Yoshitake Mano

Cloning and complete nucleotide sequence of mouse immunoglobulin γ1 chain gene

Abstract The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.

Cytoplasmic regulation and cyclic variation in protein synthesis in the early cleavage stage of the sea urchin embryo

Abstract Protein synthesis in intact sea urchin embryos in the early cleavage stage showed cyclic variation superimposed on an increasing basal rate of synthesis. The cyclic variations in protein synthesis seem to be correlated with mitotic division, and may be directly related to the mitotic cycle. The length of the cycles in intact embryos was inversely correlated with the temperature, but the amount of protein synthesized during each cycle was approximately the same regardless of the temperature. Cyclic fluctuation in protein synthesis, initiated by fertilization, was observed even in the presence of actinomycin D. Cycles of protein synthesis were observed even in the absence of nuclear and cytoplasmic division due to treatment with colchicine, puromycin, and cycloheximide. Similar cyclic variation, but in mirror image to that of protein synthesis, was observed in proteolytic activity. Cell-free systems (12,000 g supernatant), where practically no protease activity was found, of sea urchin embryos at various stages were shown to carry out protein synthesis at rates varying in a cyclic fashion without increase in the basal level of synthesis. These cyclic changes were not observed in intact unfertilized eggs or in cell-free systems prepared from them. The periodic appearance and disappearance of polyribosomes corresponding to the cell-free cycle was observed on incubation of the 12,000 g supernatant from fertilized eggs. It was suggested that the cytoplasm may regulate protein synthesis, resulting ultimately in mitotic division. Continuous and discontinuous protein synthesis in this stage and the nature of cyclic protein synthesis are discussed.

The mode of action of aphidicolin on DNA synthesis in isolated nuclei

Abstract Aphidicolin (a specific inhibitor of DNA polymerase-α) inhibited DNA synthesis in isolated nuclei from sea urchin embryos but ddTTP (an inhibitor of DNA polymerases-β and -γ) did not, indicating that DNA polymerase-α was responsible for DNA synthesis in isolated nuclei. DNA synthesis in isolated nuclei was inhibited by aphidicolin noncompetitively with respect to each of dNTPs indicating that properties of in situ DNA polymerase activity in isolated nuclei are different from those of the purified DNA polymerase-α which was inhibited by aphidicolin competitively with respect to dCTP and noncompetitively with respect to the other 3 dNTPs. Similar results were obtained using HeLa cell nuclei.

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I‐cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.

Thymidine kinase, thymidylate kinase and 32Pi and [14C]thymidine incorporation into DNA during early embryogenesis of the sea urchin

Abstract In the early embryogenesis of the sea urchin, the relationship between the activity of thymidine kinase (ATP:thymidine 5′-phosphotransferase, EC 2.7.1.21) and thymidylate kinase (ATP:thymidinemonophosphotransferase, EC 2.7.4.9) and of DNA synthesis was investigated. Thymidine kinase and thymidylate kinase activities were found in the homogenate and in the crude extract of unfertilized sea-urchin eggs and embryos. Maximal activity of the kinases was found in unfertilized eggs and the activity was decreased after fertilization. Thereafter, the activities changed periodically in parallel during the cell cycle and their maxima appeared at a defined stage of the cell cycle. [2- 14 C]Thymidine and 32 P i incorporation into the DNA fraction was found usually just after each division, with the exception of the first division cycle, which occurred at the stage when pronuclear fusion may take place. DNA synthesis was observed after the appearance of the peak of activity of these two kinases in the division cycles. The DNA synthesis system was not included in one division cycle (the division took place between the time of the appearance of the activity peak of DNA synthesis and that of the two kinases). In the early embryogenesis of the sea urchin, the mechanism of regulation of thymidine kinase activity was investigated and the following results were obtained. Puromycin and ethionine depressed thymidine kinase activity in vivo . Thus, the increase of thymidine kinase activity may be due mainly to enzyme synthesis de novo . Although thymidine kinase was observed to have a low stability, the enzyme had a similar lability in the homogenates of unfertilized eggs and of embryos. Thymidine protected thymidine kinase activity from heat inactivation. dTTP was a feedback inhibitor, but dCDP was not an activator.

Cytoplasmic location of DNA polymerase-α and -β of sea urchin eggs

Abstract Unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus , were quantitatively separated into nucleate and anucleate halves by isopycnic centrifugation. Approximately 32 % of DNA polymerase-α and 34 % of DNA poly-merase-β activities were found in the anucleate halves. From the diameters, the volume of the anucleate half was calculated to be 24.5 % that of the egg. It is concluded that nearly all DNA polymerase-α and -β are localized in the cytoplasm and it is suggested that DNA polymerases are bound to a cytoplasmic granule.

Identification of γ-like DNA polymerase from sea urchin embryos

Abstract A γ-like DNA polymerase devoid of DNA polymerase-α and -β activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) · oligo (dT)12–18 followed by poly(dA) · oligo(dT)12–18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-γ.

ENZYMATIC STUDIES ON THE METABOLISM OF URONIC AND ALDONIC ACIDS RELATED TO L‐ASCORBIC ACID IN ANIMAL TISSUES*

The biosynthesis of L-ascorbic acid from D-glucose in animal tissues has been studied by many workers, affording evidence that D-glucuronic acid, L-gulonic acid, or their lactones are converted to L-ascorbic On the other hand, it was proved that L-xylulose is formed from L-gulonic and the glucuronic acid cycle was prop~sed .~ Thus it seems to be necessary to study the metabolism of these uronic and aldonic acids enzymatically to clarify the possible pathway as a multienzyme system of reactions. The degradation of L-ascorbic acid in animal tissues has been studied for many years. It was proved that L-ascorbic acid can be metabolized back to D-glucoseloJl and that the degradation of L-ascorbic acid takes place to form 1,-lyxonic acid1* or L-xylose.Io These findings suggest the importance of studying the degradation of 2,3-diketo-~-gulonic acid formed by the oxidation of L-ascorbic acid in animal tissues. Thus the chief object of this paper is to study the enzymatic reactions concerning the metabolism of D-glucuronic and L-gulonic acids and their lactones, as well as of 2,3-diketo-~-gulonic acid. The enzymes studied are: triphosphopyridine nucleotide (TPN) L-hexonate dehydrogenase, which oxidizes Lgulonic acid to D-glucuronic acid; diphosphopyridine nucleotide (DPN) Lgulonate dehydrogenase, which oxidizes L-gulonic acid to L-xylulose with carbon dioxide evolution; lactonase I and 11, which hydrolyze the lactone ring of Dglucuronolactone and L-gulonolactone ; L-gulonolactone oxidizing enzyme, which converts L-gulonolactone to L-ascorbic acid; and 2,3-diketoaldonate decarboxylase, which decomposes 2,3-diketo-~-gulonic acid to L-lyxonic and L-xylonic acids with evolution of carbon dioxide (FIGURE 1). These enzymes are all present in the soluble fraction of liver homogenate with the exception of lactonase I1 and L-gulonolactone oxidizing enzyme, which are present in the microsomes.

Participation of the sulfhydryl groups of a protein in the cyclic variation in the rate of protein synthesis in a cell-free system from sea urchin cells

Evidence is presented that in a cell-free system from sea urchin cells during early development cyclic variation in the rate of protein synthesis is controlled by a concomitant cyclic fluctuation in the SH-content of a particular protein species. Cyclic fluctuation of the SH-content of the TCA-soluble protein fraction was observed in homogenates of fertilized and unfertilized cells without changes in the amount of protein. The SH-cycle seemed to start on fertilization in homogenates of fertilized eggs and at the time of homogenization in homogenates of unfertilized eggs. A similar fluctuation was also observed in the SH-content of the KCl-soluble protein fraction in cell-free systems from unfertilized eggs. Stimulation of the rate of protein synthesis by the 0.6 m KCl-soluble protein fraction was proportional to the content of protein-bound SH-groups in this fraction. This stimulatory effect was found to be due to the specific action of the SH-groups of a particular protein. A series of KCl-soluble protein fractions prepared at various stages from the supernatant fractions of unfertilized eggs induced various levels of protein synthesis in the supernatant fraction of other unfertilized cells when supplemented with maternal messenger RNA and a low-molecular-weight factor, this induction being proportional to the SH-content of these fractions. The protein responsible for the stimulation was purified to a practically homogenous state by gel filtration on Sephadex. Little species specificity was found in the stimulation of protein synthesis by purified proteins. Using a sea urchin transhydrogenase preparation, reversible electron transfer was observed between this protein and Ca 2+ -insoluble proteins. This seems to be the only protein species acting as a pacemaker of the cyclic variation in the rate of protein synthesis in the cell-free system by fluctuation in its SH-content, so it is suggested that cyclic protein synthesis is guided by cyclic fluctuation in the SH-content of the KCl-soluble protein.

Immunoglobulin γ1 heavy chain gene: Structural gene sequences cloned in a bacterial plasmid

Abstract Immunoglobulin γ1 heavy chain cDNA was cloned into an Escherichia coli plasmid pCR1 and its nucleotide sequence was determined. The hybrid plasmid contained approx. 900-bases-long γ1 chain cDNA sequence, including the complete sequence of the C H2 and C H3 domains and the 3′ untranslated region, and a partial sequence of the C H1 domain. The nucleotide sequence predicts an extra lysine at the carboxyl-terminus of the γ1 chain. Comparison of the nucleotide sequence of 3′ untranslated regions of the immunoglobulin γ1 chain and κ light chain showed a significant homology although lengths are quite divergent.