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F Grummt

Publications and source records attributed to F Grummt.

At least 19 recordsLinked to original sources
PubMed1999

Identification and characterization of mouse homologue to yeast Cdc7 protein and chromosomal localization of the cognate mouse gene Cdc7l.

The Cdc7 kinase is required for the G1/S-phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae. Here, we report the identification of a mouse cDNA, MmCdc7, whose product is closely related in sequence to Saccharomyces cerevisiae Cdc7 as well as their human, Xenopus and Schizosaccharomyces pombe homologues. The MmCdc7p contains the conserved subdomains common to all protein-serine/threonine kinases and three kinase inserts that are characteristic of members of the Cdc7 protein family. We have mapped the locus of the MmCdc7 gene to chromosome 5, band 5E. Conservation of structures among members of the Cdc7-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes.

Amino Acid Sequence↗
PubMed1999

Identification and characterization of MmORC4 and MmORC5, two subunits of the mouse origin of replication recognition complex.

Two new members of the mouse origin recognition complex (ORC) have been cloned that are closely related to Saccharomyces cerevisiae ORC4 and ORC5 as well as to their human homolog. Both MmORC4p and MmORC5p have a putative nucleotide triphosphate binding motif. Transcription of MmORC4 and MmORC5 is not suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. The transcription levels of both ORC genes are constantly high in all phases of the cell cycle. A screen based on the two-hybrid approach suggests that the product of the ORC4 gene interacts with the ORC2, but not with the ORC1 protein. The conservation of structure among members of the ORC4- and ORC5-related family of proteins suggests that these proteins play a key role in the initiation of DNA replication in all eukaryotes.

3T3 Cells↗
PubMed1999

Identification, characterization and chromosomal localization of the cognate human and murine DBF4 genes.

The kinase Dbf4p/Cdc7p is required for the G1/S phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae. Here, we report the identification and characterization of mouse and human cDNAs whose products are related in sequence to Saccharomyces cerevisiae DBF4 cDNA. Both mammalian Dbf4 proteins contain a putative site for phosphorylation by CDK, PEST protease cleavage sites, nuclear localization signals and a short-looped zinc finger-like domain. Transcription of MmDBF4 is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. Upon replenishment of the medium, transcript levels increase during progression through G1, peaking as cells enter S phase. MmDbf4p interacts physically with Cdc7p and Mcm2p in vivo. Using fluorescence in situ hybridization (FISH), the human DBF4 gene was localized to chromosome 7 (q21.3), whereas FISH mapped the murine counterpart to band A2 on chromosome 5. The results of chromosome mapping indicate that in both mouse and human the gene is present as a single copy. The structural conservation between Dbf4-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes.

3T3 Cells↗
PubMed1999

Identification and characterization of a mouse homolog to yeast Cdc6p.

Periodic expression of the Cdc6 protein is essential for the entry of budding yeast cells into S phase, and also for participating in checkpoint controls that ensure that DNA replication is completed before mitosis is initiated. We have identified a mouse protein closely related to Cdc6p (MmCdc6p) as well as to its human and Xenopus homologs. The gene coding for MmCdc6p (Cdc6) is located at band D on murine chromosome 11. Analysis of its genomic region revealed that the 13-kb Cdc6 gene is divided into 12 exons by 11 introns. MmCdc6p has putative cyclin-dependent phosphorylation sites, a destruction box, nuclear localization signals, a nucleotide triphosphate-binding motif, and a potential leucine zipper. None of these consensus motifs except the leucine-zipper and the destruction box overlaps an intron. Expression of MmCdc6 mRNA and protein is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. Upon replenishment of the medium, transcript and protein levels increase during progression through G(1), peaking as cells enter S phase. MmCdc6p is phosphorylated in vitro by cdk1/cyclin B, cdk4/cyclin D, cdk2/cyclin E, and cdk2/cyclin A, respectively at serine-residues. In vivo however, phosphorylation of MmCdc6p is carried out by cdk2/cyclin A at serine-residues exclusively. Conservation of structures among members of the Cdc6-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes. These results strongly suggest, that Cdc6p plays an important role in cell cycle regulation and replication licensing.

Amino Acid Sequence↗
PubMed1998

Activation of SV40 DNA replication in vivo by amplification-promoting sequences of the mouse ribosomal gene cluster.

Mouse genomic DNA sequences from the region upstream of the ribosomal genes reported to contain amplification-promoting activity (APS1 and APS2) were tested for their ability to support SV40 replication in vivo. Both APSs can functionally replace the auxiliary sequence 2 on the late side of the SV40 origin, which facilitates initiation of replication. This stimulatory activity on the SV40 origin was orientation independent and was exerted over a distance of at least 0.9 kb. However, the APS2 element can not substitute for the auxiliary sequence 1 on the early side of the origin. Although both APS elements contain various prospective transcription factor binding sites, they do not trans-activate transcription from a minimal promoter. In this respect they differ from the enhancer element of the auxiliary sequence 2 of the SV40 origin. These results suggest that the rDNA sequences tested contain genetic information facilitating replication initiation on a viral core origin.

Animals↗
PubMed1998

Mouse homolog of the yeast origin recognition complex subunit ORC1 and chromosomal localization of the cognate mouse gene Orc1.

ORC1 encodes a subunit of the eukaryotic origin recognition complex in yeast, which has been shown to play a crucial role in chromosomal DNA replication and transcriptional silencing. We have identified a mouse protein closely related to Saccharomyces cerevisiae ORC1 as well as to the human, Xenopus and Drosophila homologs. MmORC1 has putative cyclin-dependent phosphorylation sites and a nucleotide triphosphate-binding motif. We have mapped the gene locus of the murine Orc1 gene to chromosome 4, band D. Conservation of structures among members of the ORC1-related proteins suggests that these proteins play a key role in the intiation of DNA replication in all eukaryotes.

Amino Acid Sequence↗
PubMed1997

Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I.

A replication fork barrier (RFB) at the 3' end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3' terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I-specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

3T3 Cells↗
PubMed1997

Mapping replication origins by nascent DNA strand length.

The mapping of replication origins by nascent DNA strand length determination is a very sensitive generally applicable method that identifies even single-copy origins in mammalian chromosomes. A major advantage of this procedure is that there is no need for synchronization of cells or treatment with metabolic agents, which allows the origin to be studied under physiological conditions. This technique is based upon the amplification of specific sequence markers on nascent DNA strands that initiated replication within the region of the putative origin. Therefore, this method requires detailed sequence information of the locus to be analyzed. As a first step, nascent DNA of proliferating cells is pulse-labeled with BrdU followed by size fractionation and purification with anti-BrdU antibodies. The position of putative origins can then be determined via identification of the shortest nascent strands that can be amplified by PCR and hybridized to probes homologous to the amplified segments. Here, we give a detailed description of the theory behind the method and a full recipe for its application. Advantages and limitations of the procedure are discussed.

Animals↗
PubMed1997

Temporal order of replication of mouse ribosomal RNA genes during the cell cycle.

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences.

3T3 Cells↗
PubMed1997

p53 activates Fanconi anemia group C gene expression.

The tumor suppressor protein p53 (wtp53) can bind to specific target sequences and activate transcription of genes adjacent to these DNA elements. Two p53 binding sites are present in the gene coding for the Fanconi anemia complementation group C (FAC), one in the promoter region (from -1295 to -1266) and one in the coding region of FAC (from +1828 to +1848). Gel shift experiments show that wtp53 binds to the p53 target sequence in the promoter region of the FAC gene. We have investigated whether binding of p53 to these target sites may affect expression of the FAC gene. Transfection experiments show that overexpression of wtp53 in human diploid fibroblasts and lymphoblasts augments transcription of the FAC gene up to three-fold. The transfection efficacy was approximately 15% for both cell types. The FAC expression activity per transformed cell was stimulated to an estimated level of 18- to 21-fold upon overexpression of p53. The tumor-derived p53 mutants, His175 and His273, that fail to bind DNA showed only a reduced stimulatory activity on FAC transcription. Luciferase assays demonstrated that interaction of p53 with its target site in the FAC promoter does not modulate the promoter activity. We suggest that the p53 binding site contributes to, but may not be an absolute prerequisite for p53-directed transcriptional activation. We conclude that the FAC gene can be added to the list of genes that interact with p53.

Binding Sites↗
PubMed1997

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.

Animals↗
PubMed1996

p53 inhibits JC virus DNA replication in vivo and interacts with JC virus large T-antigen.

The onset of DNA replication is an important step within the life cycle of the human neurotropic polyomavirus JC. In this report, evidence that both the human and the murine tumor suppressor protein p53 strongly inhibit JCV DNA replication in vivo is presented. This inhibition is dose-dependent and not a secondary effect of a decreased expression of JCV large T-antigen in response to p53. Using deletion mutants of murine p53 and tumor-derived point mutations of human p53, the basis of the suppression of JCV DNA replication by p53 was dissected. Deletion of either the amino- or the carboxy-terminal domain of murine p53 did not interfere with the repression of JCV DNA replication. However, deletion of the highly conserved central region of p53 abolished the inhibitory effect on replication. The tumor-derived human mutant p53(His273) inhibited JCV DNA replication significantly, whereas another tumorigenic mutant, p53(His175), had no inhibitory effect Concomitantly, a direct protein-protein interaction between p53 and JCV large T-antigen was lost in mutants which did not affect JCV DNA replication. These results strongly suggest that p53 inhibits JCV DNA replication by interacting with JCV large T-antigen.

Animals↗
PubMed1996

Mapping of replication initiation sites in the mouse ribosomal gene cluster.

We have used nascent strand determination analysis to map start sites of DNA replication in the mouse ribosomal gene cluster in which individual copies of the ribosomal genes are separated by intergenic spacer regions. One origin of bidirectional replication (OBR) was localized within a 3 kb region centered about 1.6 kb upstream of the rDNA transcription start site. At least one additional initiation site is situated near the 3' end of the transcription unit. Adjacent to the OBR at the transcription start site are located two amplification-promoting sequences, i.e., APS1 and APS2. Nuclease-hypersensitive sites were identified in both of the two APSs as well as in the OBR region, thus indicating that these sequences have an altered chromatin structure. In the OBR an intrinsically bent region, a purine-rich element and other prospective initiation zone components are found.

3T3 Cells↗
PubMed1996

Repression of interleukin-2 and interleukin-4 promoters by tumor suppressor protein p53.

Interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by activated but not by resting mature T cells are pleiotropic cytokines affecting growth and differentiation of diverse cell types, such as T cells, B cells, and mast cells. There is little information about the molecular basis for the constitutive repression of IL-2 and IL-4 gene expression in unstimulated T cells. We investigated the possibility that wild-type (wt) p53, a nuclear tumor suppressor protein, might serve to repress IL-2 and IL-4 gene expression in murine E14 T lymphoma and in human Jurkat cells. We transiently cotransfected these cells with constitutive simian virus 40 (SV 40) early promoter expression plasmids overproducing wt or mutant murine p53 and with appropriate luciferase (luc) reporter plasmids containing the promoter elements of murine IL-2 and IL-4 genes to evaluate the effect of various p53 species on these promoters. Murine wt p53 derived from pSG5p53cD strongly repressed the IL-2 and IL-4 promoters in both cell lines induced by the phorbol ester TPA and the Ca2+ ionophore ionomycin but not, however, in uninduced cells. In similar transient transfection experiments with lymphoma cells, overexpression of deletion mutant species of murine p53 revealed that the N-terminal and C-terminal domains are crucial for inhibition of both IL-2 and IL-4 gene expression. These parts of p53 comprise the transactivation domain at the amino terminal side, which has previously also been shown to interact with the TATA-box binding-protein TBP and the carboxy-terminal oligomerization domain. Additionally, it was shown that a previously described inhibitory protein, the high-mobility-group protein HMG-I/Y, does not functionally interact with p53. Cotransfection of expression plasmids for both p53 and HMG-I/Y did not alter the extent of inhibition by the individual proteins. These data suggest that p53 can downmodulate both IL-2 and IL-4 gene expression and that both the transactivation and oligomerization domains of the tumor suppressor protein are essential for this transcriptional repression.

Animals↗
PubMed1995

An intrinsically bent region upstream of the transcription start site of the rRNA genes of Arabidopsis thaliana interacts with an HMG-related protein.

Intrinsically bent DNA structures are thought to play crucial roles as functionally important modules in promoter/enhancer elements. Here we show the existence of a DNA bending centre within the intergenic region of the tandemly arranged rRNA genes of Arabidopsis thaliana. By use of a circular permutation assay, this bending centre was localized to position -284 to -256 upstream of the transcription start site. A 143 bp fragment containing the bent region was shown to interact with several nuclear proteins. Evidence is presented that one of these proteins is related to the high-mobility-group (HMG) proteins, a group thought to be involved in transcription and replication processes.

Arabidopsis↗
PubMed1995

Functional interaction between the POU domain protein Tst-1/Oct-6 and the high-mobility-group protein HMG-I/Y.

The POU domain protein Tst-1/Oct-6 is a transcriptional activator of human papovavirus JC virus in transient transfections. Because of its endogenous expression in myelinating glia, Tst-1/Oct-6 might also be an important determinant for the glia specificity of JC virus in vivo. Activation of viral early and late genes depends on the ability of Tst-1/Oct-6 to interact with an AT-rich element within the viral regulatory region. Here, we show that this element not only is bound by Tst-1/Oct-6 but, in addition, serves as a binding site for the high-mobility-group protein HMG-I/Y. In the presence of HMG-I/Y, Tst-1/Oct-6 exhibited an increased affinity for this AT-rich element. The specificity of this effect was evident from the fact that no stimulation of Tst-1/Oct-6 binding was observed on a site that did not allow binding of HMG-I/Y. In addition, both proteins interacted with each other in solution. Direct contacts were identified between the POU domain of Tst-1/Oct-6 and a short stretch of 10 amino acids in the central portion of HMG-I/Y. These results point to an accessory role for HMG-I/Y in the activation of JC viral gene expression by the POU domain protein Tst-1/Oct-6. In agreement with such a role, HMG-Y synergistically supported the function of Tst-1/Oct-6 in transient transfections, measured on the early promoter of JC virus or on an artificial promoter consisting of only a TATA box and the common binding element for Tst-1 and HMG-I/Y.

Base Sequence↗
PubMed1995

A novel type of unstable homogeneously staining region with a head-to-tail arrangement: spontaneous decay and reintegration of DNA elements into a plethora of new chromosomal sites.

After transfection of amplification-promoting DNA elements into mammalian cells, homogeneously staining regions (HSRs) are formed by high copy numbers of transfected DNA arranged in head-to-tail polymers. Here, we wanted to evaluate the stability of this type of HSR during prolonged cultivation of transfected cells in selective medium. Thymidine kinase-deficient mouse L cells were transfected with pAPR4tk DNA harboring the amplification-promoting element 4 (APR4) linked to the gene for thymidine kinase (TK) or, alternatively, transfected with a DNA construct (pARP4t-PA) carrying, in addition, the expression cassette for human tissue-type plasminogen activator (t-PA). After transfection, one or two HSRs per cell were formed that disintegrated spontaneously after 25-40 wk of continuous cultivation in the presence of selective HAT (hypoxanthine-aminopterin-thymidine) medium. Unexpectedly, plasmid DNA reinserted into a plethora of new chromosomal sites, as revealed by in situ hybridization and Southern blot analysis. Coincidently, secretion of t-PA decreased to 10-20% of its original level. After transfection of pAPR4tk DNA lacking the t-PA expression cassette, HSR decay and reintegration of plasmid constructs into multiple chromosomal sites were also observed, whereas the ptk vector without an amplification-promoting DNA element did not form an HSR after transfection. We conclude that, in contrast to the pattern of known structures with head-to-tail arrangements, the HSR formed by amplification-promoting DNA elements represents a novel type of HSR that disintegrates by transposition into a plethora of new chromosomal integration sites. This process is mediated by the amplification-promoting DNA element itself and can be observed even when selective pressure is maintained.

Animals↗