The maternal histone H1 variant, H1M (B4 protein), is the predominant H1 histone in Xenopus pregastrula embryos.

The Xenopus laevis H1M protein (formerly called B4) is a maternally inherited histone H1 subtype restricted in its expression to early development. Levels of H1M, as well as of the somatic histones H1A, H1B, and H1C, were determined during early embryogenesis using subtype-specific anti-peptide antisera. H1M accumulates late in oogenesis to a titer of approximately 1 ng/unfertilized egg.

Glycogen breakdown in cleaving Xenopus embryos is limited by ADP.

Xenopus eggs contain large stores of glycogen, but this glycogen is not glycolytically processed during cleavage. The Embden-Meyerhof pathway is inhibited by the absence of pyruvate kinase activity in vivo, and lactate and pyruvate are present at relatively low levels. In the late blastula, just preceding gastrulation, lactate levels increase, indicating the onset of glycogen breakdown and glycolytic flux.

Synthesis and modification of D7 protein during Xenopus oocyte maturation.

The Xenopus maternal mRNA D7 is translationally repressed during oogenesis, only becoming recruited into polysomes during oocyte maturation, with D7 protein being detectable for the first time prior to germinal vesicle breakdown (GVBD). The synthesis of D7 protein was found to be induced by a variety of maturation-promoting agents including cyclin, c-mos and crude preparations of MPF. D7 protein induced by all these agents is post-translationally modified and exists as a number of variants of differing molecular weight.

The maternal gene product D7 is not required for early Xenopus development.

The Xenopus D7 gene codes for a novel protein whose expression is restricted to early development. D7 protein is synthesized for the first time during oocyte maturation (1988, Genes Dev. 2, 1296-1306).

The involvement of mitochondria in carbon metabolism in cleavingXenopus embryos.

The major carbon sources inXenopus oocytes and cleavage-stage embryos appear to be amino acids, which are oxidized to form pyruvate (to support the Krebs cycle) and phosphoenolpyruvate (for anabolic processes). Metabolism of various metabolites in vitro into aspartate or glutamate, and then partially into phosphoenolpyruvate, requires the presence of mitochondria, suggesting that metabolism in vivo utilizes mitochondrial enzymes. The rate limiting step in metabolism in the stage VI oocyte appears to be uptake and/or metabolism of compounds by the mitochondria; the rate of metabolism increases during maturation.

Carbon metabolism in early amphibian embryos.

Xenopus embryos undergoing cleavage utilize amino acids as their main carbon source for metabolism. Glycolysis (from stored glycogen) begins near the onset of gastrulation. Thus, a major transition in the metabolism of the early embryo occurs before morphological differentiation.

Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments.

Microinjection of oligodeoxynucleotides (ODNs) complementary to cellular mRNAs has been advanced as an experimental approach to degrade target mRNAs in vivo and thereby obtain information as to the function of their cognate proteins. It is shown here that ODNs can induce a variety of aberrations in cell metabolism and structure when injected into Xenopus oocytes. Examination of histological sections of ODN-injected oocytes revealed the frequent abnormal accumulation of heavily staining basophilic material in the area of the germinal vesicle (gv).

Functions of maternal mRNA in early development.

In this review, the types of mRNAs found in oocytes and eggs of several animal species, particularly Drosophila, marine invertebrates, frogs, and mice, are described. The roles that proteins derived from these mRNAs play in early development are discussed, and connections between maternally inherited information and embryonic pattern are sought. Comparisons between genetically identified maternally expressed genes in Drosophila and maternal mRNAs biochemically characterized in other species are made when possible.

Regulation of carbon flux from amino acids into sugar phosphates in Xenopus embryos.

Xenopus laevis oocytes and embryos are glycogenic cells, metabolizing sugar phosphates into glycogen. These cells have very low pyruvate kinase activity in vivo and, consequently, make little pyruvate and lactate through glycolysis. Nevertheless, oocytes and embryos do contain significant pyruvate and lactate levels.

Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.

Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from approximately 90 to 200 adenylate residues.

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs.

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs.

Metabolic regulation during early frog development: glycogenic flux in Xenopus oocytes, eggs, and embryos.

32P-labeled glucose 6-phosphate and phosphoenolpyruvate were injected into oocytes, fertilized eggs, and early embryos of Xenopus laevis, and the 32P label was followed into glycolytic enzymes and acid-soluble metabolites. The kinetics of labeling of phosphoglucomutase and phosphoglyceromutase and the formation of specific metabolites were used to measure carbon flux through glycolytic intermediates in these cells. In full-grown stage VI oocytes, fertilized eggs, and cells of cleaving embryos, carbon metabolism is in the glycogenic direction.

Destruction of a translationally controlled mRNA in Xenopus oocytes delays progesterone-induced maturation.

The maternal mRNA D7 is a moderately abundant transcript in Xenopus laevis whose expression is highest in, and perhaps restricted to, oogenesis and early embryogenesis. The nucleotide sequence of cloned D7 cDNA was determined and shown to have the capacity to code for a 31-kD protein. This amino acid sequence was searched against a protein data base, and no homologous proteins were found.

Expression of a histone H1-like protein is restricted to early Xenopus development.

Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes.

Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence.

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined.

Metabolic regulation during early frog development. Identification of proteins labeled by 32P-glycolytic intermediates.

When 32P-labeled phosphoenolpyruvate is injected into Xenopus laevis oocytes, a 50-60-kDa protein of subunit size Mr 29,000 is rapidly labeled, followed by a second (monomeric) protein of 66 kDa concomitant with the loss of label from the first protein. We have identified these proteins as, respectively, the glycolytic enzymes phosphoglyceromutase and phosphoglucomutase. The phosphoglyceromutase is labeled at a histidine and the phosphoglucomutase at a serine, presumably at their active sites during the gluconeogenic transformation of phosphoenolpyruvate into glycogen.

Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis.

1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2.

Tumorigenic Xenopus cells express several maternal and early embryonic mRNAs.

Recombinant cDNA libraries were constructed from poly(A)+ RNA isolated from different stages of oogenesis and embryogenesis from the clawed toad Xenopus laevis. Hybridization analyses were used to describe the accumulation of specific RNAs represented by these cDNA clones in oocytes, embryos, adult liver, a cell line derived from Xenopus borealis embryos (Xb693), and a tumorigenic substrain of that cell line (Xb693T). It was found that from 550 cDNA clones analysed, six sequences accumulate to higher titers in poly(A)+ RNA isolated from the tumorigenic cell line compared with the non-tumorigenic cell line.

Localization of specific mRNA sequences in Xenopus laevis embryos by in situ hybridization.

In situ hybridization of cloned cDNA probes to frozen sections of Xenopus laevis stage-42 tadpoles has been used to determine the tissue localization of several mRNAs. Nine out of sixteen probes tested hybridized to most or all tadpole tissues; seven probes exhibited tissue-specific hybridization. The non-tissue-specific sequences hybridized to RNA species that are also present in maternal RNA while the tissue-specific sequences hybridized to embryonic RNA species induced after gastrulation and undetectable in maternal RNA.

Changes in RNA titers and polyadenylation during oogenesis and oocyte maturation in Xenopus laevis.

The titers of over 90 sequences isolated by cDNA cloning of oocyte poly(A)+RNA were examined during oogenesis in Xenopus laevis. The relative titers of most sequences in unfertilized eggs are established in pre-lamp brush oocytes and persist throughout oogenesis. We have identified several sequences whose titers decrease significantly during the growth phase of oogenesis as well as a few sequences whose titers increase slightly during this period.

Mobilization of specific maternal RNA species into polysomes after fertilization in Xenopus laevis.

Unfertilized eggs of many species contain large amounts of maternal mRNA that are used to support protein synthesis during the first few hours of development, before the onset of embryonic transcription. We have examined the accumulation of nonpolysomal maternal RNAs in polysomes after fertilization in Xenopus laevis by measuring the distributions of specific sequences in nonpolysomal and polysomal fractions. In an arbitrary selection of 18 maternal sequences that are largely nonpolysomal in the full-grown oocyte, 13 became enriched in polysomes by the 16-cell cleavage stage.

Constancy of DNA organization of polymorphic and nonpolymorphic genes during development in Xenopus.

A study was undertaken to test for the occurrence of DNA rearrangements or amplifications during embryonic development in Xenopus laevis. DNA isolated from testes and liver was digested with four restriction enzymes, separated on agarose gels, transferred to nitrocellulose, and hybridized with over 50 cloned cDNA probes generated from embryonic poly (A)+ RNA. No qualitative or quantitative differences were detectable in the DNA hybridization patterns of testes and liver DNA, suggesting that, at least during liver development, selective amplifications or rearrangements occur rarely if at all.

The accumulation of prominent tadpole mRNAs occurs at the beginning of neurulation in Xenopus laevis embryos.

Cloned cDNA probes have been used to measure the sizes and titers of transcripts in total RNA preparations during early development in Xenopus laevis. Of more than 20 different sequences derived from abundant and moderately abundant RNA which were present in full-grown oocytes and persisted during early development, the transcript sizes of all but 3 of these sequences were invariant. Two transcripts were of a higher molecular weight in oocytes than in embryos, but their titers in oocytes were less than 5% their titers in embryos and thus these larger maternal transcripts do not significantly contribute to embryonic, polysomal mRNA.

Multiple ubiquitin mRNAs during Xenopus laevis development contain tandem repeats of the 76 amino acid coding sequence.

A cDNA clone, pXlgC20, was isolated from a library constructed from poly(A)+ RNA from stage 10 X. laevis gastrulae. This sequence hybridizes with up to nine different RNA species ranging in size from 1600 to 3500 nucleotides, regularly spaced at intervals of about 230 nucleotides.

Construction of expression plasmids producing high levels of human leukocyte-type interferon in Escherichia coli.

An expression plasmid was constructed, consisting of the promoter/operator region of the tryptophan operon from Serratia marcescens and a synthetic ribosome-binding site ligated into pBR322. Leukocyte-type interferon gene fragments (IFN-alpha A and IFN-alpha C) isolated from a cDNA library from human lymphoblastoid (Namalwa) cells were inserted into the unique HindIII site of the expression plasmid, and the resulting recombinant plasmids directed the synthesis of up to 5 X 10(5) units of A-type preinterferon, 2 X 10(7) units of A-type mature interferon and 8 X 10(5) units of C-type mature interferon per liter culture.