Showing papers by "Cold Spring Harbor Laboratory published in 1994"
TL;DR: Consistent with models claiming a role for long-term potentiation (LTP) in memory, LTP in hippocampal slices from CREB mutants decayed to baseline 90 min after tetanic stimulation, however, paired-pulse facilitation and posttetanic potentiation are normal.
1,832 citations
TL;DR: It is shown that p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK, and blocks the ability of PCNA to activateDNA polymerase δ, the principal replicative DNA polymerase.
Abstract: The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. Furthermore, p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.
1,703 citations
TL;DR: Using an inducible transgene that expresses a dominant negative member of the fly CREB family, LTM was specifically and completely blocked only after induction, while ARM and learning were unaffected, suggesting that LTM formation requires de novo gene expression probably mediated by CREBfamily genes.
1,002 citations
TL;DR: The crystal structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.
904 citations
TL;DR: It is shown that consolidated memory of conditioned (learned) odor avoidance 1 day after extended training consisted of two genetically distinct, functionally independent memory components: anesthesia-resistant memory (ARM) and long-term memory (LTM).
880 citations
TL;DR: The structure of PTP1B should serve as a model for other members of the PTP family and as a framework for understanding the mechanism of tyrosine dephosphorylation.
Abstract: Protein tyrosine phosphatases (PTPs) constitute a family of receptor-like and cytoplasmic signal transducing enzymes that catalyze the dephosphorylation of phosphotyrosine residues and are characterized by homologous catalytic domains. The crystal structure of a representative member of this family, the 37-kilodalton form (residues 1 to 321) of PTP1B, has been determined at 2.8 A resolution. The enzyme consists of a single domain with the catalytic site located at the base of a shallow cleft. The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. This site is formed from an 11-residue sequence motif that is diagnostic of PTPs and the dual specificity phosphatases, and that contains the catalytically essential cysteine and arginine residues. The position of the invariant cysteine residue within the phosphate binding site is consistent with its role as a nucleophile in the catalytic reaction. The structure of PTP1B should serve as a model for other members of the PTP family and as a framework for understanding the mechanism of tyrosine dephosphorylation.
728 citations
TL;DR: It is shown that p21 does not block PCNA-dependent nucleotide-excision repair, in contrast to its inhibition of simian virus 40 DNA replication, and the short gap-filling DNA synthesis by PC NA-dependent DNA polymerases δ and ɛ is less sensitive to inhibition by p21 than is long primer-extension synthesis.
Abstract: In mammalian cells, DNA damage increases the levels of the nuclear tumour-suppressor p53, resulting in elevated synthesis of p21, an inhibitor of cyclin-dependent kinases (CDK). p21 may also directly block DNA replication by inhibiting the proliferating-cell nuclear antigen (PCNA), an essential DNA replication protein. However, PCNA is also required for nucleotide-excision repair of DNA, an intrinsic part of the cellular response to ultraviolet irradiation. Using an in vitro system, we now show that p21 does not block PCNA-dependent nucleotide-excision repair, in contrast to its inhibition of simian virus 40 DNA replication. Furthermore, the short gap-filling DNA synthesis by PCNA-dependent DNA polymerases delta and epsilon is less sensitive to inhibition by p21 than is long primer-extension synthesis. The ability of p21 to inhibit the role of PCNA in DNA replication but not in DNA repair rationalizes in vivo data showing that genetic damage leads to inactivation of chromosomal replication while allowing damage-responsive repair.
668 citations
TL;DR: It is shown that P encodes a Myb homolog that recognizes the sequence CCT/AACC, in sharp contrast with the C/TAACGG bound by vertebrate Myb proteins.
629 citations
TL;DR: Variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.
Abstract: The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.
621 citations
TL;DR: Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins to replicate both strands of the double helix conjointly.
Abstract: Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.
543 citations
TL;DR: The structure of an Oct-1 POU domain-octamer DNA complex has been solved at 3.0 A resolution and the novel arrangement raises important questions about cooperativity in protein-DNA recognition.
TL;DR: The addition of the purified MAP kinase-specific phosphatase MKP-1 demonstrates thatMAP kinase activity is required for both the establishment and maintenance of the mitotic arrest induced by spindle depolymerization.
TL;DR: Cloning of C. elegans genes has shown that two of them encode proteins with similarity to vertebrate cell death genes and has revealed that nematodes and mammals share a common pathway for programmed cell death.
TL;DR: These results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis, suggesting thatDNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.
TL;DR: This review will focus on several specific examples that highlight the interplay between PTPs and PTKs in cell signaling.
TL;DR: It is proposed that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.
Abstract: hnRNP A1 is a pre-mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5' splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP-1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA-protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre-mRNA binding or RNA annealing activity. The C-terminal Gly-rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly-rich domain, binds more strongly to pre-mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre-mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.
TL;DR: In this article, the use of vectors containing the Tn903 kanr gene conferring G418-resistance was reported, which showed that copy number showed a tight correlation with drug resistance.
Abstract: Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.
TL;DR: It is suggested that postsynaptic CaMKII activity is necessary and sufficient to generate LTP.
Abstract: Calcium-calmodulin-dependent protein kinase II (CaMKII) is a necessary component of the cellular machinery underlying learning and memory. Here, a constitutively active form of this enzyme, CaMKII(1-290), was introduced into neurons of hippocampal slices with a recombinant vaccinia virus to test the hypothesis that increased postsynaptic activity of this enzyme is sufficient to produce long-term synaptic potentiation (LTP), a prominent cellular model of learning and memory. Postsynaptic expression of CaMKII(1-290) increased CaMKII activity, enhanced synaptic transmission, and prevented more potentiation by an LTP-inducing protocol. These results, together with previous studies, suggest that postsynaptic CaMKII activity is necessary and sufficient to generate LTP.
TL;DR: The yeast two-hybrid data suggest that scd1, scd2, cdc42sp, and ras1, in its GTP-bound state, act cooperatively to form a protein complex.
TL;DR: Observations suggest that neurotrophins may contribute to phenotypic differentiation of GABAergic neurons in the developing striatum through their influence on GABAergic properties of developing striatal neurons.
TL;DR: The selection of 5' splice sites in alternative splicing is closely related to that in constitutive splicing, and uses the same components in somewhat different ways.
TL;DR: The results suggest that STE5 is a scaffolding protein that facilitates interactions between components of the pheromone-responsive mitogen-activated protein kinase module of Saccharomyces cerevisiae, and it is proposed that such scaffolding proteins serve to inhibit cross-talk between functionally unrelated mitogen -activated protein Kinase modules within the same cell.
Abstract: We present genetic evidence for complex formation of STE5 and the STE11, STE7, and FUS3 protein kinases, the pheromone-responsive mitogen-activated protein kinase module of Saccharomyces cerevisiae. Interaction between STE5 and STE11 is not dependent on STE7, and interaction between STE5 and STE7 does not require STE11. The N-terminal regulatory domain of STE11 is both necessary and sufficient for interaction with STE5. Interaction between STE7 and STE11 is bridged by STE5, suggesting the formation of a multiprotein complex. We also demonstrate biochemical interaction between STE5 and STE11 by using a combination of bacterially expressed fusion proteins and extracts prepared from yeast. Our results suggest that STE5 is a scaffolding protein that facilitates interactions between components of the pheromone-responsive mitogen-activated protein kinase module. We further propose that such scaffolding proteins serve to inhibit cross-talk between functionally unrelated mitogen-activated protein kinase modules within the same cell.
TL;DR: Tropomyosins are a family of actin filament binding proteins identified in many organisms, including yeast, nematodes, Drosophila, birds and mammals.
TL;DR: The observed effects in vivo on neuropeptide expression in various brain regions by injecting BDNF into the cerebroventricle of newborn rats suggest that BDNF regulates the development of neuropeptic expression in the CNS in a plastic manner.
Abstract: Brain-derived neurotrophic factor (BDNF) specifically enhances and maintains the expression of neuropeptide Y (NPY) and somatostatin (SOM) in cultured neocortical neurons (Nawa et al., 1993). In this article, we examined its effects in vivo on neuropeptide expression in various brain regions by injecting BDNF into the cerebroventricle of newborn rats. Repeated administration (2x) of BDNF increased contents of NPY- like immunoreactivity (NPY-LI) and substance P (SP)-LI most markedly in the anterior neocortex by 11- and 24-fold, respectively, in comparison to values in the animals receiving control injection. A smaller but significant increase was also observed in immunoreactivity for somatostatin (SOM), enkephalin (ENK), and cholecystokinin (CCK). mRNA for NPY, SP, and SOM was similarly upregulated in the anterior neocortex, suggesting that BDNF enhances peptide synthesis rather than inhibiting peptide release or degradation. Among the brain regions examined, however, peptidergic responses to BDNF were different with respect to their spatial distribution and time course. Induction of SP- LI, NPY-LI, and SOM-LI around the injection site was most pronounced in cortical layers II/III, layers IV-VI, and layer VI, respectively. Peptidergic immunoreactivity was also enhanced in other brain regions ipsilateral to the injection site, for example, NPY-LI in the hippocampus, thalamic nuclei, and striatum, and SOM-LI in the striatum. A single injection of BDNF elevated SP-LI to a plateau level within 12 hr while NPY-LI and SOM-LI reached maximum levels at 48 hr, and then all returned to control levels at 68 hr. In contrast, the same dose of NGF had no influences on the neuropeptide levels at 48 hr. These observations suggest that BDNF regulates the development of neuropeptide expression in the CNS in a plastic manner.
TL;DR: It is observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement.
Abstract: We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.
TL;DR: Observations suggest that DEP-1 may contribute to the mechanism of contact inhibition of cell growth by occurring gradually with increasing cell contact and is initiated before saturation cell density is reached.
Abstract: cDNA encoding a receptor-like protein-tyrosine-phosphatase (PTP) termed DEP-1 was isolated from a HeLa cell library. The cDNA predicts an enzyme consisting of an extracellular segment containing eight fibronectin type III repeats, a single transmembrane segment, and a single intracellular PTP domain. Following expression of DEP-1 cDNA in COS cells a glycoprotein of 180 kDa was detected and PTP activity was demonstrated in immunocomplexes with a C-terminal peptide antiserum. Endogenous DEP-1 was detected in WI-38 human embryonic lung fibroblasts by immunoblotting and immunocomplex PTP assays. Immunoblot analysis of DEP-1 expression in WI-38 cells revealed dramatically increased levels and activity of the PTP in dense cultures relative to sparse cultures. Also, DEP-1 activity, detected in PTP assays of immunocomplexes, was increased in dense cell cultures. In contrast, the expression levels of PTP-1B did not change with cell density. This enhancement of DEP-1 expression with increasing cell density was also observed in another fibroblast cell line, AG1518. The increase in DEP-1 occurs gradually with increasing cell contact and is initiated before saturation cell density is reached. These observations suggest that DEP-1 may contribute to the mechanism of contact inhibition of cell growth.
TL;DR: An essential role for activation of MAP kinases in the transition from the quiescent to the DNA replication phase of the eukaryotic cell cycle is suggested.
Abstract: Mitogen-activated protein kinases (MAP kinases) are common components of signaling pathways induced by diverse growth stimuli. Although the guanidine nucleotide-binding Ras proteins are known to be upstream activators of MAP kinases, the extent to which MAP kinases directly contribute to the mitogenic effect of Ras is as yet undefined. In this study, inhibition of MAP kinases by the MAP kinase phosphatase MKP-1 blocked the induction of DNA synthesis in quiescent rat embryonic fibroblast REF-52 cells by an activated mutant of Ras, V12Ras. These results suggest an essential role for activation of MAP kinases in the transition from the quiescent to the DNA replication phase of the eukaryotic cell cycle.
TL;DR: Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases.
Abstract: The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-A resolution. The protein folds into an alpha/beta-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.