scispace - formally typeset
Search or ask a question

Showing papers by "Cold Spring Harbor Laboratory published in 1995"


Journal ArticleDOI
06 Jul 1995-Nature
TL;DR: A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.
Abstract: The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme, named apopain, is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.

4,096 citations


Journal ArticleDOI
01 Sep 1995-Science
TL;DR: Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity, and cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings.
Abstract: Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

2,305 citations


Journal ArticleDOI
01 Jun 1995-Nature
TL;DR: It is reported that a high proportion of synapses in hippocampal area CA1 transmit with NMDA receptors but not AMPA receptors, making these synapses effectively non-functional at normal resting potentials.
Abstract: Long-term potentiation (LTP) is an enhancement of synaptic strength that can be produced by pairing of presynaptic activity with postsynaptic depolarization. LTP in the hippocampus has been extensively studied as a cellular model of learning and memory, but the nature of the underlying synaptic modification remains elusive, partly because our knowledge of central synapses is still limited. One proposal is that the modification is postsynaptic, and that synapses expressing only NMDA (N-methyl-D-aspartate) receptors before potentiation are induced by LTP to express functional AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors. Here we report that a high proportion of synapses in hippocampal area CA1 transmit with NMDA receptors but not AMPA receptors, making these synapses effectively non-functional at normal resting potentials. These silent synapses acquire AMPA-type responses following LTP induction. Our findings challenge the view that LTP in CA1 involves a presynaptic modification, and suggest instead a simple postsynaptic mechanism for both induction and expression of LTP.

1,372 citations


Journal ArticleDOI

1,078 citations


Journal ArticleDOI
TL;DR: The development of an efficient insertional mutagenesis system in Arabidopsis that permits identification of genes by their patterns of expression during development and suggests that the identification and cloning ofArabidopsis genes expressed in any developmental process is feasible using this system.
Abstract: The crucifer Arabidopsis thaliana has been used widely as a model organism for the study of plant development. We describe here the development of an efficient insertional mutagenesis system in Arabidopsis that permits identification of genes by their patterns of expression during development. Transposable elements of the Ac/Ds system carrying the GUS reporter gene have been designed to act as enhancer traps or gene traps. A novel selection scheme maximizes recovery of unlinked transposition events. In this study 491 plants carrying independent transposon insertions were generated and screened for expression patterns. One-half of the enhancer trap insertions and one-quarter of the gene trap insertions displayed GUS expression in seedlings or flowers, including expression patterns specific to organs, tissues, cell types, or developmental stages. The patterns identify genes that act during organogenesis, pattern formation, or cell differentiation. Transposon insertion lines with specific GUS expression patterns provide valuable markers for studies of Arabidopsis development and identify new cell types or subtypes in plants. The diversity of gene expression patterns generated suggests that the identification and cloning of Arabidopsis genes expressed in any developmental process is feasible using this system.

780 citations


Journal ArticleDOI
TL;DR: It is suggested that there is tissue-specific regulation of mouse telomerase during development and aging in vivo, and most mouse tissues had active telomerases, compared to human tissues, which may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.
Abstract: Telomere shortening and telomerase activation in human somatic cells have been implicated in cell immortalization and cellular senescence. To further study the role of telomerase in immortalization, we assayed telomere length and telomerase activity in primary mouse fibroblasts, in spontaneously immortalized cell clones, and in mouse tissues. In the primary cell cultures, telomere length decreased with increased cell doublings and telomerase activity was not detected. In contrast, in spontaneously immortalized clones, telomeres were maintained at a stable length and telomerase activity was present. To determine if telomere shortening occurs in vivo, we assayed for telomerase and telomere length in tissues from mice of different ages. Telomere length was similar among different tissues within a newborn mouse, whereas telomere length differed between tissues in an adult mouse. These findings suggest that there is tissue-specific regulation of mouse telomerase during development and aging in vivo. In contrast to human tissues, most mouse tissues had active telomerase. The presence of telomerase in these tissues may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.

707 citations


Journal ArticleDOI
24 Feb 1995-Cell
TL;DR: Results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha- Ras-induced mammalian cell transformation.

674 citations


Journal ArticleDOI
07 Apr 1995-Cell
TL;DR: An enhancement of LTM formation is reported after induced expression of an activator isoform of dCREB2 of CREB2, which depends on phosphorylation of the activator transgene.

647 citations


Journal ArticleDOI
23 Jun 1995-Science
TL;DR: The crystal structures of a cysteine-215-->serine mutant of protein tyrosine phosphatase 1B complexed with high-affinity peptide substrates corresponding to an autophosphorylation site of the epidermal growth factor receptor were determined.
Abstract: The crystal structures of a cysteine-215-->serine mutant of protein tyrosine phosphatase 1B complexed with high-affinity peptide substrates corresponding to an autophosphorylation site of the epidermal growth factor receptor were determined. Peptide binding to the protein phosphatase was accompanied by a conformational change of a surface loop that created a phosphotyrosine recognition pocket and induced a catalytically competent form of the enzyme. The phosphotyrosine side chain is buried within the period and anchors the peptide substrate to its binding site. Hydrogen bonds between peptide main-chain atoms and the protein contribute to binding affinity, and specific interactions of acidic residues of the peptide with basic residues on the surface of the enzyme confer sequence specificity.

571 citations


Journal ArticleDOI
05 Jan 1995-Nature
TL;DR: It is shown that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9, and that distinct degradation signals or regulated interaction with the ubiquitIn-protein ligase system may determine the cell-cycle specificity of cyclin proteolysi.
Abstract: CELL cycle progression in eukaryotes is controlled by the p34cdc21CDC28 protein kinase and its Short-lived, phase-specific regulatory subunits called cyclins1,2. In Xenopus oocytes, degradation of M-phase (B-type) cyclins is required for exit from mitosis and is mediated by the ubiquitin-dependent proteolytic system3. Here we show that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9. Repression of UBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA. In ubc9 mutants both CLB5, an S-phase cyclin4,5, and CLB2, an M-phase cyclin6,7, are stabilized. In wild-type cells the CLB5 protein is unstable throughout the cell cycle, whereas CLB2 turnover occurs only at a specific cell-cycle stage8. Thus distinct degradation signals or regulated interaction with the ubiquitin-protein ligase system may determine the cell-cycle specificity of cyclin proteolysi

511 citations


Journal ArticleDOI
04 May 1995-Nature
TL;DR: Evidence is presented that the cytostatic effect of NGF is mediated by nitric oxide (NO), a second messenger molecule with both para- and autocrine properties that can diffuse freely and act within a restricted volume.
Abstract: Arrest of cell division is a prerequisite for cells to enter a program of terminal differentiation. Mitogenesis and cytostasis of neuronal cell precursors can be induced by the same or by different growth or trophic factors. Response of PC12 cells to nerve growth factor (NGF) involves a proliferative phase that is followed by growth arrest and differentiation. Here we present evidence that the cytostatic effect of NGF is mediated by nitric oxide (NO), a second messenger molecule with both para- and autocrine properties that can diffuse freely and act within a restricted volume. We show that NGF induces different forms of nitric oxide synthase (NOS) in neuronal cells, that nitric oxide (NO) acts as a cytostatic agent in these cells, that inhibition of NOS leads to reversal of NGF-induced cytostasis and thereby prevents full differentiation, and that capacity of a mutant cell line to differentiate can be rescued by exogenous NO. We suggest that induction of NOS is an important step in the commitment of neuronal precursors and that NOS serves as a growth arrest gene, initiating the switch to cytostasis during differentiation.

Journal ArticleDOI
TL;DR: A multiple sequence alignment of 42 amino-Mtases revealed nine conserved motifs, corresponding to the motifs I to VIII and X previously defined in C5-cytosine Mtases, which appear to be more closely related than has been appreciated.


Journal ArticleDOI
01 Sep 1995-Science
TL;DR: The shorter template regions of the mouse and other rodent telomerase RNAs could account for the shorter distribution of products (processivity) generated by the mouse enzyme relative to the human telomersase.
Abstract: Telomerase synthesizes telomeric DNA repeats onto chromosome ends de novo. The mouse telomerase RNA component was cloned and contained only 65 percent sequence identity with the human telomerase RNA. Alteration of the template region in vivo generated altered telomerase products. The shorter template regions of the mouse and other rodent telomerase RNAs could account for the shorter distribution of products (processivity) generated by the mouse enzyme relative to the human telomerase. Amounts of telomerase RNA increased in immortal cells derived from primary mouse fibroblasts. RNA was detected in all newborn mouse tissues tested but was decreased during postnatal development.

Journal ArticleDOI
30 Jun 1995-Cell
TL;DR: Chromatin assembly factor I (CAF-I) from human cell nuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system as mentioned in this paper.

Journal ArticleDOI
02 Jun 1995-Cell
TL;DR: It is suggested that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

Journal ArticleDOI
TL;DR: The results suggest that the sustained increase of BDNF protein in these limbic structures is involved in prolonged post‐seizure phenomena, including peptidergic alterations.
Abstract: Messenger RNA for brain-derived neurotrophic factor (BDNF) is distributed in many brain regions and regulated by excitatory neuronal activity. Despite numerous studies of BDNF mRNA, the distribution and regulation of BDNF protein are poorly understood because of the difficulty of its quantitative measurement. We have established a two-site enzyme immunoassay that detects trace amounts of BDNF protein (> 1 pg/assay) but not other neurotrophins or growth factors. The highest levels of BDNF in adult rat brain were found in the hippocampus, followed by the hypothalamus, neocortex, cerebellum, thalamus and striatum. This pattern is similar, but not identical, to the distribution of BDNF mRNA. A similar disparity between BDNF protein and mRNA levels was observed in their changes after hilus lesion-induced limbic seizures. In limbic structures, BDNF concentrations remained elevated 4 days after seizure onset, whereas BDNF mRNA has been reported previously to return to basal levels within 46 h. The temporal and spatial differences between the dynamics of protein and mRNA levels suggest the importance of post-translational and/or subcellular processes for BDNF production. The persistence of the increases in BDNF content was also reflected in its biological activity, e.g. peptidergic differentiation activity. After limbic seizures, neuropeptide Y content was most markedly and persistently elevated in the entorhinal/amygdaloid region, where the most sustained up-regulation of BDNF protein was observed. These results suggest that the sustained increase of BDNF protein in these limbic structures is involved in prolonged post-seizure phenomena, including peptidergic alterations.

Journal ArticleDOI
01 Oct 1995-Yeast
TL;DR: PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps and is used to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.
Abstract: Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura+ phenotype. Finally, selection for Ura- cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has 'popped out' the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.

Journal ArticleDOI
TL;DR: It is demonstrated that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues, suggesting that P TPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/ catenin complex in vivo.
Abstract: The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu, possesses an MAM domain, an immunoglobulin domain, and four fibronectin type-III repeats. It binds homophilically, i.e., PTPmu on the surface of one cell binds to PTPmu on an apposing cell, and the binding site lies within the immunoglobulin domain. The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins. In cadherins, this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton. In this article, we demonstrate that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues. Greater than 80% of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu; however, the complex is dissociated when lysates are prepared in more stringent, SDS-containing RIPA buffer. In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin, but not to alpha- or beta-catenin. Consistent with their ability to interact in vivo, PTPmu, cadherins, and catenins all localized to points of cell-cell contact in MvLu cells, as assessed by immunocytochemical staining. After pervanadate treatment of MvLu cells, which inhibits cellular tyrosine phosphatase activity including PTPmu, the cadherins associated with PTPmu are now found in a tyrosine-phosphorylated form, indicating that the cadherins may be an endogenous substrate for PTPmu. These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/catenin complex in vivo.

Journal ArticleDOI
TL;DR: Structural work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reaction and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes.
Abstract: In prokaryotes, the major role of DNA methylation is to protect host DNA against degradation by restriction enzymes. In eukaryotes, DNA methylation has been implicated in the control of several cellular processes, including differentiation, gene regulation, and embryonic development. Structural work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reaction and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes. Structural comparison of three enzymes, HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase, reveals a striking similarity in protein folding and indicates that many SAM-dependent methyltransferases have a common catalytic-domain structure. This feature permits the prediction of tertiary structure for other DNA, RNA, protein, and small-molecule methyltransferases from their amino acid sequences, including the eukaryotic CpG methyltransferases.

Journal ArticleDOI
TL;DR: These studies support the application of reverse-genetic strategies, including the use of temporally specific agonists and antagonists, to advance the functional dissection of memory formation.

Journal ArticleDOI
02 Jun 1995-Cell
TL;DR: The purification of telomerase and the cloning of cDNAs encoding two protein subunits from the ciliate Tetrahymena are described and two proteins of 80 and 95 kDa copurified and coimmunoprecipitated with telomersase activity and the previously identified TetrahYmena telomerases RNA are identified.

Journal ArticleDOI
TL;DR: In this article, a biochemical rationale for the hypothesis that carriers of certain p16(INK4) mutations are at increased risk of developing melanoma has been presented, along with biochemical analyses of the missense mutations and a single somatic mutation detected in these families.
Abstract: Cell division is controlled by a series of positive and negative regulators which act at sequential points throughout the cell cycle. Disturbance of these checks could contribute to cancer by allowing excessive cell proliferation. The point in G1 at which cells irrevocably commit to DNA synthesis is controlled by protein complexes consisting of cyclin-dependent kinases (CDK4 or CDK6) and cyclins (D1, D2 or D3). These complexes are inhibited by low molecular weight proteins, such as pl6(INK4) (refs 1,2), p15(INK4B) (ref. 3) and pl 8 (ref. 4). Deletion or mutation of these CDK- inhibitors could lead to unchecked cell growth, suggesting that members of the p16(INK4) family may be tumour suppressor genes. The recent detection of p16(INK4) (MTS1) mutations in familial melanoma kindreds, many human tumour cell lines, and primary tumours is consistent with this idea. Previously, we described eight germline p16(INK4) substitutions in 18 familial melanoma kindreds. Genetic analyses suggested that five mutations predisposed carriers to melanoma, whereas two missense mutations had no phenotypic effect. We now describe biochemical analyses of the missense germline mutations and a single somatic mutation detected in these families. Only the melanoma-predisposing mutants were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 and cyclin D1/CDK6 complexes in vitro. Our data provide a biochemical rationale for the hypothesis that carriers of certain p16(INK4) mutations are at increased risk of developing melanoma.

Journal ArticleDOI
17 Nov 1995-Cell
TL;DR: The cloning of the genes encoding the 120 kDa (ORC1, 62 kDa(ORC3), and 56 kDa) subunits of ORC and the reconstitution of the complete complex after expression of all six subunits in insect cells are reported.

Journal ArticleDOI
12 May 1995-Science
TL;DR: The PROLIFERA (PRL) gene, identified by gene trap transposon mutagenesis in Arabidopsis, is required for megaga-metophyte and embryo development and was expressed in dividing cells throughout the plant.
Abstract: Gene trap transposon mutagenesis can identify essential genes whose functions in later development are obscured by an early lethal phenotype. In higher plants, many genes are required for haploid gametophyte viability, so that the phenotypic effects of their disruption cannot be readily observed in the diploid plant body. The PROLIFERA (PRL) gene, identified by gene trap transposon mutagenesis in Arabidopsis, is required for megaga-metophyte and embryo development. Reporter gene expression patterns revealed that PRL was expressed in dividing cells throughout the plant. PRL is related to the MCM2-3-5 family of yeast genes that are required for the initiation of DNA replication.

Journal ArticleDOI
TL;DR: Cl cloning of the Saccharomyces cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5 is reported, and eight segments of conserved amino acid sequences that define a family of related proteins are identified.
Abstract: Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.

Journal ArticleDOI
08 Dec 1995-Science
TL;DR: It is suggested that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication.
Abstract: The origin recognition complex (ORC), a multisubunit protein identified in Saccharomyces cerevisiae, binds to chromosomal replicators and is required for the initiation of cellular DNA replication Complementary DNAs (cDNAs) encoding proteins related to the two largest subunits of ORC were cloned from various eukaryotes The cDNAs encoding proteins related to S cerevisiae Orc1p were cloned from the budding yeast Kluyveromyces lactis, the fission yeast Schizosaccharomyces pombe, and human cells These proteins show similarity to regulators of the S and M phases of the cell cycle Genetic analysis of orc1+ from S pombe reveals that it is essential for cell viability The cDNAs encoding proteins related to S cerevisiae Orc2p were cloned from Arabidopsis thaliana, Caenorhabditis elegans, and human cells The human ORC-related proteins interact in vivo to form a complex These studies studies suggest that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication

Journal ArticleDOI
TL;DR: The cloning of a Drosophila NOS gene, dNOS, is reported, located at cytological position 32B, and structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.
Abstract: Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.

Journal ArticleDOI
TL;DR: The comparable protein folding and the existence of equivalent amino acids in similar secondary and tertiary positions indicate that many (if not all) AdoMet-dependent methyltransferases have a common catalytic domain structure, which permits tertiary structure prediction of other DNA, RNA, protein, and small-molecule Ado metases from their amino acid sequences.

Journal ArticleDOI
TL;DR: Mutation of nucleotides in the B1 element suggests that this element has other functions in the initiation of DNA replication besides participating in the ORC-DNA interaction, supported by DNase I footprint analyses.
Abstract: Replicators are genetically defined elements within chromosomes that determine the location of origins of DNA replication. In the yeast Saccharomyces cerevisiae, the ARS1 replicator contains multiple functional DNA elements: an essential A element and three important B elements--B1, B2, and B3. Functionally similar A, B1, and B2 elements are also present in the ARS307 replicator. The B3 element binds a replication and transcription enhancer protein Abf1p, whereas the A element is required for binding the origin recognition complex (ORC). The function of the B1 and B2 elements remains to be defined. We have used a gel-based DNA binding assay to study the interaction between replicators and the putative initiator protein ORC. In addition to the established requirements for ATP and the A element for ORC-DNA interaction, the new data demonstrate that sequences in the B1 element are also important for ORC-DNA association. This conclusion is supported by DNase I footprint analyses and demonstrates that ORC binds to a bipartitite recognition element within the DNA. Furthermore, mutation of nucleotides in the B1 element suggests that this element has other functions in the initiation of DNA replication besides participating in the ORC-DNA interaction.