Showing papers by "Cold Spring Harbor Laboratory published in 1989"
TL;DR: The results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.
Abstract: Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.
2,941 citations
TL;DR: It is reported here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation, suggesting that induction of angiogenesis is an important step in carcinogenesis.
Abstract: It is now well established that unrestricted growth of tumours is dependent upon angiogenesis. Previous studies on tumour growth, however, have not revealed when or how the transition to an angiogenic state occurs during early tumour development. The advent of transgenic mice carrying oncogenes that reproducibly elicit tumours of specific cell types is providing a new format for studying multi-step tumorigenesis. In one of these models, transgenic mice expressing an oncogene in the beta-cells of the pancreatic islets heritably recapitulate a progression from normality to hyperplasia to neoplasia. We report here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. A novel in vitro assay confirms that hyperplasia per se does not obligate angiogenesis. Rather, a few hyperplastic islets become angiogenic in vitro at a time when such islets are neovascularized in vivo and at a frequency that correlates closely with subsequent tumour incidence. These findings suggest that induction of angiogenesis is an important step in carcinogenesis.
1,995 citations
TL;DR: The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.
Abstract: The telomerase enzyme of Tetrahymena synthesizes repeats of the telomeric DNA sequence TTGGGG de novo in the absence of added template. The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.
1,623 citations
TL;DR: In this paper, the authors look for changes in p105-RB that may regulate its activity during the cell cycle, and generate synchronized cell populations and follow their progression through the cell-cycle.
1,004 citations
TL;DR: The results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A, including p105-RB, the product of the retinoblastoma susceptibility gene.
728 citations
TL;DR: The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described, and the reaction described herein reflects the process of chromatin formation during DNA replication in vivo.
633 citations
TL;DR: It is suggested that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.
606 citations
TL;DR: It is proposed that Tat acts through TAR to increase initiation complex formation on the HIV-1 promoter and to stabilize complexes during elongation to reduce the chance of transactivation by E1A and eliminate trans-activation by Tat.
546 citations
TL;DR: Five highly conserved motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides, and can be used to discriminate the known 5- methylcytOSine forming methyltransferases from all other methyl transferases of known sequence, and from allother identified proteins in the PIR, GenBank and EMBL databases.
Abstract: Thirteen bacterial DNA methyltransferases that catalyze the formation of 5-methylcytosine within specific DNA sequences possess related structures. Similar building blocks (motifs), containing invariant positions, can be found in the same order in all thirteen sequences. Five of these blocks are highly conserved while a further five contain weaker similarities. One block, which has the most invariant residues, contains the proline-cysteine dipeptide of the proposed catalytic site. A region in the second half of each sequence is unusually variable both in length and sequence composition. Those methyltransferases that exhibit significant homology in this region share common specificity in DNA recognition. The five highly conserved motifs can be used to discriminate the known 5-methylcytosine forming methyltransferases from all other methyltransferases of known sequence, and from all other identified proteins in the PIR, GenBank and EMBL databases. These five motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides. By searching the unidentified open reading frames present in the GenBank and EMBL databases, two potential 5-methylcytosine forming methyltransferases have been found.
463 citations
TL;DR: It is concluded that the cyclic synthesis of PCNA in cycling HeLa cells maintainsPCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.
431 citations
TL;DR: In vivo inhibition of tyrosine dephosphorylation by exposure of cells to a phosphatase inhibitor is associated with G2 arrest, which is reversible upon the removal of the phosphat enzyme inhibitor.
TL;DR: It is demonstrated that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.
TL;DR: A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb, which was observed to result from a single point mutation within a splice acceptor sequence in J 82 genomic DNA.
Abstract: The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.
TL;DR: It is concluded that the βγ-subunit of G-proteins activates IK.ACH by stimulating the production of lipoxygenase-derived second messengers.
Abstract: Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers.
TL;DR: The herpes simplex virus transactivator VP16 par-ticipates in the formation of a multiprotein-DNA complex with the ubiquitous octamer-motif-binding factor Oct-1, indicating that this structure is an ancient target for protein-protein interactions mediating transcriptional control.
Abstract: The herpes simplex virus transactivator VP16 participates in the formation of a multiprotein-DNA complex with the ubiquitous octamer-motif-binding factor Oct-1. Complex formation is dependent on specific amino acids in the Oct-1 homoeodomain which are in positions analogous to positive control mutations in helix 2 of the lambda phage repressor helix-turn-helix motif, indicating that this structure is an ancient target for protein-protein interactions mediating transcriptional control.
TL;DR: The system capable of replicating plasmid DNAs containing the SV40 origin of DNA replication in vitro is a paradigm for studies on the replicate of other virus DNAs and the replication of cellular chromosomes.
Abstract: Recent advances in our understanding of the mechanism and regulation of eukaryotic DNA replication have been expedited by the use of cell-free systems capable of initiation of DNA replication. The system capable of replicating plasmid DNAs containing the SV40 origin of DNA replication in vitro is a paradigm for studies on the replication of other virus DNAs and the replication of cellular chromosomes. This review outlines some of the contemporary issues and developments related to this complex problem.
TL;DR: Observations suggest that the cdc13+-encoded cyclin acts to regulate both the catalytic properties and the localization of the protein kinase of which it is a subunit.
TL;DR: In human cells, the large T antigens of SV40 or JC virus also form complexes with 107K, suggesting that these associations may represent another component of a common mechanism for transformation between adenoviruses and polyoma viruses.
TL;DR: The strategies and methods used by the QUEST system for two-dimensional gel analysis are described, and the performance of the system is evaluated.
TL;DR: The identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae is reported and it is shown that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication.
Abstract: Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.
TL;DR: In both infected and uninfected cells, p60 was found in a complex with the cdc2 protein kinase, suggesting that each might play a distinct role in regulation of the cell cycle.
TL;DR: It is suggested that co‐ordinated synthesis of these strands requires dynamic protein‐protein interactions between these replication factors and the two DNA polymerases.
Abstract: DNA synthesis by two eukaryotic DNA polymerases, alpha and delta, was studied using a single-strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase alpha or delta, DNA synthesis was limited and short DNA strands of approximately 100 bases were produced. Addition of replication factors RF-A, PCNA and RF-C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF-A and RF-C independently stimulated DNA polymerase alpha activity 4- to 6-fold, yielding relatively short DNA strands (less than 1 kb) and PCNA had no effect. In contrast, polymerase delta activity was stimulated co-operatively by PCNA, RF-A and RF-C approximately 25- to 30-fold, yielding relatively long DNA strands (up to 4 kb). Neither RF-C nor RF-A appear to correspond to known polymerase stimulatory factors. RF-A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases alpha and delta is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co-ordinated synthesis of these strands requires dynamic protein-protein interactions between these replication factors and the two DNA polymerases.
TL;DR: It is shown that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays, and that a double-stranded oligonucleotide carrying the major c- fos CRE is sufficient to block induction of the endogenous c-Fos gene by cAMP.
Abstract: Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.
TL;DR: Four suppressor genes that permit transcription of the Saccharomyces cerevisiae HIS4 gene in the absence of GCN4, BAS1, and BAS2, trans-acting proteins normally required for activation of HIS4 transcription are identified.
TL;DR: This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.
Abstract: Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.
TL;DR: It is shown that transcription from the U6 promoter is dependent on a sequence similar to the U2 proximal element and on an AT-rich element centered around position -27 that determines the RNA polymerase specificity of snRNA promoters and hence the site of transcription termination.
TL;DR: It is shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene.
Abstract: A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.
TL;DR: Results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin, and it is suggested that even though the two polymerases function asymmetrically, they normally progress coordinately.
Abstract: Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.
TL;DR: The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI, consistent with a model in which activation requires the interaction of two molecules of DAI with ds RNA, followed by intermolecular autophosphorylation of the latent enzyme.
Abstract: The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.
TL;DR: Based on a variety of biochemical and immunological criteria, grp 75 is shown to be a member of the hsp 70 family of stress proteins, while hsp 58 represents the mammalian equivalent of the bacterial groEL protein.