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G J Pruijn

Publications and source records attributed to G J Pruijn.

65 records · Page 4Linked to original sources
PubMed1991

Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes.

The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.

Base Composition↗
PubMed1991

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

Adenoviridae↗
PubMed1991

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren's syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren's syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren's syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.

Antibody Specificity↗
PubMed1988

An ACCC-containing protein-binding sequence in the neighbourhood of the decanucleotide recognition site of the immunoglobulin gene promoter.

The decanucleotide (dc) box TNATTTGCAT is an important functional element in the K light chain gene promoter and was shown to bind nuclear proteins. We now describe the binding of nuclear factor III to dc containing oligonucleotides and define a second protein binding site in the neighbourhood of dc which turned out to be recognized by a partially purified NFIII preparation but not by highly purified NFIII (abbreviations ref. 1). This site comprises an ACCC-containing sequence element. It was further characterized by the hydroxyl radical footprinting method which is applicable to 40 bp long oligonucleotides. The significance of the ACCC-containing sequence and of the previously described 15mer sequence pd is discussed.

Adenine↗
PubMed1988

Interaction between the octamer-binding protein nuclear factor III and the adenovirus origin of DNA replication.

Nuclear factor III (NFIII) is a HeLa sequence-specific DNA-binding protein that stimulates initiation of adenovirus DNA replication in vitro and may be involved in regulation of transcription of several cellular and viral genes. We have studied the interaction between NFIII and the binding site in the adenovirus type 2 (Ad2) origin in detail by methidiumpropyl-EDTA.iron(II) and hydroxyl radical footprinting and by alkylation interference experiments. Our results indicate that (i) the core of the recognition sequence is 5'-TATGATAAT-3'; (ii) both major and minor groove base contacts are detected, and all base pairs in the core are involved in binding; (iii) many backbone contacts are observed divided into a large domain coinciding with the core and a small domain; (iv) contact points are not confined to one side of the DNA helix in contrast to the nuclear factor I (NFI)-binding site; (v) the binding site overlaps the NFI-binding site for at least one nucleotide. A number of Ad2 mutants as well as related binding sites in the origins of other adenovirus serotypes were systematically compared for binding with NFIII. The results are in good agreement with the contact point studies and show that at least one AT base pair is commonly required by NFI and NFIII for optimal binding. The strongest binding site, which contains the octamer/decanucleotide motif (ATGCAAAT[NA]), was found in the Ad4 origin, which lacks an NFI-binding site. Stimulation of in vitro DNA replication of Ad2, Ad4, and Ad12 by NFIII showed that the maximal level of stimulation is dependent on the affinity of NFIII for the origin.

Adenoviruses, Human↗
PubMed1987

Promoter and enhancer elements containing a conserved sequence motif are recognized by nuclear factor III, a protein stimulating adenovirus DNA replication.

Nuclear factor III (NFIII) is a protein from HeLa cells that stimulates the initiation of adenovirus type 2 (Ad2) DNA replication by binding to a specific nucleotide sequence in the origin, adjacent to the nuclear factor I recognition site. DNA sequences sharing a high degree of homology to the NFIII binding site in Ad2 were found in a number of transcription regulatory elements, all containing the octanucleotide sequence ATGCAAAT. We have analysed the interaction between NFIII and the octamer-containing sequences in a histone H2B promoter, immunoglobulin light and heavy chain promoters, an immunoglobulin heavy chain enhancer, a U2 snRNA enhancer and the SV40 enhancer as well as Ad4. All sequences were recognized by NFIII as indicated by gel retardation assays, DNase I footprinting and methylation protection experiments. A comparison of the relative binding affinities using competition assays indicated that mutations in the octanucleotide sequence reduced the binding affinity considerably. Small but significant differences in affinity were also observed depending on the sequences bordering the conserved octanucleotide. The methylation protection patterns indicate that both major and minor groove contacts are involved in NFIII binding. The data suggest that NFIII could function both in adenovirus DNA replication and in the transcriptional control of several groups of genes sharing the octanucleotide sequence.

Adenoviruses, Human↗
PubMed1986

Inhibition of adenovirus DNA replication in vitro by autoimmune sera.

Sera from patients suffering from autoimmune diseases were analyzed for the presence of antibodies that inhibit adenovirus DNA replication in vitro. DNA replication was studied in a reconstituted system containing purified viral proteins (DNA binding protein, DNA polymerase and the precursor to the terminal protein) and a crude nuclear extract from HeLa cells. About half the autoimmune sera analyzed inhibited DNA replication by more than 50% while only 2 out of 31 control sera showed strong inhibition. The inhibition was caused by the IgG fractions of the sera and was most frequently observed with sera from scleroderma patients. Several lines of evidence indicate that the inhibition is not due to anti-DNA antibodies. The mechanism of inhibition of two strongly inhibitory sera was further investigated. The IgG fractions from these sera blocked DNA chain elongation more than 80% but had no effect on the initiation step or the synthesis of the first 26 nucleotides. Using a dot blot assay and different incubation conditions, evidence was obtained that the inhibition is due to immunorecognition of a nuclear factor from HeLa cells. Two nuclear proteins are known to be required for adenovirus DNA replication, nuclear factors I and II. DNA replication in the presence of purified nuclear factor I instead of a crude nuclear extract was only slightly inhibited by the antisera. In agreement with this, immunorecognition of nuclear factor I could not be detected using a dot blot assay. Since nuclear factor II is not required in our assay system, these results suggest the existence of another nuclear component involved in adenovirus DNA replication which is neutralized by these antibodies.

Adenoviridae↗
PubMedDate not supplied

Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication.

Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.

Adenoviridae↗