PubMed Health⌕ Search

Biomedical subjects

S Dalton

Publications and source records attributed to S Dalton.

At least 19 recordsLinked to original sources
PubMed1997

Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex.

In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.

Animals↗
PubMed1996

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.

We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.

Amino Acid Sequence↗
PubMed1995

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Amino Acid Sequence↗
PubMed1995

Cdc54 belongs to the Cdc46/Mcm3 family of proteins which are essential for initiation of eukaryotic DNA replication.

CDC54 is a gene essential for initiation of DNA replication in Saccharomyces cerevisiae, and which is known to genetically interact with other regulators of the S-phase, including CDC46. We describe the isolation and sequencing of CDC54 and show that it encodes a protein structurally related to Cdc46p, Mcm2p and Mcm3p by the presence of a conserved domain of 145 amino acids which is internal to each polypeptide. This conserved domain resembles the DEAD box of RNA helicases and is similar to the conserved domain associated with a group of transcription and replication factors with known or assumed DNA-dependent ATPase activity, suggesting it may be involved in nucleic-acid recognition. Comparison of Cdc54p to related proteins from other species revealed that it closely resembles cdc21p from Schizosaccharomyces pombe.

Amino Acid Sequence↗
PubMed1995

Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor.

Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae. We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation. Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutation which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo. A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex. A point mutation in the CLB2 UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control of CLB2 transcription. We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.

Base Sequence↗
PubMed1995

Human shoulder tendon biopsy samples in organ culture produce procollagenase and tissue inhibitor of metalloproteinases.

OBJECTIVE: To investigate the production of the matrix metalloproteinase (MMP), collagenase (MMP-1), and its natural inhibitor, the tissue inhibitor of metalloproteinases (TIMP) by diseased human tendon samples in organ culture. METHODS: Portions of tendons were excised from the shoulders of patients undergoing shoulder surgery, classified as either proximal to the lesion (abnormal) or distal to the lesion (normal) according to their macroscopic appearance at surgery, and placed in organ culture for periods of up to 28 days. The release of collagenase and TIMP activity in the conditioned culture medium was measured. RESULTS: Procollagenase and TIMP were both produced by all the tendon samples for an extended period of time. The levels of enzyme and inhibitor varied between patients, but in most of them TIMP levels were greater than collagenase levels. In one sample of calcified tendon, procollagenase levels were greater than those of TIMP. The mean level of collagenase produced by tendon proximal to the lesion and tendon distal to the lesion were not significantly different (95.2 (SD 106.8) U/g and 34.0 (45.3) U/g, respectively), while the corresponding figures for TIMP were 109.7 (62.3) U/g and 53.0 (27.9) U/g (p = < 0.05), although there was considerable variation in some samples. Western blotting and collagen fragment analysis confirmed that the collagenolytic activity detected was attributable to the metalloproteinase fibroblast collagenase (MMP-1). CONCLUSIONS: Tendon tissue can actively secrete procollagenase, an enzyme that, once activated, is capable of remodelling collagen, the major connective tissue component of tendon. Collagenase is produced even in unstimulated cultures, although the concentrations of TIMP are usually greater than that of collagenase in most samples. Some activation of collagenase appeared to have occurred. These results indicate that tendon tissue cells are capable of producing a remodelling response, even in end stage tendon disease.

Adult↗
PubMed1994

Mapping of the human SAP1 (SRF accessory protein 1) gene and SAP2, a gene encoding a related protein, to chromosomal bands 1q32 and 12q23, respectively.

SAP1, SAP2, and ELK1 form a related subgroup of ETS-domain proteins that can form ternary complexes with the transcription factor SRF at the c-fos serum response element (SRE). SAP1 was identified by a genetic screen for proteins interacting with SRF expressed in yeast, and SAP2 by its homology with SAP1; ELK1 was previously identified by its homology to the ETS domain. cDNA probes were used to isolate cosmid and phage clones harboring genes encoding SAP1 and SAP2. These clones were subsequently used to map the genes to 1q32 and 12q23, respectively, by fluorescence in situ hybridization.

Chromosome Mapping↗
PubMed1993

Isolation and characterization of SRF accessory proteins.

Many genes which are regulated by growth factors contain a common regulatory element, the serum response element (SRE). Activation of transcription by the SRE involves a ternary complex formed between a ubiquitous factor, serum response factor (SRF), and a second protein, p62/TCF. We used a yeast genetic screen to isolate cDNAs encoding a protein, SAP-1, with the DNA binding properties of p62/TCF. The SAP-1 sequence contains three regions of homology to the previously uncharacterized Elk-1 protein, which also acts as an SRF accessory protein. Only two of these regions are required for cooperative interactions with SRF in the ternary complex. The third contains several conserved sites for the MAP kinases, whose activity is regulated in response to growth factor stimulation. We discuss the potential role of these proteins in regulation of the c-fos SRE.

Amino Acid Sequence↗
PubMed1993

Functional analysis of a growth factor-responsive transcription factor complex.

Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory factor, Elk-1. We constructed altered-binding specificity derivatives of SRF and Elk-1 that form a ternary complex at a mutated, inactive SRE; like Elk-1, the Elk-1 variant only binds its target as part of a ternary complex with SRF. Simultaneous expression of these SRF and Elk-1 derivatives restores serum-regulated activity to the mutated SRE in transfected cells. Efficient transcriptional activation is dependent on the regulated phosphorylation of Elk-1 C-terminal MAP kinase sites and requires the C-terminal sequences of SRF as well as SRF sequences that mediate ternary complex formation. These experiments provide direct evidence that SRF and Elk-1 functionally cooperate in the ternary complex at the SRE to regulate transcription.

3T3 Cells↗
PubMed1993

Factors predicting death, survival and functional outcome in a prospective study of early rheumatoid disease over fifteen years.

Sixty-four survivors from a prospective study of early rheumatoid disease were assessed again at a mean of 15.2 years from presentation and their status compared with 29 patients who had died. Eleven of the dead and only two of the survivors had been treated with steroids. There was a small increase in mortality due to the disease itself but only one death was directly caused by it. As might be expected, those who died were older. In the first year of disease, they had lower haemoglobin levels, a lower body mass, higher sedimentation rates and higher levels of blood urea. One-fifth at entry to the study and two-fifths by the time of death, had poor functional capacity. Of 64 survivors, six had poor functional capacity at entry and nine after 15 years. Discriminant analysis was performed to identify the most powerful combination of early features predicting a poor functional outcome. A combination including early erosive change, seropositivity, poor grip strength and cervical subluxation predicted the outcome correctly in 73% of survivors. Almost 60% of survivors remained with or improved to normal function at 15 years suggesting that morbidity is not as bad as has been suggested in the past.

Adult↗
PubMed1993

Obesity surgery: dietary and psychosocial expectations and reality.

Psychosocial and dietary habits have been compared in patients considering (Pre n = 33) or who had (Post n = 32) surgery for morbid obesity. Failure of conventional diets was attributed to lack of self-discipline (Pre 92% [22/24] vs. Post 87% [20/23], NS). Consumption of fast foods fell (Pre 70% [23/33] vs. Post 16% [5/32], p < 0.0001). Preoperative patients had unrealistic social expectations. They exaggerated the prospect of improved friendship (Pre 67% [22/32] vs. Post 34% [11/21], p < 0.05), erroneously anticipated better sex (Pre 78% [25/32] vs. Post 50% [15/30], p < or = 0.05), predicted better acceptance at work (Pre 85% [23/27] vs. Post 50% [15/30], p < or = 0.05), and misanticipated improved relationship with their partner (Pre 77% [20/26] vs. Post [47% 8/17], p < or = 0.05). Two factors predicted becoming employed following surgery: age (became employed [n = 5] 28 +/- 2 years vs. remained unemployed [n = 12] 44 +/- 4 years, p < 0.05) and percentage of excess weight lost (became employed 76 +/- 11 vs. remained unemployed 51 +/- 7, p < 0.05). The free support group was "useful" (17/17), yet only 5% attended regularly. Patients considering obesity surgery had specific unrealistic psychosocial expectations. They infrequently availed themselves of postoperative professional help. We identify the features associated with gaining employment.

Adult↗
PubMed1992

Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max.

The c-myc protein (Myc) contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix-leucine zipper (bHLH-Z) domain that directs dimerization of Myc with its partner, the max protein (Max), and promotes DNA binding to sites containing a CACGTG core consensus sequence. Despite these characteristics and the observation that Myc can modulate gene expression, a direct role for Myc or Max as transcription factors has never been demonstrated. Here we use Saccharomyces cerevisiae as an in vivo model system to show that the Myc protein is a sequence-specific transcriptional activator whose DNA binding is strictly dependent on dimerization with Max. Transactivation is mediated by the amino-terminal domain of Myc. We find that Max homodimers bind to the same DNA sequence as Myc+Max but that they fail to transactivate and thus can antagonize Myc+Max function. We also show that the Max HLH-Z domain has a higher affinity for the Myc HLH-Z domain than for itself, and suggest that the heterodimeric Myc+Max activator forms preferentially at equilibrium.

Base Sequence↗
PubMed1992

Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element.

We used a yeast genetic screen to isolate cDNAs that encode a protein, SRF accessory protein-1 (SAP-1), that is recruited to the c-fos serum response element (SRE) as part of a ternary complex that includes serum response factor (SRF). SAP-1 requires DNA-bound SRF for ternary complex formation and makes extensive DNA contacts to the 5' side of SRF, but does not bind DNA autonomously. Ternary complex formation by SAP-1 requires only the DNA-binding domain of SRF, which can be replaced by that of the related yeast protein MCM1. We isolated cDNAs encoding two forms of SAP-1 protein, SAP-1a and SAP-1b, which differ at their C termini. Both SAP-1 proteins contain three regions of striking homology with the elk-1 protein, including an N-terminal ets domain. Ternary complex formation by SAP-1 requires both the ets domain and a second conserved region 50 amino acids to its C-terminal side. SAP-1 has similar DNA binding properties to the previously characterized HeLa cell protein p62/TCF.

Amino Acid Sequence↗