Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis

1989 ◽  
Vol 67 (9) ◽  
pp. 612-631 ◽  
Author(s):  
Michèle Denis-Duphil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Regulatory Gene ◽  
The Other ◽  
Transcriptional Level ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
Multifunctional Enzyme ◽  
Pathway Gene

There are six enzymatic steps in the de novo biosynthesis of uridine monophosphate (UMP). In yeast, six structural genes (ura2, ura4, ura1, ura5, ura10, and ura3) and one regulatory gene (PPR1) are involved in this metabolic pathway. Gene ura2 codes for a multifunctional protein that carries the first two enzymatic activities of the pathway, i.e., carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Gene ura2 has been cloned and sequenced, revealing the presence of three open reading frames, one of which codes for the multifunctional protein, a polypeptide of 2212 amino acids, with a mRNA of 7 ± 0.3 kilobases. Expression of gene ura2 is regulated at the transcriptional level. As I indicate here, it could also be controlled at the posttranscriptional level since all the consensus sequences for a 1.2-kilobases intron are present in the coding sequence of the open reading frame. The deducted amino acid sequence has allowed the identification of four domains. Starting from the amino terminus of the protein, these are glutamine amido transferase, CPSase, a domain that resembles dihydroorotase (DHOase-like) but does not have DHOase activity, and ATCase. There are also two sites of interest that match known concensus phosphorylation sites; one is located in the distal part of the CPSase domain, the other in the connector region between DHOase-like and ATCase domains. The protein has been purified as a multienzyme aggregate and as a multifunctional protein. The latter form, when isolated from a protease B deficient strain of Saccharomyces cerevisiae, contained mostly polypeptide chains of 220 kilodaltons. Work is currently in progress to determine the site(s) of phosphorylation of this protein in vitro. ATCase activity of both wild-type and protease-deficient strains has been found to be localized in the nucleus. Channeling of carbamyl phosphate, the first intermediate in the pathway, has been demonstrated both in vitro and in permeabilized cells. The other genes of UMP biosynthesis, except for ura5, are regulated by induction of their transcription by the combined action of the product of the ppr1 gene and the inducer, dihydroorotate. Dihydroorotate dehydrogenase activity was found in the cytoplasm. Two isoenzymes of orotate phosphoribosyl transferase have been found, coded for by ura5 and ura10. The products of genes ura10 and ura3 are proposed to participate in the channeling of orotidine monophosphate. The discussion considers the problem posed by the isolation of both multienzyme complexes and multifunctional proteins resulting from the expression of the same cluster genes. I suggest that regulation by processing at the posttranscriptional and posttranslational levels could be regarded as an alternative explanation for these observations, which were previously explained in terms of proteolysis.Key words: yeast, pyrimidines, multifunctional enzyme, phosphorylation, proteolysis.

Transcriptional regulation of the human CAD gene during myeloid differentiation

1987 ◽  
Vol 7 (5) ◽  
pp. 1961-1966
Author(s):  
G N Rao ◽  
E S Buford ◽  
J N Davidson
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Morphological Changes ◽  
Transcriptional Level ◽  
Carbamoyl Phosphate ◽  
Aspartate Transcarbamylase ◽  
Trans Retinoic Acid ◽  
Beta Actin ◽  
Cad Gene ◽  
Cad Protein

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics

2011 ◽  
Vol 435 (3) ◽  
pp. 771-781 ◽  
Author(s):  
Tatu J. K. Haataja ◽  
M. Kristian Koski ◽  
J. Kalervo Hiltunen ◽  
Tuomo Glumoff
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Structural Features ◽  
Full Length ◽  
Multifunctional Enzyme ◽  
Domain Composition ◽  
Substrate Channelling

All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure–function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Exogenous dTMP utilization by a novel tup mutant of Saccharomyces cerevisiae

1982 ◽  
Vol 152 (1) ◽  
pp. 111-119
Author(s):  
L F Bisson ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Culture Medium ◽  
De Novo ◽  
The Other ◽  
Normal Cells ◽  
Pyrimidine Bases

The rate and extent of entry of dTMP were measured in strains of Saccharomyces cerevisiae carrying two new tup mutations (tup5 and tup7) and most of the other tup mutations which have been reported previously by others. The tup7 mutation allowed dramatically greater accumulation of dTMP than any of the other mutations tested. Specific labeling of DNA by [CH3-3H]dTMP, fate of the dTMP pool inside of the cells, and degradation of the dTMP in the culture medium were investigated in strains carrying the tup7 mutation. The extracellular dTMP was not appreciably degraded, and that accumulated intracellularly was readily phosphorylated to dTDP and dTTP. Under optimum labeling conditions, 60 to 80% of the total thymidylate residues in newly synthesized DNA were derived from the exogenously provided dTMP, even in the absence of a block in de novo dTMP biosynthesis. An apparent Km for entry of 2 mM dTMP was found. The tup7 mutation increased permeability to dTMP (and some other 5'-mononucleotides), but did not affect uptake of nucleosides and purine and pyrimidine bases. Uptake of dTMP could be almost completely inhibited by moderate concentrations of Pi. These findings and other observations suggest that entry of dTMP in strains carrying the tup7 mutation is mediated by a permease whose function in normal cells is the transport of Pi.

scholarly journals Selective inhibition of Ph-positive ALL cell growth through kinase-dependent and -independent effects by CDK6-specific PROTACs

Blood ◽  
2020 ◽  
Vol 135 (18) ◽  
pp. 1560-1573 ◽  
Author(s):  
Marco De Dominici ◽  
Patrizia Porazzi ◽  
Youcai Xiao ◽  
Allen Chao ◽  
Hsin-Yao Tang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Enzymatic Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Regulatory Gene ◽  
Selective Inhibition ◽  
Hematologic Malignancies ◽  
Hematopoietic Progenitors

Abstract Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6–targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor–resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide “proof of principle” that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the “CDK6 dependence” of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.

84 MARKERS OF DNA METHYLATION IN OVINE UTERO–PLACENTAL TISSUES DURING EARLY PREGNANCY: EFFECTS OF ASSISTED REPRODUCTIVE TECHNOLOGY

2012 ◽  
Vol 24 (1) ◽  
pp. 154
Author(s):  
A. T. Grazul-Bilska ◽  
M. L. Johnson ◽  
P. P. Borowicz ◽  
D. A. Redmer ◽  
L. P. Reynolds
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technology ◽  
Early Pregnancy ◽  
De Novo ◽  
Reproductive Technology ◽  
The Other ◽  
Gravid Uterus ◽  
Natural Breeding ◽  
De Novo Methylation

Compromised pregnancies can be caused by genetic, epigenetic, environmental and/or other factors. Assisted reproductive technology (ART) may have profound effects on placental and fetal development, leading eventually to compromised pregnancy. DNA methylation, regulated by DNA methyltransferases (Dnmt) and other factors, plays an important role during embryonic, including placental, development. Altered DNA methylation in the trophoblast and, subsequently, the placenta has been reported for compromised pregnancies and may contribute to embryonic/fetal loss. Little is known, however, about DNA methylation processes in placental tissues during early stages of normal or compromised pregnancies in any species. Thus, we hypothesised that ART would affect the expression of 5 methylcytosine (5mC; a marker of global methylation) and mRNA for Dnmt1, 3a and 3b in utero-placental tissues during early pregnancy in sheep. Pregnancies (n = 7 per group) were achieved through natural breeding (NAT, control), or transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF) or in vitro activation (IVA; parthenogenetic clones). On Day 22 of pregnancy, caruncle (CAR; maternal placenta) and fetal membranes (FM; fetal placenta) were snap-frozen separately for RNA extraction followed by quantitative real-time PCR. In addition, cross sections of gravid uterus were fixed and then used for immunohistochemical detection and image analysis of 5 mC in FM. In FM, expression of mRNA for Dnmt3a was ∼2-fold greater (P < 0.01) in IVA compared with the other groups and was similar in NAT, NAT-ET and IVF groups. Expression of 5 mC was ∼2- to 3-fold greater (P < 0.02) in IVF and IVA than in NAT. In CAR, mRNA expression for Dnmt1 was ∼1.5-fold greater (P < 0.04) in IVA compared with the other groups, but Dnmt3a expression was less (P < 0.04) in NAT-ET and IVA than NAT. Expression of mRNA for Dnmt1 in FM and 3b in FM and CAR was similar in all groups. In IVA and/or IVF pregnancy, increased expression of Dnmt3a mRNA and/or 5 mC in FM may indicate de novo methylation in the fetal placenta. Furthermore, in pregnancies created through ART, decreased expression of Dnmt3a in CAR may indicate reduced de novo methylation in maternal placenta. Thus, in sheep, ART may have specific effects on growth and function of utero-placental and fetal tissues through regulation of DNA methylation and likely other mechanisms. These data provide a foundation for determining the basis for altered DNA methylation of specific genes in placental and embryonic tissues in compromised pregnancies. In addition, these data will help us to better understand placental regulatory mechanisms in compromised pregnancies and to identify strategies for rescuing such pregnancies. Supported by Hatch Project ND01712; USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR and NSF-MRI-ARRA grant to ATGB.

scholarly journals Exploitation of De Novo Helper-Lipids for Effective Gene Delivery.

2009 ◽  
Vol 11 (4) ◽  
pp. 56 ◽  
Author(s):  
Tomoaki Kurosaki ◽  
Takashi Kitahara ◽  
Mugen Teshima ◽  
Koyo Nishida ◽  
Junzo Nakamura ◽  
...  
Keyword(s):  
Gene Delivery ◽  
De Novo ◽  
Transfection Efficiency ◽  
The Other ◽  
Penetration Enhancers ◽  
Cell Toxicity ◽  
In Vivo Transfection ◽  
In Vitro Transfection

Purpose: In gene delivery, a fusogenic lipid such as dioleyl phosphatidylethanolamine (DOPE) which is a component of cationic liposomal vector is important factor for effective transfection efficiency. We investigated the effect of penetration enhancers as alternative helper-lipids to DOPE. Methods: Transdermal penetraion enhancers such as N-lauroylsarcosine (LS), (R)-(+)-limonene (LM), vitamin E (VE), and phosphatidyl choline from eggs (EggPC) were used in this experiments as helper-lipids with N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethlylammonium chloride (DOTMA) and cholesterol (CHOL). We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of plasmid DNA/cationic liposomes complexes. Results: In transfection experiments in vitro, the cationic lipoplexes containing LS had highest transfection efficiency among the other lipoplexes independently of FBS. Furthermore, the lipoplexes containing LS had lowest cell toxicity among the other lipoplexes in the presence of FBS. As the results of erythrocytes interaction experiment, DOTMA/LS/CHOL, DOTMA/VE/CHOL, and DOTMA/EggPC/CHOL lipoplexes showed extremely lower hematotoxicity. On the basis of these results, the in vivo transfection efficiencies of the lipoplexes were examined. The lipoplexes containing LS had the highest transfection activity among the other lipoplexes. Conclusion: In conclusion, several transdermal penetration enhancers are available for alternative helper-lipids to DOPE in cationic liposomal vectors. Among them, DOTMA/LS/CHOL lipoplexes showed superior characteristics in in vitro transfection efficiency, cell toxicity, hematotoxicity, and in vivo transfection efficiency.

Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes

1984 ◽  
Vol 4 (8) ◽  
pp. 1521-1527
Author(s):  
T E Torchia ◽  
R W Hamilton ◽  
C L Cano ◽  
J E Hopper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Null Mutation ◽  
Null Mutant ◽  
Wild Type

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

scholarly journals The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis

1999 ◽  
Vol 338 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Evelyne RAUX ◽  
Treasa McVEIGH ◽  
Sarah E. PETERS ◽  
Thomas LEUSTEK ◽  
Martin J. WARREN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Biosynthetic Pathways ◽  
Subtle Difference ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
His Tag ◽  
Cobalamin Biosynthesis

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.

scholarly journals Localization of the multifunctional protein CAD in astrocytes of rodent brain.

1991 ◽  
Vol 39 (5) ◽  
pp. 695-700 ◽  
Author(s):  
W Cammer ◽  
M Downing
Keyword(s):  
Carbonic Anhydrase ◽  
Glutamine Synthetase ◽  
Active Sites ◽  
De Novo ◽  
Third Ventricle ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
Normal Rat ◽  
Myelin Deficient ◽  
In Cells

The CAD multidomain protein, which includes active sites of carbamyl phosphate synthetase II (CPS II, glutamine-dependent), aspartate transcarbamylase, and dihydroorotase, was immunostained in normal rat brains, the gliotic brains of myelin-deficient mutant rats, and brains from normal weanling hamsters. In each of these tissues CAD was observed in cells resembling astrocytes. In hamster brain, CAD immunofluorescence was also found in cells closely related to astrocytes, i.e., the Bergmann glia in cerebellum and the tanycytes surrounding the third ventricle. The astrocytic identity of the CAD-positive cells in rat brain was confirmed by double immunofluorescence staining with antibodies against glial fibrillary acidic protein (GFAP). The two enzymes carbonic anhydrase and glutamine synthetase occur in the cytoplasm of normal astrocytes in gray matter and of reactive astrocytes during gliosis. Products of each enzyme, i.e., bicarbonate and glutamine, are required for the CPS II reaction, which is the first step in the biosynthesis of pyrimidines. Therefore, the present results suggest roles for carbonic anhydrase and glutamine synthetase, as well as CAD, in pyrimidine biosynthesis in brain and a role for the astrocytes in the de novo synthesis of pyrimidines.

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