Biochemical research on oogenesis. RNA accumulation during oogenesis of the dogfish Scyliorhinus caniculus

Four biochemical mechanisms have been shown to operate in the oocytes of amphibians and teleosts: (1) amplification of the 28 S and 18 S genes, (2) noncoordinate accumulation of 5 S RNA and 28 S + 18 S RNA, (3) storage of 5 S and transfer RNA made in excess by small oocytes within nucleoprotein particles, (4) expression of different 5 S genes in oocytes and somatic cells. We have tried to extend these observations to another group of vertebrates, i.e., selacians (Chondrichthya). Our data suggest that ribosomal gene amplification is low or absent in the oocytes of the dogfish Scyliorhinus caniculus. However, previtellogenic oocytes of this species accumulate more 5 S RNA than needed for ribosome assembly. Transfer and 5 S RNA present in small oocytes are probably not free in the cell sap. A substantial fraction of these RNAs sediments at 10 S when homogenates of immature ovaries are centrifuged in sucrose density gradients. In contrast to what we observed in amphibians and teleosts, 5 S RNA from ovaries of S. caniculus is identical in sequence to 5 S RNA from liver. Among the four mechanisms mentioned above, the second and probably the third one are used by the oocytes of S. caniculus. Mechanism (4) is absent in this species. No definitive conclusion can be drawn concerning mechanism (1), i.e., ribosomal gene amplification.     

Biochemical research on oogenesis. Comparison between the proteins of the ribosomes and the proteins of the 42 S particles from small oocytes of Xenopus laevis

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown.The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles.The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.     

Fructose bisphosphatase of Saccharomyces cerevisiae. Cloning, disruption and regulation of the FBP1 structural gene

Fructose bisphosphatase catalyzes a key reaction of gluconeogenesis. We have cloned the fructose bisphosphatase (FBP1) structural gene from Saccharomyces cerevisiae by screening a genomic library for complementation of an Escherichia coli fbp deletion mutation. The cloned DNA expresses in E. coli a fructose bisphosphatase activity which is precipitable with antibodies specific for the yeast enzyme and is sensitive to inhibition by fructose 2,6-bisphosphate. Evidence is presented demonstrating that the entire gene, including all cis-acting regulatory sequences, has been cloned. A substitution mutation that disrupts FBP1 was incorporated into the yeast genome by transplacement to construct a fructose bisphosphatase null mutation. The fbp1 mutant strain is a hexose auxotroph, otherwise growing normally. Southern blot hybridization analysis confirmed the structure of the transplacement and demonstrated that FBP1 is present in single copy in the haploid genome. Northern blot hybridization analysis revealed an mRNA of about 1350 nucleotides, whose presence was repressible by glucose in the medium. Fructose bisphosphatase activity was not greatly overproduced when the FBP1 gene was present on a multicopy vector in yeast.     

The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids

We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotin-avidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy.When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.     

The effect of X chromosome heterozygosity on the structure of the ribosomal genes in Drosophila melanogaster.

Sucrose gradient analysis of DNA isolated from detergent-pronase lysates of adult flies has been used to look for ribosomal genes not integrated into the DNA of the chromosome in genotypes containing various combinations of inversions having breakpoints in the proximal heterochromatin of the X chromosome. Unintegrated genes are found in females heterozygous for inversions which have one breakpoint between the nucleolus organizer and the centromere. Homozygotes and males do not have unintegrated genes. The results suggest that unintegrated ribosomal genes result from an interaction between homologues having different arrangements of the proximal heterochromatin. In addition, data from a series of stocks carrying duplications of the X heterochromatin provide independent evidence for the size of the DNA on our gradients.     

Patterns of gene expression during teleost embryogenesis: Lactate dehydrogenase isozyme ontogeny in the medaka (Oryzias iatipes)

The ontogeny of the lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes during medaka (Oryzias latipes) embryogenesis was determined after the genetic and molecular bases of this multilocus isozyme system were established. Three LDH loci are differentially expressed among the tissues of the adult medaka. The LDH-A locus was expressed almost exclusively in the white skeletal muscle, the LDH-B locus in all tissues examined, and the LDH-C locus in the eye and brain. The contribution of each of these LDH loci was quantitatively determined throughout early medaka embryogenesis by using a combination of electrophoretic, immunochemical, and spectrophotometric procedures. LDH-B₄ is present throughout embryogenesis and is the predominant LDH isozyme during this period. LDH-C subunit activity was first detected 146 hr after fertilization (26°C), 142 hr prior to hatching. LDH-A subunit activity, however, was not detected until after hatching and, then, only as heterotetramers containing LDH-B subunits. The pattern of LDH gene expression during medaka embryogenesis was compared with the patterns of LDH gene expression during early development in five other teleost species. Some common patterns of differential LDH gene expression appear to exist among the teleosts. In all species examined, isozymes encoded in at least one LDH locus, A and/or B, were present throughout development. Those isozymes present continually during embryogenesis also tend to be active in a wide variety of differentiated tissues in the adult fish. Conversely, LDH isozymes which are active in a restricted number of adult tissues are detected only later in embryogenesis. The initiation of LDH-C gene expression, however, is closely coupled with morphological and functional differentiation of those cells in which this locus is predominantly expressed in the adult.     

The effect of pH and temperature on the kinetics of native and altered glycogen phosphorylase.

Data on the effect of pH and temperature on the kinetics of rabbit muscle phosphorylases a and b and reduced phosphorylase b (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) with glycogen as the saturating and inorganic phosphate the variable substrate are presented. The kinetic profiles as a function of pH are similar for these enzyme species except that the positions of the pH-maximal velocity profiles for reduced phosphorylase b are relatively invariant in the 15 °–30 ° range, whereas the “native” phosphorylases exhibit a substantial shift of the lower pH limb of the profile toward the acid side when the temperature is lowered from 30 to 15 °C. It is proposed that a group with a pK near 6.0 at 30 °C determines the acid limb of maximal velocity profiles. The phosphoryl moiety of enzyme bound pyridoxal 5′-phosphate is suggested for this group. A conformational transition in the protein, which is somehow modified when the aldimine bond between protein and pyridoxal 5′-phosphate is reduced, is invoked to account for the large decrease of this acid side apparent pK for the ternary complex of native phosphorylases when the temperature is lowered. A group with a pK near 7.1 and a heat of ionization of about 8000 cal/mol determines the alkaline limb of maximal velocity profiles at 30 °C. An imidazoyl ring ionization of an enzyme histidyl group is proposed to account for this behavior. In the enzyme-glycogen binary complex, the apparent heat of ionization of this group has an anomalous value of about ?10,000 cal/ mol. It is suggested that a neighboring amino or arginyl guanidinium group is able to interact with the imidazoyl ring in the absence of bound inorganic phosphate to cause this anomalous behavior. The effect of pH on K_m for inorganic phosphate is simply explained by a group with a pK of 6.56 and low heat of ionization. The data are interpreted to indicate that the dianion of inorganic phosphate is the true substrate for all forms of phosphorylase. The kinetic results of this report are closely compared with other kinetic data in the literature on mammalian, plant, and bacterial α-glucan phosphorylases and general overall similarity is demonstrated. Various methods for analyzing pH-kinetic data for enzymes are briefly discussed, and the crucial difference in conclusions the choice of method can make is demonstrated with our data.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

The requirement for biotin and fatty acids in the cytotoxic T-cell response.

Dialyzed fetal calf serum (FCS) was a poor source of serum supplement for in vitro cytotoxic T lymphocyte (CTL) generation. Serum dialysate or biotin fully restored dialyzed FCS to activities comparable to FCS. It was concluded that the active principal in serum dialysate was biotin because its further dialysis was prevented by addition of avidin, a biotin binding protein. Avidin inhibited CTL generation only when added during the early stages of mixed lymphocyte cultures, whereas biotin could restore activity even if added at a later time. When FCS enriched in a fatty acid mixture, or in palmitic acid alone, was used as the serum supplement, avidin-mediated inhibition of CTL generation was markedly reduced. Avidin also inhibited CTL generation in cultures containing killed macrophages as the stimulating cell, and supplemented with Con-A-induced spleen cell supernatant, a source of helper factor(s). These experiments suggest that fatty acid biosynthesis and the attendant synthesis of structural lipids of appropriate fatty acid composition play a prominent role in the generation of CTL     

The biosynthesis of progesterone by cultured mouse midgestation trophoblast cells.

The ability of cultured midgestation mouse trophoblast cells to synthesize progesterone from pregnenolone has been monitored by radioimmunoassay or chromatography and crystallization. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of Δ⁵,3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Since there is little or no further metabolism of the progesterone formed, the ability of trophoblast cells to convert pregnenolone to progesterone in vitro is an accurate reflection of the activity of 3β-HSD in these cells.Midgestation cultures of giant trophoblast cells have a substantially higher 3β-HSD specific activity than the smaller ectoplacental cone cells. Neither giant trophoblast nor ectoplacental cone cell cultures show an increased 3β-HSD specific activity in response to a variety of hormones, including gonadotrophins. In fact, regardless of the gestation age at which the trophoblast cultures are initiated, 3β-HSD activity inevitably follows the same temporal pattern observed in vivo. Taken together, these facts suggest that the levels of 3β-HSD in trophoblast cells are intrinsically controlled and that, unlike the ovary, progesterone production by trophoblast cells in vivo is not influenced by gonadotrophic hormone levels.     

The origin of CSF choline and its relation to acetylcholine metabolism in brain.

Elevation of serum choline levels in the rabbit either by intravenous injection of choline or by the pharmacological action of oxotremorine results in a rise in cisternal CSF choline levels. It was excluded that the oxotremorine induced rise in CSF choline levels can be ascribed to its action on the CNS. Therefore changes in CSF choline levels can be merely the result of changes in peripheral choline stores and do not necessarily reflect changes in the cholinergic activity of the CNS. From isotopic labelling experiments the contribution of serum choline to CSF choline was found to be 42%.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.     

Evidence for a single catalytic site on the "beta-D-glucosidase-beta-D-galactosidase" of almond emulsin.

An isoenzyme of glycosidase obtained from almond emulsin, which is both a β-d-glucosidase and a β-d-galactosidase, has now been shown to possess β-D-fucosidase activity. It has been concluded that all three activities reside in a single catalytic site for the following reasons. (i) d-Glucosylamine, d-galactosylamine, and d-fucosylamine (a newly discovered potent inhibitor of this enzyme) each act competitively against all three of the substrates. (ii) Any given inhibitor exhibits the same K_i value when tested in the presence of any of the three substrates, (iii) When the enzyme is incubated with any two of the p-nitrophenyl glycoside substrates, at or above their respective K_m values, the rate of p-nitrophenol formation is not additive, but rather is equal to the value calculated on the basis of the individual K_m values and relative maximum velocities.     

The ventromedial nucleus of the hypothalamus and the hormonal arousal of sexual behaviors in the female rat

Bilateral lesions of the ventromedial nucleus of the hypothalamus interfered with the estrogenic induction of sexual receptivity in the female rat, but seemingly did not affect the ability of female rats to show lordosis following combined stimulation with estrogen and progesterone. In addition, ventromedial hypothalamic lesions did not affect the ability of females to show male-like sexual activity in response to exogenous androgenic stimulation.     

Studies on the heat-precipitated phosphoenzyme complex of eel electric organ (Na + K).ATPase.

When the hydrolytic reaction between eel electric organ (Na + K) · ATPase and [γ-³²P]ATP is terminated at neutral pH by heat precipitation, a phosphoenzyme complex is formed which reaches maximal levels in the simultaneous presence of Mg, Na, and K. After formation of a steady-state level of phosphoenzyme in the presence of Mg and Na, a pulse of K increases the level of the heat-precipitated phosphoenzyme (while decreasing the level of the acid-precipitated phosphoenzyme). The formation of the heat-precipitated phosphoenzyme is clearly inhibited by ouabain only when the phosphoenzyme is formed in the presence of Mg, Na, and K. Inorganic phosphate decreases the level of the heat-precipitated phosphoenzyme, but not that of the acid-precipitated phosphoenzyme (in the presence of Mg and Na or in the presence of Mg, Na, and K). Moreover, a heat-precipitated, ouabain-sensitive phosphoenzyme forms in the reaction between the eel (Na + K) · ATPase and ³²P_i with or without ATP. The pH stability of the heat-precipitated phosphoenzyme complex is maximal at pH 6 to 8, and this complex shows little or no reactivity with neutral hydroxylamine, suggesting that the phosphate is not bound to an acyl residue of the protein. These experiments indicate that both heat-resistant and acid-resistant phosphoenzymes are formed during the (Na + K) · ATPase reaction at pH 7.4.