Eva Dworkin-Rastl

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. Clone pXlgC20 contains two complete repeats of a 228 bp sequence as well as part of a third repeat, all adjacent and in the same orientation. One possible translational reading frame in pXlgC20 completely spans the repeat sequences, coding for a protein composed of tandem 76 amino acid units. The amino acid sequence of each unit completely matches that of human ubiquitin. Ubiquitin is translated in the form of a multimeric precursor molecule containing several units. We show that genomic DNA fragments exist that contain at least 12 of these units in tandem and propose that the different mRNA size classes vary in their number of ubiquitin coding sequences.

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. Among 21 sequences analyzed by RNA gel blots, all remained unchanged in titer during oocyte maturation. A significant fraction of early oocyte RNA does not bind to oligo(dT)cellulose, but by the end of oogenesis transcripts for many RNA species examined are detected exclusively in the poly(A)+RNA fraction. During oocyte maturation a slight size shift or a broadening of the hybridizing band can be seen for many sequences, indicative of poly(A) elongation or degradation.

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. Glycolytic intermediates injected into these cells were metabolized into UDP-glucose and then presumably into glycogen. Carbon flow between phosphoenolpyruvate and glucose 6-phosphate does not utilize fructose 1,6-bisphosphatase; rather, it may depend largely on enzymes of the pentose phosphate pathway. Maturation and fertilization of the oocyte did not result in a change in the qualitative pattern of metabolites formed. Pyruvate kinase, although abundant in oocytes and embryos, is essentially inactive in these cells. Pyruvate kinase also appears to be inactive in small previtellogenic stage II oocytes; however, in these cells injected glycolytic intermediates were not metabolized to UDP-glucose.

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. The enzymology that supports the carbon metabolism of the cleaving amphibian embryo resembles that of many mammalian tumor cells.

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. To determine the source of carbon for sugar phosphates and pyruvate, 14C-labeled intermediary metabolites were injected into fertilized eggs and their metabolism examined by thin-layer chromatography. Alanine, pyruvate, and lactate form a pool of carbon that fluxes into sugar phosphates. Cytosolic (nonmitochondrial) aspartate, oxaloacetate, and malate form a pool of carbon which is largely blocked in the short-term from entering the smaller alanine/pyruvate/lactate pool. The data indicate that the major source of carbon for sugar phosphates in fertilized eggs and rapidly cleaving embryos is the alanine/pyruvate/lactate pool. Pyruvate from this pool is converted in the mitochondria to phosphoenolpyruvate, which in turn is metabolized outside the mitochondria to sugar phosphates. A key enzyme in regulating flux from amino acid carbon to pyruvate is malic enzyme. Three malic enzyme isozymes, one soluble and two mitochondrial, were partially isolated and kinetically characterized from total ovarian tissue. Full-grown oocytes and eggs, however, have very low soluble malic enzyme activity, which results in the separation of the cytosolic aspartate/oxaloacetate/malate and alanine/pyruvate/lactate pools.

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. The oocyte transcripts and the embryonic transcripts of one of these sequences are transcribed from different though cross-hybridizing genes. Cellular titers of a number of RNA sequences have also been studied and show that increases in the cellular titers of several poly(A)+RNA species are the result of de novo transcription and not simply polyadenylation. A number of sequences abundant in tadpole RNA but absent or very rare in eggs have also been examined. All of these sequences first appear in development in substantial titers in the late gastrula or early neurula, 12-15 hr after fertilization. Many other sequences already present in eggs which persist during development show an increase in titer 12-15 hr after fertilization. These data suggest that this late gastrula transcriptional event may be a major transition of gene expression that accompanies the cellular differentiation and morphogenesis that begin at this developmental time.

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.

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. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.

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. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of microM concentrations of fructose-1,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.

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). Injection of D7 RNA into the full-grown oocyte and its subsequent translation into D7 protein neither induced oocyte maturation nor affected the kinetics of hormone-induced maturation. Overexpression of D7 protein by 20-fold in the early Xenopus embryo by injection of D7 RNA into fertilized eggs did not affect subsequent development. Oocytes specifically lacking D7 mRNA were generated by oligodeoxynucleotide-mediated RNA destruction within the oocyte. Unfertilized eggs generated from such oocytes lacked detectable D7 protein, but nevertheless could be activated and fertilized. Embryos generated from such eggs, estimated to contain less than 5% of wildtype levels of D7 protein, developed normally up to the tailbud stage. Thus the D7 protein, the product of a maternal mRNA that is under strict translational repression in oocytes, appears not to be required for oocyte maturation, activation, fertilization or early embryonic development in Xenopus.