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.     

Synthesis of lens crystallins in Xenopus oocytes as determined by quantitative immunoprecipitation.

Total poly(A)-containing calf lens mRNA was microinjected into Xenopus oocytes and synthesis of alpha, beta, and gamma-crystallins was demonstrated. By a method of quantitative immunoprecipitation the rate of translation of purified 14S alphaA2-crystallin mRNA was compared with translation of 9-S rabbit globin mRNA. Maximal response of oocytes was obtained with virtually the same molar amounts of mRNA, taking into account the larger size of the alphaA2-crystallin mRNA. Kinetics of translation were also very similar and both mRNAs were translated with similar rate and efficiency for at least two days. It was estimated that 20-30 polypeptide chains per hour per mRNA molecule were synthesized.     

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Characterization of myelin proteolipid mRNAs in normal and jimpy mice.

A clone specific for the rat myelin proteolipid protein (PLP) was isolated from a cDNA library made in pUC18 from 17-day-old rat brain stem mRNA. This clone corresponded to the carboxyl-terminal third of the PLP-coding region. The clone was used to identify PLP-specific mRNAs in mouse brain and to establish the time course of PLP mRNA expression during mouse brain development. Three PLP-specific mRNAs were seen, approximately 1,500, 2,400, and 3,200 bases in length, of which the largest was the most abundant. During brain development, the maximal period of PLP mRNA expression was from 14 to 25 days of age, and this was a similar time course to that for myelin basic protein mRNA expression. When the jimpy mouse, an X-linked dysmyelination mutant, was studied for PLP mRNA expression, low levels of PLP mRNA were seen which were approximately 5% of wild-type levels at 20 days of age. When jimpy brain RNA was analyzed by Northern blotting, the PLP-specific mRNA was shown to be 100 to 200 bases shorter than the wild-type PLP-specific mRNA. This size difference was seen in the two major PLP mRNAs, and it did not result from a loss of polyadenylation of these mRNAs.     

Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH,and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro

Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine whether they would be useful markers of oocyte competence. In serum-free media, only 1000 ng/ml pFSH induced marked cumulus expansion, but the effect of 100 ng/ml pFSH was amplified in the presence of 10% serum. In contrast, cumulus expansion occurred with 1 ng/ml rhFSH in the absence of serum. FSHr mRNA was highest at 0–6 h of maturation, then abundance decreased. Similarly, Cx43 mRNA expression was highest from 0–6 h but decreased by 24 h of maturation. However, the relative abundance of LHr mRNA did not change from 6–24 h of maturation. Decreased levels of FSHr, LHr and Cx43 mRNAs were detected in COCs of poorer quality. In conclusion, expansion of bovine cumulus occurred at low doses of rhFSH in serum-free media. In summary, FSHr, LHr and Cx43 mRNA abundance reflects COC quality and FSHr and Cx43 mRNA expression changes during in vitro maturation; these genes may be useful markers of oocyte developmental competence.