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R Knippers

Publications and source records attributed to R Knippers.

At least 73 records · Page 4Linked to original sources
PubMed1992

Replication of SV40 minichromosomes in vitro.

We operationally define two forms of SV40 minichromosomes, a 75S-form, prepared at low salt concentration, referred to as native minichromosomes, and a 50S-form, obtained after treatment with 0.5 M potassium acetate, the salt-treated minichromosomes. Both preparations of minichromosomes serve well as templates for replication in vitro. Their respective replication products are strikingly different: replicated native minichromosomes contain a densely packed array of the maximal number of nucleosomes whereas replicated salt-treated minichromosomes carry, on average, half of the maximal number. We conclude that in both cases parental nucleosomes are transferred to progeny DNA, and, in addition, that an assembly of new nucleosomes occurs during the replication of native minichromosomes. This is apparently due to the presence of a nucleosome assembly factor as a constituent of native minichromosomes that dissociates upon treatment with salt. We further show that preparations of minichromosomes usually contain significant amounts of copurifying hnRNP particles and SV40 virion precursor particles. However, these structures do not detectably affect the replication and the chromatin assembly reactions.

Acetates↗
PubMed1992

The interaction of SV40 large T antigen with unspecific double-stranded DNA: an electron microscopic study.

T antigen, an early protein encoded by simian virus 40 (SV40), is a specific DNA-binding protein with high affinity for elements in the viral origin of replication where it forms a double-hexameric complex as a prerequisite for DNA untwisting and, in the presence of ATP hydrolysis, for DNA unwinding. Like other specific DNA-binding proteins, T antigen also associates with DNA strands of random sequence albeit at reduced affinity. In addition, T antigen is able to unwind unspecific DNA sequences starting from internal binding sites. This property could be a step in the pathway leading to the chromosomal rearrangements that are frequently observed in SV40-transformed cells. This possibility prompted us to investigate the binding of T antigen to unspecific DNA using electron microscopy. We observed that the protein binds randomly to many unspecific DNA sites excluding a preference for particular DNA sequences or structural features. Addition of ATP to the binding buffer induces the formation of oligomeric, possibly hexameric, T antigen complexes that frequently align to form long arrays of DNA-bound protein. Magnesium salts induce the formation of tightly packed T antigen aggregates which bind to DNA to form many DNA branches and loops that emanate from the aggregated protein core. Upon ATP hydrolysis, aggregated T antigen catalyzes the unwinding of DNA duplices.

Adenosine Triphosphate↗
PubMed1991

Binding of human glutaminyl-tRNA synthetase to a specific site of its mRNA.

The human glutaminyl-tRNA synthetase is able to bind to its own mRNA. The enzyme contains two binding regions. One is located in the central section of the enzyme which includes its most hydrophilic portion with ten lysine residues in a block of 20 amino acids. This part of the enzyme binds unspecifically to all RNA sequences tested. A second binding region is located in that part of the enzyme which shows high degrees of sequence similarities with the bacterial and yeast glutaminyl-tRNA synthetases, and which is most likely responsible for the charging of tRNA with glutamine. This second RNA binding region specifically interacts with a site in the 3' noncoding region of the synthetase's mRNA. The binding site in the mRNA is characterized by an extended secondary structure that includes elements of the 'identity set' of nucleotides recognized by the enzyme when interacting with tRNA. We discuss possible physiological implications of the interaction between glutaminyl-tRNA synthetase and its mRNA.

Base Sequence↗
PubMed1991

Purification and characterization of a novel 5' exodeoxyribonuclease from the yeast Saccharomyces cerevisiae.

We have isolated from yeast cells an exonuclease which preferentially attacks double-stranded DNA from the 5' ends producing 5'-mononucleotides as reaction products. A second typical product is a full-length single-stranded DNA complement, suggesting that the enzyme hydrolyzes one DNA strand in a processive manner before it associates with another DNA substrate to initiate a new reaction cycle. Its biochemical properties suggest that the enzyme is unlike the yeast exonucleases which have been reported so far. However, the new exonuclease is strikingly similar to the well characterized 5' exonuclease of bacteriophage lambda.

Centrifugation, Density Gradient↗
PubMed1991

Structure of the human type I DNA topoisomerase gene.

We describe the molecular organization of the human gene coding for type I DNA topoisomerase. The coding sequence is split into 21 exons distributed over at least 85 kilobase pairs (kb) of human genomic DNA. The sizes of the 20 introns vary widely between 0.2 and at least 30 kb and all contain the sequence elements known to be required for pre-mRNA splicing. Several of the intron sequences separate exons encoding parts of the enzyme that are highly conserved between human and yeast suggesting that at least some of the exons may code for individual, structurally, or functionally important domains of the enzyme. We also describe the promoter sequence of the human topoisomerase I gene and show that it is composed of distinct functional elements.

Amino Acid Sequence↗
PubMed1991

The primary structure of human glutaminyl-tRNA synthetase. A highly conserved core, amino acid repeat regions, and homologies with translation elongation factors.

We describe the nucleotide sequences of several overlapping cDNA clones specific for human glutaminyl-tRNA synthetase. The identified open reading frame indicates that the enzyme is composed of 1440 amino acids. A stretch of about 360 amino acids of the human enzyme is highly conserved in bacterial and yeast glutaminyl-tRNA synthetases. However, the human enzyme is three times larger than the bacterial and twice as large as the yeast enzyme suggesting that a considerable part of human glutaminyl-tRNA synthetase has evolved to perform functions other than the charging of tRNA. The sequence outside of the conserved core region includes three 57-amino acid repeats followed by a consecutive stretch of 11 charged amino acids. A computer assisted search of two protein data banks reveals that the human glutaminyl-tRNA synthetase shares small blocks of amino acid similarities with several other synthetases of different amino acid specificities. Interestingly, the enzyme also possesses some regions of similarities with eukaryotic translation elongation factor EF-1 but not with any other sequence stored in the protein data banks. The coding regions of human and mouse glutaminyl-tRNA synthetase cDNAs are identical at 94% of the codons. However, the 3'-noncoding regions of mouse and human mRNAs are more divergent (approximately 68%) but both possess the potential to form stable secondary structures of similar general architecture.

Amino Acid Sequence↗
PubMed1991

Simian-virus-40 large-T-antigen-catalyzed DNA and RNA unwinding reactions.

Simian virus 40 large T antigen is a helicase separating the complementary strands of double-stranded DNA in the presence of hydrolyzable ATP and of double-stranded RNA in the presence of non-ATP nucleotides (GTP, CTP or UTP). We have constructed partially single-stranded nucleic acid substrates consisting of RNA or DNA strands hydrogen bonded to either RNA or DNA complements. We found that ATP is utilized as a cofactor for the T-antigen-catalyzed unwinding reaction when the substrates contain overhanging single-stranded DNA, regardless of whether the double-stranded region is DNA or hybrid DNA.RNA. Conversely, non-ATP nucleotides are used when the overhanging single strand is RNA. Based on these and additional findings, we propose that the bound nucleic acid induces a conformational change in T antigen resulting in a proper orientation of both nucleic acid and nucleotide relative to the active center of the ATPase/helicase domain of the enzyme. The implications of our conclusion for the roles which T antigen may play in vivo are discussed.

Antibodies, Monoclonal↗
PubMed1991

The Q300 gene: a novel transcription unit induced in simian virus 40-infected and -transformed mouse cells.

The Q300 element is a single-copy 233-bp genomic mouse DNA fragment carrying a high-affinity binding site for the simian virus 40 (SV40) large T antigen. This element was used to screen an EMBL3 mouse genomic library. We could identify a genomic clone containing an approximately 500-bp transcribed region flanking the Q300 element. The transcribed region, termed the Q300 transcription unit or Q300 gene, is overexpressed in acutely SV40-infected or SV40-transformed mouse and rat cells. The Q300 gene includes an open reading frame which has the coding potential for a small polypeptide with an extremely hydrophobic N terminus and a hydrophilic C terminus. The deduced polypeptide has some similarity with the papillomavirus E5 oncoprotein.

Amino Acid Sequence↗
PubMed1991

Transfer of nucleosomes from parental to replicated chromatin.

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.

Binding, Competitive↗
PubMed1990

Structural characterization of the human DNA topoisomerase I gene promoter.

We have isolated a genomic DNA fragment from HeLa cells containing the promoter region and the first two exons of the human gene encoding DNA topoisomerase I (hTOP1). Transcription of hTOP1 mRNA initiates at multiple sites which are clustered 247 nucleotides and 210 nucleotides upstream of the translation-initiation site of the protein coding region. The nucleotide sequence of the region preceding the transcription-initiation sites is G/C rich and contains sequence motifs which are known binding sites of the transcription factors Oct1 (octameric transcription factor 1), Sp1 and AP2 (activator protein 2). Furthermore, one cAMP-responsive element is present 50 nucleotides upstream of the transcription-initiation site nearest the 5' end. Neither TATA nor CAAT boxes were found in the promoter region of the hTOP1 gene. A 918-bp fragment containing the sequence elements described above drives the transient expression of a chloramphenicol acetyl transferase (CAT) gene sequence in transfected HeLa and 293 cells. In addition we analyzed a 10-kb fragment containing the promoter and exons 1 and 2 for regions of DNase I hypersensitivity. We detected one prominent DNase-I-hypersensitive region in the promoter close to the putative transcription-factor-binding sites and several weaker regions in intron 2.

Autoradiography↗
PubMed1990

Glutaminyl-tRNA synthetase as a component of the high-molecular weight complex of human aminoacyl-tRNA synthetases. An immunological study.

The human glutaminyl-tRNA synthetase is three times larger than the corresponding bacterial and twice as large as the yeast enzyme. It is possible that the additional sequences of the human glutaminyl-tRNA synthetase are required for the formation of the multienzyme complex which is known to include several of aminoacyl-tRNA synthetases in mammalian cells. To address this point we prepared antibodies against three regions of the human glutaminyl-tRNA synthetase, namely against its enzymatically important core region, and against two sections in its large C-terminal extension. In intact multienzyme complexes the core region was accessible to specific antibody binding. However, the C-terminal sections became available to specific antibody binding only when certain components of the multienzyme complex were either absent or degraded. These findings allow first conclusions as to the relative position of some components in the mammalian aminoacyl-tRNA synthetase complex.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Molecular structures of two human DNA topoisomerase I retrosequences.

We have isolated recombinant lambda-phage clones that contain sequences complementary to the 3' half of the cDNA encoding human topoisomerase I (hTOP1). These lambda clones belong to three distinct classes: class-I clones contain sequences from the active gene located on human chromosome 20. Class-II and class-III clones contain sequences corresponding to the cDNA encoding hTOP1 from nucleotide (nt) 2208 to 3434 and from nt 1639 to 3434, respectively. These sequences exhibit the characteristic features of retroposons or retrosequences. They are most likely derived from truncated mRNA transcripts of the active gene. We propose to designate the truncated hTOP1 sequence located on chromosome 1 as the pseudogene 1 (psi 1-hTOP1) and the sequence on chromosome 22 as the pseudogene 2 (psi 2-hTOP1). Pseudogene psi 1-hTOP1 has two unique properties: it is flanked by upstream sequences which display promoter activity in transient expression assays, and it contains an open reading frame which could code for the 211 C-terminal amino acids of hTOP1. Pseudogene psi 2-hTOP1 is located within an AluI repetitive element and is flanked on one side by a (CA)21 stretch.

Base Sequence↗
PubMed1990

The human QARS locus: assignment of the human gene for glutaminyl-tRNA synthetase to chromosome 1q32-42.

We have used a cDNA encoding the core region of the human glutaminyl-tRNA synthetase to determine the chromosomal localization of the corresponding gene. Southern blots of restricted DNA from a panel of rodent-human cell lines and in situ chromosome hybridization gave identical results showing that the human gene locus for glutaminyl-tRNA synthetase resides on the distal long arm of chromosome 1. There are now nine mapped aminoacyl-tRNA synthetase genes in the human genome.

Animals↗
PubMed1990

An SV40 large T antigen binding site in the cellular genome is part of a cis-acting transcriptional element.

A genomic mouse DNA fragment (Q300), containing a high affinity binding site for SV40 large T antigen, serves as a cis-acting transcriptional element in in vivo and in in vitro studies. We have performed experiments to investigate whether bound T antigen could modulate the promoter-enhancer activity of the Q300 element. In vivo studies showed a negative effect of T antigen on the Q300 driven expression of the chloramphenicol acetyl transferase reporter gene. Band shift and DNAase I protection experiments demonstrated that T antigen and a nuclear protein, probably a CCAAT-binding factor, can simultaneously bind to closely adjacent sites on the Q300 DNA. Transcription studies in vitro showed that bound T antigen suppresses the transcriptional enhancer effect of the Q300 element. We interpret the results of these model studies to indicate that T antigen, bound to DNA, is able to affect the function of a cellular cis-acting transcriptional element. Bound T antigen may influence the activity of a cellular transcription factor at a closely adjacent DNA site.

Animals↗
PubMed1989

Effects of the cellular p53 protein on Simian-virus-40-T-antigen-catalyzed DNA unwinding in vitro.

It is known that large T antigen, the regulatory protein encoded by Simian virus 40 (SV40), forms tight complexes with the cellular p53 protein in SV40-transformed rodent cells. Using immunoaffinity procedures we have purified large T antigen and, in separate experiments, the cellular p53 protein. The two proteins formed complexes in vitro which bound well to double-stranded DNA fragments although in a sequence-unspecific manner. Free, uncomplexed T antigen readily converted double-stranded DNA into a single-stranded form whereas in-vitro-formed p53-T-antigen complexes were inactive in this reaction. We conclude that one function of p53 in SV40-transformed mouse cells could be the inhibition of the replication initiating activity of T antigen.

Animals↗
PubMed1989

RNA unwinding activity of SV40 large T antigen.

Large T antigen, the regulatory protein encoded by simian virus 40, has DNA helicase activity and unwinds double-stranded DNA at the expense of ATP. T antigen also functions as an RNA helicase separating duplex regions in partially double-stranded RNA substrates. Surprisingly, T antigen RNA helicase activity requires UTP, CTP, or GTP as a cofactor, whereas ATP is an inefficient energy source for the RNA unwinding reaction. Accordingly, T antigen has both an intrinsic non-ATP NTPase activity that is stimulated by single-stranded RNA and an ATPase activity stimulated by single-stranded DNA. Thus, it appears that the bound nucleotide determines whether T antigen acts as an RNA helicase or as a DNA helicase.

Antigens, Polyomavirus Transforming↗
PubMed1989

DNA binding properties and replication activity of the T antigen related D2 phosphoprotein.

According to earlier genetic experiments, a region within the N-terminal 50-100 amino acids may be important for the replication function of T antigen, the initiator protein of simian virus 40 (SV40). We have investigated this possibility using the T antigen related D2 protein in several biochemical assay systems. D2 protein, a phosphoprotein coded for by the adeno-SV40 hybrid virus Ad2+D2, shares its 594 C-terminal amino acids with authentic T antigen and its 104 N-terminal amino acids with an adenovirus structural protein. We confirmed earlier studies showing that D2 protein appeared to bind well to specific binding sites in the SV40 origin of replication. We found, however, that D2 protein was rather inefficient, inducing the unwinding of the double-stranded origin region, and was much less active than authentic T antigen as an initiator of in vitro SV40 DNA replication. We interpret these findings to indicate that D2 protein molecules associate with the origin to form an aberrant complex that is quite inefficient, inducing DNA unwinding and the establishment of replication forks. The possibility that the N-terminus may be required for an optimal arrangement of T antigen at the origin was supported by results of dephosphorylation studies. Dephosphorylation of N-terminal phosphoamino acids had significant effects on the stability of D2 protein-origin complexes.

Antigens, Viral, Tumor↗