The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase G-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifles with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-speciflic DNA polymerase a-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.Replication of simian virus 40 (SV40) DNA requires only one virus-encoded protein, large tumor antigen (T antigen); initiates within a unique, well-defined origin sequence; proceeds bidirectionally; and terminates in a manner thought to be analogous to that utilized by the host chromosome (1, 2). Replication occurs on a template that is associated with nucleosomes in a structure resembling cellular chromatin (3). Thus, the study of SV40 and presumably cellular DNA replication should be facilitated by the recent development of in vitro systems that reproduce many key aspects of SV40 DNA replication in vivo (4)(5)(6)(7)(8). By using such systems, the DNA sequences required 'for origin function in vitro have been identified (9, 10), the roles of the complex of DNA polymerase a (pol a) and primase in viral replication and host species specificity have been investigated (11), and a system has been described whereby newly replicated DNA is assembled into a chromatin-like structure (12).Genetic and biochemical analyses of prokaryotic systems have revealed a number of activities directly involved in the enzymatic process of DNA replication: origin-specific binding activity, priming and deoxynucleotide polymerizing activities, helix unwinding activity, single-stranded DNA binding protein (SSB) to maintain the DNA in an unwound configuration, primer removal activity, DNA ligase, activities that relieve torsional strain accumulating ahead of the replication fork and resolve daughter molecules, and factors that modify either the template or one of the activities mentioned above to increase their efficiency (13). With these studies in mind, we have purified enzymes thought to be required for SV40 (and cellular) DNA replication in vivo and used the...

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for ␦-sarcoglycan (␦-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5 upstream of the second exon of ␦-SG gene, and its 5 upstream region of more than 27.4 kb, including the authentic first exon of ␦-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of ␦-SG transcripts with the consequent loss of ␦-SG protein in all the CM hamsters, despite the fact that the protein coding region of ␦-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including ␦-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of ␦-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of ␦-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.Cardiomyopathy (CM) manifests dyspnea, cardiac failure, or sudden death, causing serious morbidity and mortality. Clinical features and molecular genetic studies of CM demonstrate a wide variety of possible genetic causes of this disease but the causative genes and pathogenesis are poorly understood (1-3). Medical treatment for this progressive disease are only palliative with poor prognosis. Syrian hamsters with CM are known to inherit both CM and muscular dystrophy as an autosomal recessive trait but the genetic cause still remains to be elucidated (4-6). Recent studies on muscular dystrophy revealed the genetic importance of sarcoglycans (SGs), a subcomplex of dystrophin-associated glycoprotein complex (DAGC), in this disease (7-10).Distinct sublines of Syrian hamster manifesting hypertrophic CM (HCM; BIO 14.6 and its descendant UMX7.1) or dilated CM (DCM; TO-2) have been established from the original line BIO1.50 (5, 6). We have reported to the DDBJ (DNA Data Base of Japan) that no mutation exists in the coding regions of cDNAs of BIO14.6 for ␣-, ␤-, or ␥-SGs, all of which are lost in cardiac and skeletal muscles of this animal, where dystrophin is normally expressed (11). Our latest study revealed that these SGs are also deficient in UMX7.1 and TO-2 (vide infra), suggesting a hypothesis that both HCM and DCM share the loss of SG subcomplex as a common causative feature in hamster. In addition, ␦-SG, which was identified recently, seemed to constitute DAGC together with ␣-, ␤-, and ␥-SGs (12). These facts prompted us to identify the causative gene common to HCM and DCM wit...

The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it-to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurifiled with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen- This sequence of events is analogous to the steps proposed for the initiation of replication in prokaryotic systems. For instance, initiation of Xdv (8) and Escherichia coli origin (oriC) replication (9) is believed to depend upon precise protein interactions with the replication origin. These interactions enable the DnaB protein, a DNA helicase (10), to unwind the duplex. Furthermore, when a plasmid containing the E. coli oriC sequence was incubated with proteinsDnaA, DnaB, and DnaC, protein HU, single-strand DNA binding protein (SSB), and DNA gyrase-a DNA form of unusual electrophoretic mobility was produced (11). This form consisted of highly underwound covalently closed circles, and it was suggested that the helicase and DNA gyrase drive the unwinding.We have detected a similar origin-specific unwinding of DNA in a eukaryotic system, using SV40 origin-containing DNA. The protein requirements for the assay are T antigen, SSB, and a topoisomerase (topo) to relax the resultant supercoils. The reaction is also dependent on ATP, an ATP regenerating system, and Mg2". These observations suggested that T antigen is associated with a DNA helicase activity and that T antigen is responsible for the duplex DNA unwinding that is likely to initiate replication. While this work was in progress Stahl et al. (12) reported that SV40 T antigen contains a DNA helicase activity. MATERIALS AND METHODSAll reagents, enzyme preparations, and assay procedures used were as previously described (3) 16The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

We have identified a strong candidate cDNA for the mouse reeler gene. This 5 kb transcript encodes a 99.4 kD protein consisting of 881 amino acids and possessing two EGF-like motifs. We assayed two independent mutant alleles--'Jackson reeler', which has a deletion of the entire gene, and 'Orleans reeler' which exhibits a 220 bp deletion in the open reading frame, including the second EGF-like motif and resulting in a frame shift. In situ hybridization reveals that the transcript is detected exclusively in the pioneer neurons which guide neuronal cell migration along the radial array. Our findings offer an explanation for how the reeler mutant phenotype causes a disturbance of the complex architecture of the neuronal network.

The role of DNA polymerase a (pol a) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol a and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol a immunoaffinity column resulted in the loss of replication activity. The addition of purified pol a-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol a-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol a-primase complex isolated from HeLa cells activated mouse cell extracts. pol a and primase from HeLa cells were extensively purified and separated by a one-step immunoaffmity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol a or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol a alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol a, displace the non-template strand, and allow the enzyme to replicate through duplex regions.The replication of simian virus 40 (SV40) DNA is an important model system for studying eukaryotic DNA replication because it requires only one virus-encoded protein, the SV40 large tumor antigen (T antigen); all the other components involved in this process are supplied by host cells. Our interest is in the isolation of factors that are involved in mammalian DNA replication. For this purpose, we have developed an in vitro replication system that is analogous to those described by others (1-3), using a salt extract of exponentially growing HeLa cells and plasmid DNA containing the SV40 origin sequence (4, 5). Replication with these extracts was shown to be totally inhibited by aphidicolin, a specific inhibitor of DNA polymerase a (pol a). In this communication we describe the role of pol a and DNA primase in the replication system. pol a plays a key role in mammalian DNA replication (6-8). We have found that pol a and primase activity are essential for the in vitro replication of DNA containing the SV40 origin. We have also found that the cellular source of these activities plays an important role in determining whether SV40 DNA replication can be catalyzed by extracts in vitro. Those cells that support replication in vivo yield extracts that replicate SV40 DNA in vitro; cells that do not support replication yield extracts that are inactive. The pol a-primase complex isolated from permissive cell...

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In vitro replication of DNA containing the polyoma (Py) virus orign of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase a in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. Cell-free extracts of monkey and human cells that replicate SV40 DNA have been described (4-7). The in vitro replication of SV40 DNA requires, in addition to suitably prepared cell extracts, circular DNA containing the viral replication origin (ori) and purified SV40 large tumor (T) antigen. Since SV40 T antigen and DNA containing the SV40 replication origin are the only viral components required, this system should be useful for identifying and characterizing the eukaryotic proteins involved in DNA replication. In previous reports (7-9), it was shown that DNA polymerase a (pol a) and DNA primase are essential for the replication of SV40 DNA in vitro and that the source of these enzymes is important in determining whether SV40 DNA replication will occur. In contrast to extracts of human cells, mouse cell extracts supplemented with SV40 T antigen did not support replication of DNA containing the SV40 origin unless supplemented with the pol a-primase complex from HeLa cells.In this report we describe the establishment of a system that replicates Py DNA in vitro; it requires mouse cell extract, the Py replication origin, and Py T antigen. In addition, we have confirmed and further defined the important role played by pol a-primase in determining the species specificity of papovavirus DNA replication. MATERIALS AND METHODSPurification of Py T Antigen. Py T antigen was purified from CV-1 cells infected with the helper-dependent recombinant adenovirus vector Ad-SVR587 (10) as follows. Cells were infected with wild-type adenovirus [2-5 plaque-forming units (pfu) per cell] and Ad-SVR587 recombinant virus (a gift from S. Mansour, T. Grodzicker, and R. Tjian) (5-10 pfu per cell). After incubation for 36 hr at 370C, cells were scraped from thirty 150-mm plates into -3 ml of cold Dulbecco's phosphate-buffered saline (PBS: Ca2+-and Mg2+-free), washed twice with cold PBS, suspended in 10 volumes of pH 9.0 buffer [20 mM Tris HCl, pH 9.0/0.2 M NaCl/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol/1% (vol/vol) Nonidet P-40 (NP-40)/1 mM phenylmethylsulfonyl fluoride (PMSF)], and lysed on ice for 10: min. The lysate was centrifuged for 10 min at 2000 rpm in a Sorvall H-6000A rotor, and the supernatant was then centrifuged for 10 min at 20,000 rpm in a Sorvall SS34 rotor. The supernatant was mixed with 0.5 volume of pH 6.8 buffer (0.1 M Tris'HCl, pH 6.8/1 mM EDTA/1 mM dithiothre...