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R M Gronostajski

Publications and source records attributed to R M Gronostajski.

At least 37 records · Page 2Linked to original sources
PubMed1988

Stimulation of transcription in vitro by binding sites for nuclear factor I.

Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus DNA in vitro and in vivo. We have examined the effect of natural and synthetic binding sites for NFI (FIB sites) on RNA synthesis in HeLa whole cell extracts. The natural binding site used is the 26bp FIB-2 site previously isolated from the human genome. When present upstream of the TATA box of the adenovirus major late promoter, the FIB-2 site stimulates RNA synthesis 3 to 5-fold. This stimulation occurs with either orientation of the FIB-2 site. A point mutation in FIB-2 that decreases NFI binding at least 100-fold reduces, but does not completely abolish, the stimulation of transcription. A number of synthetic binding sites for NFI were tested for the ability to increase RNA synthesis. The strongest binding sites stimulated transcription the most, while the weakest sites had the least effect. These studies strongly suggest a role for NFI and cellular FIB sites in the control of RNA synthesis.

Binding Sites↗
PubMed1987

Site-specific DNA binding of nuclear factor I: effect of the spacer region.

Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus type 2 DNA in vitro and in vivo. To study sequence requirements for the interaction of NFI with DNA, we have measured the binding of the protein to a variety of synthetic sites. Binding sites for NFI (FIB sites) were previously shown to contain a consensus sequence composed of 2 motifs, TGG (Motif 1), and GCCAA (Motif 2), separated by a 6 or 7bp spacer region. To assess conserved sequences in the spacer region and flanking sequences which affect NFI binding, we have isolated clones from oligonucleotide libraries that contain the two motifs flanked by 3 degenerate nucleotides and separated by degenerate spacer regions of 6 or 7 nucleotides. With a 6bp spacer region, a strong bias exists for a C or A residue in the first position of the spacer. Sites with a 7bp spacer region contain a G and C or A residue at the first and second positions, respectively, of the spacer, but also possess conserved residues at other positions of the site.

Base Sequence↗
PubMed1986

Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides.

Nuclear factor I (NFI) binds tightly to DNA containing the consensus sequence TGG(N)6-7GCCAA. To study the role of the spacing between the TGG and GCCAA motifs, oligonucleotides homologous to the NFI binding site FIB-2 were synthesized and used for binding assays in vitro. The wild-type site (FIB-2.6) has a 6bp spacer region and binds tightly to NFI. When the size of this spacer was altered by +/- 1 or 2bp the binding to NFI was abolished. To further assess the role of the spacer and bases flanking the motifs, two oligonucleotide libraries were synthesized. Each member of these libraries had intact TGG and GCCAA motifs, but the sequence of the spacer and the 3bp next to each motif was degenerate. The library with a 6bp spacer bound to NFI to 40-50% the level of FIB-2.6. The library with a 7bp spacer bound to NFI to only 4% the level of FIB-2.6 and some of this binding was weaker than that of FIB-2.6 DNA. This novel use of degenerate DNA libraries has shown that: 1) the structural requirements for FIB sites with a 7bp spacer are more stringent than for sites with a 6bp spacer and 2) a limited number of DNA structural features can prevent the binding of NFI to sites with intact motifs and a 6bp spacer region.

Base Sequence↗
PubMed1985

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene.

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.

Amino Acid Sequence↗
PubMed1985

Determination of DNA sequences essential for FLP-mediated recombination by a novel method.

The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site-specific recombination across the two FLP-binding sites of the plasmid. We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event. A linear DNA containing two FLP sites in a direct orientation was treated with the double-strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites. The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro. FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA. Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP-treated DNA showed to the nucleotide the duplex DNA sequence required for FLP-mediated recombination. To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7. The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP. A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins.

Base Sequence↗
PubMed1985

The FLP protein of the 2-micron plasmid of yeast. Inter- and intramolecular reactions.

We have studied the mechanism of reaction of the FLP protein of the yeast 2-micron plasmid on linear substrates. The products of the reaction are dependent upon the concentration of FLP protein. At low concentrations of FLP, products resulting from intramolecular recombination between two FLP target sites accumulate. At higher concentrations of FLP, intermolecular recombination results in the accumulation of products which are larger than the starting substrate. At higher concentrations still, FLP-promoted recombination is inhibited. Potassium chloride (0.15 M) inhibits the intermolecular reaction and also prevents the inhibition of FLP-mediated recombination caused by high concentrations of FLP protein. We present a model that explains these findings.

DNA Nucleotidyltransferases↗
PubMed1985

The ATP dependence of the degradation of short- and long-lived proteins in growing fibroblasts.

To characterize the system(s) responsible for degradation of short-lived and long-lived proteins in mammalian cells, we compared the concentrations of ATP required for the degradation of these classes of proteins in growing hamster fibroblasts. By treating CHEF-18 cells with increasing concentrations of dinitrophenol and 2-deoxyglucose, it was possible to reduce their steady-state ATP content by different amounts (up to 98%). These treatments caused a rapid decrease in the degradation of both short- and long-lived proteins. Removal of the inhibitors led to a prompt restoration of ATP and proteolysis. As ATP content fell below normal levels (about 3.1 mM), rates of proteolysis decreased in a graded biphasic fashion. Reduction in ATP by up to 90% (as may occur in anoxia or injury) decreased proteolysis up to 50%; and with further loss of ATP, protein breakdown fell more sharply. Degradation of both classes of proteins was inhibited by 80% when ATP levels were reduced by 98%. The levels of ATP required for the breakdown of short- and long-lived proteins were indistinguishable. Protein synthesis was much more sensitive to a decrease in ATP content than protein breakdown and fell by 50% when ATP was reduced by only 15%. Chloroquine, an inhibitor of lysosome function, did not reduce the degradation of either class of proteins in growing cells, but it did inhibit the enhanced degradation of long-lived proteins upon removal of serum (in accord with previous studies). Thus, in growing fibroblasts, an ATP-dependent nonlysosomal process appears responsible for the hydrolysis of both short- and long-lived proteins.

Adenosine Triphosphate↗
PubMed1985

The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

Base Sequence↗
PubMed1985

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

Base Sequence↗
PubMed1984

Properties of the adenovirus DNA polymerase.

The 140,000-Da adenovirus-encoded DNA polymerase (Ad Pol) is required for viral DNA replication both in vitro and in vivo. The polymerase co-purifies in a complex with the 80,000-Da precursor (pTP) of the terminal protein (TP) found covalently attached to the 5' ends of adenovirus DNA. To better understand their function in DNA replication, we have examined the properties of the Ad Pol and the pTP X Ad Pol complex on natural and synthetic DNA templates. The pTP X Ad Pol complex utilizes a variety of homopolymer template-primer combinations including poly(dC) X oligo(dG), poly(dA) X oligo(dT), poly(dT) X oligo(dA), and poly(dT) X oligo(rA). With poly(dT) as template and oligo(rA) or oligo(dA) as primer, DNA synthesis by the pTP X Ad Pol complex is stimulated as much as 100-fold by the 59,000-Da adenovirus DNA-binding protein (Ad DBP). ATP (4 mM) can further increase the rate of DNA synthesis 3- to 10-fold. The Ad DBP does not stimulate the activity of host (HeLa cell) DNA polymerase alpha with poly(dT) X oligo(dA) (or oligo(rA)) as the template-primer, and Escherichia coli single-stranded DNA binding protein cannot substitute for the Ad DBP in the stimulation of the Ad Pol activity. Under optimal conditions, poly(dA) chains 30,000 nucleotides in length are formed indicating that the Ad Pol can be a highly processive enzyme. An exonuclease activity co-sediments with the pTP X Ad Pol complex during glycerol gradient centrifugation, and co-purifies with the 140,000-Da Ad Pol after dissociation of the pTP X Ad Pol complex with urea. The Ad Pol-associated nuclease hydrolyzes single-stranded DNA in a 3'----5' direction and is at least 10-fold more active on single-stranded DNA than on duplex DNA. The Ad Pol has no detectable endonuclease activity on single-stranded DNA or duplex circular DNA. Analysis of the products of the nuclease activity showed that 5'-deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The Ad DBP inhibits the hydrolysis of DNA by the polymerase-associated nuclease activity.

Adenosine Triphosphate↗
PubMed1984

Purification of a primase activity associated with DNA polymerase alpha from HeLa cells.

Highly purified preparations of eukaryotic DNA polymerase alpha have been shown to contain primase activity (Kaguni, L.S., Rossignol, J-M., Conaway, R.C. Banks, G.R., and Lehman, I.R. (1983) J. Biol. Chem. 258, 9037-9039; Yagura, T., Kozu, T., and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127; Shioda, M., Nelson, E.M., Bayne, M.L., and Benbow, R.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7209-7213). We have investigated the de novo synthesis of DNA by a primase-DNA polymerase alpha preparation isolated from human HeLa cells using the synthetic homopolymers poly(dT) and poly(dC) as templates. In the presence of poly(dT), synthesis of poly(dA) required ATP in addition to dATP while synthesis of poly(dG) in the presence of poly(dC) required GTP in addition to dGTP. The primase activity required a much lower GTP concentration (Km = 0.1 mM) than ATP (Km = 0.8 mM) for the synthesis of DNA. Guanosine 5'-O-(3-thiotriphosphate), 5'-guanylyl-beta, gamma-imidodiphosphate, and 5'-guanylyl methylenediphosphonate substituted for GTP but the corresponding ATP analogues did not substitute for ATP. Furthermore, ATP and ATP analogues inhibited the GTP-dependent reaction while GTP and GTP analogues inhibited the ATP-dependent reaction. DNase treatment of products labeled with [alpha-32P] GTP revealed that an RNA oligomer was covalently linked to newly synthesized DNA. Alkaline hydrolysis of these products yielded GMP and pppGp, indicating that the primer was initiated with GTP. Alkaline hydrolysis of [alpha-32P]dGTP-labeled products yielded 2'- and 3'-GMP showing that DNA chains are covalently linked to the 3' ends of RNA chains. The primase activity could not be separated from DNA polymerase alpha through a 200-fold enrichment involving phosphocellulose, DNA-cellulose, hydroxylapatite, DEAE-cellulose and glycerol gradient purification steps. However, primase activity was found to be less stable than DNA polymerase alpha activity under a variety of conditions.

Adenine Nucleotides↗
PubMed1984

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells.

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.

Animals↗
PubMed1984

The role of increased proteolysis in the atrophy and arrest of proliferation in serum-deprived fibroblasts.

When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.

Animals↗
PubMed1984

Isolation of human DNA sequences that bind to nuclear factor I, a host protein involved in adenovirus DNA replication.

Nuclear factor I is a 47,000-dalton protein isolated from human HeLa cells that is required for the in vitro replication of adenovirus DNA. This protein was previously shown to bind specifically to nucleotides 17-48 of the left-hand terminus of cloned adenovirus serotype 5 DNA. An in vitro assay for DNA sequences that compete with adenovirus DNA for the binding of nuclear factor I has been developed. With this assay, we have shown specific binding of human DNA sequences to nuclear factor I. Using the DNA binding activity of nuclear factor I, we have isolated and cloned segments of human DNA that bind tightly to this protein. One nuclear factor I binding site is present about every 100,000 base pairs in the HeLa cell genome. The binding of these DNA molecules to nuclear factor I resembles the binding of cloned adenovirus DNA to the protein and is resistant to high ionic strength. The isolation of DNA sequences from HeLa cells that bind specifically to nuclear factor I suggests that this protein interacts with host DNA in vivo.

Adenoviruses, Human↗
PubMed1984

Energy requirement for degradation of tumor-associated protein p53.

A 53,000-dalton protein (p53) present in large amounts in several types of tumorigenic cells was rapidly degraded in nontumorigenic BALB/c 3T3 fibroblasts (t 1/2, approximately 0.5 h) but not in tumorigenic methylcholanthrene-induced mouse sarcoma cells (t 1/2, greater than 2 h). In 3T3 cells, dinitrophenol and 2-deoxyglucose, agents which reduce ATP production, inhibited the rapid degradation of p53 and the slower breakdown of total cell protein. After removal of these agents, the degradation of both p53 and total cell proteins resumed at their normal rates. Inhibitors of intralysosomal proteolysis (Ep475 and chloroquine) did not reduce the rate of degradation of p53. Thus, in 3T3 cells, p53 appears to be degraded by a nonlysosomal, ATP-dependent proteolytic system similar to that previously shown to degrade short- and long-lived proteins in growing fibroblasts. The immunoreactive p53 which remained in ATP-depleted cells had the same molecular weight as the p53 in the control cells. No intermediate products of p53 degradation were detected by immunoprecipitation in either ATP-depleted or control cells. Hence, ATP seems to be required for an initial step in the degradation of p53. Although the amount of labeled p53 was increased in simian virus 40-transformed and methylcholanthrene-induced mouse sarcoma cells, the amount of p53 labeled during a 3-h pulse in Moloney virus- and Rous sarcoma virus-transformed cells and untransformed 3T3 cells was similar. Thus, an increased net rate of p53 accumulation is not a common feature of transformed tumorigenic cells.

Animals↗
PubMed1978

Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana.

The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD(+) oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K(m) values for alpha-ketoglutarate, NADH, NH(4) (+), NAD(+), and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the K(m) for alpha-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K(m) values for NADH and NAD(+) were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between alpha-ketoglutarate and an epsilon-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH(4) (+)-inducible glutamate dehydrogenase in this organism.

Journal Article↗
PubMed1978

Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana.

The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism.

Amino Acids↗