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A Jong

Publications and source records attributed to A Jong.

At least 19 recordsLinked to original sources
PubMed2001

MAT1-modulated CAK activity regulates cell cycle G(1) exit.

The cyclin-dependent kinase (CDK)-activating kinase (CAK) is involved in cell cycle control, transcription, and DNA repair (E. A. Nigg, Curr. Opin. Cell. Biol. 8:312-317, 1996). However, the mechanisms of how CAK is integrated into these signaling pathways remain unknown. We previously demonstrated that abrogation of MAT1 (ménage à trois 1), an assembly factor and targeting subunit of CAK, induces G(1) arrest (L. Wu, P. Chen, J. J. Hwang, L. W. Barsky, K. I. Weinberg, A. Jong, and V. A. Starnes, J. Biol. Chem. 274:5564-5572, 1999). This result led us to investigate how deregulation of CAK by MAT1 abrogation affects the cell cycle G(1) exit, a process that is regulated most closely by phosphorylation of retinoblastoma tumor suppressor protein (pRb). Using mammalian cellular models that undergo G(1) arrest evoked by antisense MAT1 abrogation, we found that deregulation of CAK inhibits pRb phosphorylation and cyclin E expression, CAK phosphorylation of pRb is MAT1 dose dependent but cyclin D1/CDK4 independent, and MAT1 interacts with pRb. These results suggest that CAK is involved in the regulation of cell cycle G(1) exit while MAT1-modulated CAK formation and CAK phosphorylation of pRb may determine the cell cycle specificity of CAK in G(1) progression.

Cell Division↗
PubMed2000

Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.

Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins. Although the temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studies, the molecular mechanism of the cdc6-1 mutation has been unclear. In this study, we have identified a base substitution at Gly260-->Asp, near the CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background. These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin. Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein. Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of Mcm5 from chromatin fractions. In the absence of functional Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition. Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins.

Amino Acid Substitution↗
PubMed2000

Identification and characterization of Saccharomyces cerevisiae Cdc6 DNA-binding properties.

Recent studies have shown that Cdc6 is an essential regulator in the formation of DNA replication complexes. However, the biochemical nature of the Cdc6 molecule is still largely unknown. In this report, we present evidence that the Saccharomyces cerevisiae Cdc6 protein is a double-stranded DNA-binding protein. First, we have demonstrated that the purified yeast Cdc6 can bind to double-stranded DNA (dissociation constant approximately 1 x 10(-7) M), not to single-stranded DNA, and that the Cdc6 molecule is a homodimer in its native form. Second, we show that GST-Cdc6 fusion proteins expressed in Escherichia coli bind DNA in an electrophoretic mobility shift assay. Cdc6 antibodies and GST antibodies, but not preimmune serum, induce supershifts of GST-Cdc6 and DNA complexes in these assays, which also showed that GST-Cdc6 binds to various DNA probes without apparent sequence specificity. Third, the minimal requirement for the binding of Cdc6 to DNA has been mapped within its N-terminal 47-amino acid sequence (the NP6 region). This minimal binding domain shows identical DNA-binding properties to those possessed by full-length Cdc6. Fourth, the GST-NP6 protein competes for DNA binding with distamycin A, an antibiotic that chelates DNA within the minor groove of the A+T-rich region. Finally, site-direct mutagenesis studies revealed that the (29)KRKK region of Cdc6 is essential for Cdc6 DNA-binding activity. To further elucidate the function of Cdc6 DNA binding in vivo, we demonstrated that a binding mutant of Cdc6 fails to complement either cdc6-1 temperature-sensitive mutant cells or Deltacdc6 null mutant cells at the nonpermissive temperature. The mutant gene also conferred growth impairments and increased the plasmid loss in its host, indicative of defects in DNA synthesis. Because the mutant defective in DNA binding also fails to stimulate Abf1 ARS1 DNA-binding activity, our results suggest that Cdc6 DNA-binding activity may play a pivotal role in the initiation of DNA replication.

Binding Sites↗
PubMed1999

RNA antisense abrogation of MAT1 induces G1 phase arrest and triggers apoptosis in aortic smooth muscle cells.

The human MAT1 gene (ménage à trois 1) is an assembly factor and a targeting subunit of cyclin-dependent kinase (CDK)-activating kinase. The novel mechanisms by which MAT1 forms an active CDK-activating kinase and determines substrate specificity of CDK7-cyclin H are involved in the cell cycle, DNA repair, and transcription. Hyperplasia of vascular smooth muscle cells (SMC) is a fundamental pathologic feature of luminal narrowing in vascular occlusive diseases, and nothing is yet known regarding the cell cycle phase specificity of the MAT1 gene in its involvement in SMC proliferation. To investigate such novel regulatory pathways, MAT1 expression was abrogated by retrovirus-mediated gene transfer of antisense MAT1 RNA in cultured rat aortic SMCs. We show that abrogation of MAT1 expression retards SMC proliferation and inhibits cell activation from a nonproliferative state. Furthermore, we have demonstrated that these effects are due to G1 phase arrest and apoptotic cell death. Our studies indicate a link between cell cycle control and apoptosis and reveal a potential mechanism for coupling the regulation of MAT1 with G1 exit and prevention of apoptosis.

Animals↗
PubMed1998

Saccharomyces cerevisiae Cdc6 stimulates Abf1 DNA binding activity.

In budding yeast Saccharomyces cerevisiae, an ARS binding factor 1 (Abf1) binds to the sequence-specific DNA element involved in DNA replication and transcription. We describe in this study how yeast Cdc6 protein stimulates Abf1 protein DNA binding activities. The Abf1 binding activity was reduced approximately 20-fold in a cdc6-1 mutant than in the wild-type strain. Introducing a copy of the wild-type CDC6 gene into the cdc6-1 mutant strain restored the Abf1 DNA binding activity. We demonstrated that recombinant Abf1 binds to ARS1 in vitro, and its DNA binding activity can be highly stimulated by the addition of a fusion glutathione S-transferase (GST)-Cdc6 protein. Deletion analysis revealed that the stimulating region is located at the amino terminus of the Cdc6 protein. However, we could not find the direct physical interaction between Cdc6 and Abf1. Instead, we found that the GST-Cdc6 can compete with distamycin A for binding to the DNA molecule. As distamycin A is a specific reagent that binds noncovalently to DNA at (A + T)-rich tracks, the stimulation of Abf1 DNA binding activity may be mediated by the Cdc6/DNA interaction. Our results favor a hypothesis that Cdc6 may function as an architectural factor in the assembly of a functional initiation replication complex.

Antiviral Agents↗
PubMed1996

Intracellular location of the Saccharomyces cerevisiae CDC6 gene product.

The CDC6 gene product from Saccharomyces cerevisiae is required for transition from late G1 to S phase of the cell cycle. We have investigated the subcellular localization of the CDC6 protein in yeast to explore where Cdc6p exerts its gene function (s). Using affinity-purified sera we localized Cdc6p to the cytoplasm and the nuclear matrix by both subcellular fractionation and indirect immunofluorescence microscopy. The nuclear localization was confirmed to be in the nuclear scaffold by the low-salt extraction method. The Cdc6p cannot be detected in the mitochondrial or plasma membrane fractions. Using indirect immunofluorescence, we found that a subpopulation of Cdc6p migrated into the nucleus after G1/S transition and diminished after M phase, suggesting its temporal role in nuclear DNA replication. The predicted Cdc6p polypeptide contains a conserved nuclear localization, 27PLKRKKL33, similar to that of the SV40 large T antigen and other nuclear proteins. To test whether this peptide segment plays a role in mediating nuclear transport, we have carried out site-directed mutagenesis to alter the conserved 29Lys to Thr and Arg. The wild-type nuclear localization signal of Cdc6p was found to mediate the LacZ reporter gene fused to CDC6 efficiently to the nucleus, but not the mutated versions of the nuclear localization motif. The results suggested that 29Lys is important in mediating nuclear localization, the 29Thr and 29Arg mutant versions of the CDC6 gene were also unable to complement the cdc6 temperature-sensitive mutant. However, when these mutants were expressed from a multicopy plasmid, the mutated genes could complement the mutation. Similar results were obtained in the cdc6-disrupted cells. Taken together, we suggest that (i) Cdc6p is predominantly located in the cytoplasm, (ii) the nuclear entry of Cdc6p is cell cycle dependent, and (iii) nuclear entry of Cdc6p is mediated by its nuclear localization signal. The presence of Cdc6p in both the nucleus and the cytoplasm suggests a model that Cdc6p exerts its gene function in DNA replication and mitotic restraint in the cell cycle.

Amino Acid Sequence↗
PubMed1996

Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases.

During purification of recombinant Cdc6 expressed in yeast, we found that Cdc6 interacts with the critical cell cycle, cyclin-dependent protein kinase Cdc28. Cdc6 and Cdc28 can be coimmunoprecipitated from extracts, Cdc6 is retained on the Cdc28-binding matrix p13-agarose, and Cdc28 is retained on an affinity column charged with bacterially produced Cdc6. Cdc6, which is a phosphoprotein in vivo, contains five Cdc28 consensus sites and is a substrate of the Cdc28 kinase in vitro. Cdc6 also inhibits Cdc28 histone H1 kinase activity. Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells. However, Cdc6 does not associate with Cdc28 when cells are blocked at the restrictive temperature in a cdc34 mutant, a point in the cell cycle when the B-type cyclin/Cdc28 inhibitor p40Sic1 accumulates and purified p40Sic1 inhibits the Cdc6/Cdc28 interaction. Deletion of the Cdc28 interaction domain from Cdc6 yields a protein that cannot support growth. However, when overproduced, the mutant protein can support growth. Furthermore, whereas overproduction of wild-type Cdc6 leads to growth inhibition and bud hyperpolarization, overproduction of the mutant protein supports growth at normal rates with normal morphology. Thus, the interaction may have a role in the essential function of Cdc6 in initiation and in restraining mitosis until replication is complete.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1994

Human dTMP kinase: gene expression and enzymatic activity coinciding with cell cycle progression and cell growth.

dTMP kinase (E.C.2.7.4.9.) catalyzes the phosphorylation of dTMP to the corresponding diphosphate. This enzyme is essential for DNA synthesis in vivo and is an important intermediate enzyme in the pathway of many pyrimidine analog drugs. In this report, we describe the isolation of the human dTMP kinase gene by functional complementation of a Saccharomyces cerevisiae cell cycle mutant, cdc8. The cDNA sequence revealed an open reading frame that encodes a protein with the molecular weight of 23,806. The deduced protein sequence was compared to known dTMP kinase sequences from different organisms. Although functionally complementary and structurally conserved, expressed human dTMP kinase in yeast shows little enzymatic activity. In contrast, active human dTMP kinase can be expressed from the gene cloned into the baculovirus expression system, as evidenced by increased enzymatic activity by four- to five-fold. Unlike yeast dTMP kinase, human dTMP kinase does not contain a cysteine residue after the conserved glycine-rich loop, but its enzymatic activity is still affected by the sulfhydryl inhibitor, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). The levels of dTMP kinase mRNA and its enzymatic activity fluctuate during the cell cycle, peaking at the S phase. Thus, like Saccharomyces cerevisiae CDC8 (encoding dTMP kinase), the human homolog mRNA and enzymatic activity are also cell cycle regulated. We have also examined four neuroblastoma cell lines for dTMP kinase mRNA levels and its kinase activities, which appear to vary according to cell growth rate. Our results suggest that the expression of the dTMP kinase gene and its activity coincide with various stages of cell growth. The identification of the human dTMP kinase gene and expression of its product in the baculovirus expression system should facilitate study of the mechanism of gene regulation and its role in pyrimidine metabolism.

Amino Acid Sequence↗
PubMed1993

Characteristics, substrate analysis, and intracellular location of Saccharomyces cerevisiae UMP kinase.

The yeast Saccharomyces cerevisiae SOC8 DNA fragment was isolated as a wildtype dominant suppressor of the cdc8 mutation. The SOC8 has previously been proved to be allelic with URA6, encoding the UMP kinase in yeast cells. The protein has been purified to homogeneity. In this report, we describe the characteristics of the UMP kinase from yeast. The yeast enzyme requires a divalent cation and is active over the entire range of pH from 6 to 9.5. The enzyme can use UMP and dUMP as phosphate acceptors with high activity; to a lesser extent, it can also use IMP, GMP, dGMP, 5-iodo-dUMP, XMP, and dTMP as substrates. ATP and dATP are the best phosphate donors; the enzyme could use GTP, dGTP, dCTP, and dTTP to some degree (30-50%). CTP and UTP were poor phosphate donors for the UMP kinase reaction (10-14%). Like other monophosphate kinases, UMP kinase contains a conserved nucleotide-binding site at its N-terminus following a cysteine residue, and its enzymatic activity is inhibited by sulfhydryl inhibitors such as 5,5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide. Subcellular localization studies indicate that the UMP kinase locates primarily in the cytoplasm (approximately 80%) and also in the nucleus (approximately 20%), but not in the mitochondria. These results suggest that it may exert its function in the nucleus, such as in RNA synthesis, as well as in the cytoplasm, but not in the mitochondria. The presence of UMP kinase in the nucleus might facilitate its suppression of cdc8 mutant cells, which are defective in nuclear DNA synthesis.

Amino Acid Sequence↗
PubMed1990

CDC6 mRNA fluctuates periodically in the yeast cell cycle.

Using cultures synchronized by two independent procedures, alpha-factor arrest and centrifugal elutriation, we have investigated the expression of the Saccharomyces cerevisiae CDC6 gene through the cell cycle. Our results show that the CDC6 gene is periodically expressed in the yeast cell cycle. The level of CDC6 transcripts increases in late G1, reaching a peak (approximately 10-20-fold over the initial level) at about the G1/S phase boundary. The peak of CDC6 mRNA was observed to overlap or slightly precede that of the CDC8 message, and to obviously precede that of the histone H2A message by some 25 min. Unlike histone H2A mRNA, the CDC6 mRNA as well as CDC8 mRNA were not affected by hydroxyurea treatment. These results suggest that regulation of H2A mRNA is different from that of CDC6 or CDC8. We have studied the 5'-flanking regions of CDC6 and other cell cycle-regulated genes. DNA sequence analysis of the CDC6 promoter revealed two sequences, 5'-C/GACGCGNC/G-3' and 5'-PuGNAGAAA-3' (where Pu is a purine, and N is any nucleotide), which are repeated three times each. Similar sequence elements have also been found among several cell cycle-regulated genes, including the CDC8 gene, but are not found upstream of histone genes. The possible significance of these elements is discussed.

Base Sequence↗
PubMed1989

Human deoxycytidine kinase. Sequence of cDNA clones and analysis of expression in cell lines with and without enzyme activity.

Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.

Amino Acid Sequence↗
PubMed1989

Isolation of multiple types of plasminogen activator inhibitors from vascular smooth muscle cells.

Large arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-1) bound to heparin and was eluted from the column at NaCl concentrations of 0.1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in single heparin affinity purification step.

Amino Acid Sequence↗
PubMed1986

Yeast DNA replication.

We have explored various strategies for exploiting the yeast genetic and biochemical system for understanding DNA replication. Because of the long time that has intervened between the isolation of random replication mutants of yeast and the identification of the gene products affected, an alternative approach to elucidating the molecular basis of replication has been needed. One such alternative involves purifying replication proteins, isolating the genes encoding them, and constructing the appropriate mutant from the cloned gene. We have applied this "reverse genetics" strategy to three different replication activities: DNA polymerases, single-stranded DNA binding proteins, and proteins that bind to autonomously replicating sequence (ARS) elements.

DNA Polymerase I↗
PubMed1982

The teaching of ethics in the dental hygiene curriculum.

This study describes the extent of formal instruction in ethics in dental hygiene programs. Responses from 159 programs indicated that all hygiene programs include ethics in the curriculum, but the method of teaching and the amount of time devoted to the subject varied considerably. The majority of programs included ethics as a component of a larger course such as dental hygiene practice or as a separate course in ethics and jurisprudence. Although most course instructors used the lecture format, most programs combined lecture with more active forms of learning, such as use of ethical dilemmas. In general, respondents felt satisfied with the current role of ethics in the dental hygiene curriculum, although there was some concern about their own competence in teaching the subject.

Curriculum↗