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...

A complementary DNA encoding DNA-dependent ATPase Q1 possessing DNA helicase activity, which is the major DNA-dependent ATPase in human cell extracts, was cloned from a cDNA library of human KB cells. The predicted amino acid sequence has seven consecutive motifs conserved in the RNA and DNA helicase super family and DNA helicase Q1 belongs to DEXH helicase family. A homology search indicated that helicase Q1 had 47% homology in the seven conserved regions with Escherichia coli RecQ protein. Three RNA bands of 4.0, 3.3, and 2.2 kilobases were detected in HeLa cells by Northern blotting. Analysis of the genomic DNA indicated the presence of a homologous gene in mouse cells. The DNA helicase Q1 gene was localized on the short arm of human chromosome 12 at 12p12.

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...

The synthesis of oligoribonucleotides by DNA primase in the presence of duplex DNA containing the simian virus 40 (SV40) origin of replication was examined. Small RNA chains (10-15 nucleotides) were synthesized in the presence of the four common ribonucleoside triphosphates, SV40 large tumor antigen (T antigen), the human DNA polymerase a (pol a)-DNA primase complex, the human single-stranded DNAbinding protein (HSSB), and topoisomerase I isolated from HeLa cells. The DNA primase-catalyzed reaction showed an absolute requirement for T antigen, HSSB, and pol a. The requirement for HSSB was not satisfied by other SSBs that can support the T-antigen-catalyzed unwinding of DNA containing the SV40 origin of replication. Oligoribonucleotide synthesis occurred with a lag that paralleled the lag observed in DNA synthesis. These results indicate that the specificity for the HSSB in the SV40 replication reaction is due to the pol a-primase-mediated synthesis of the Okazaki fragments. In contrast to this specificity, the elongation of Okazaki fragments can be catalyzed by a variety of different DNA polymerases, including high levels of pol a, the polymerase 6 holoenzyme, T4 polymerase holoenzyme, the Escherichia coli polymerase III holoenzyme, and other polymerases. These observations suggest that leading-strand synthesis in the in vitro SV40 replication system can be nonspecific.The replication of DNA containing the simian virus 40 (SV40) origin has been divided into a number of discrete steps (1-3). These include (a) the ATP-dependent binding of SV40 large tumor antigen (T antigen) at the core origin and the formation of hexamers on the DNA, followed by multiple changes at the origin that activate the intrinsic T-antigen DNA helicase. This results in (b) the ATP-dependent unwinding reaction, which requires a topoisomerase that relieves positive superhelicity generated by the movement of the T antigen through the duplex and a single-stranded DNA-binding protein (SSB) that binds the single strands generated by the displacement of the duplex. Prior to extensive unwinding, T antigen binds the DNA polymerase (pol) a-primase complex and facilitates (c) the initiation of small RNA primers by primase, which, coupled to (d) the action of pol a, results in the formation of small Okazaki fragments. Further unwinding leads to the continued generation of short DNA chains on the lagging strands. DNA primers can then be used for (e) the synthesis of the leading strand, which involves the addition of activator 1 (Al), also called RF-C (4), and proliferating-cell nuclear antigen (PCNA) to the 3'-hydroxyl end of the primertemplate, permitting elongation by pol 8 (4, 5). DNA synthesis continues until chains are juxtaposed; then oligoribonucleotides are removed by the combined action of RNase H and a 5' -* 3' exonuclease, and gaps are filled in by a DNA polymerase and closed by DNA ligase. The semiconserved DNA products are resolved from one another by topoisomerase II, resulting in two circular closed duplex DNA molecules.We have e...

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase 8 that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase 8 on a DNA singly primed 4X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase 6, in contrast to DNA polymerase a, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase a and 8 are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.The in vitro origin of replication-dependent simian virus 40 (SV40) DNA replication has been established by using cytosolic extracts of human or monkey cells supplemented with SV40 large tumor antigen (Tag) (1-3). SV40 Tag is a multifunctional protein possessing origin-specific DNA-binding, ATPase, and DNA helicase activities; this antigen is required for initiating DNA replication (4-7).We have described a system capable of catalyzing a Tag and SV40 origin-dependent replication reaction using purified proteins. In addition to Tag and DNA containing the SV40 origin of replication (ori'DNA), immunopurified DNA polymerase a-DNA primase complex, topoisomerase (topo) I and/or II and a multisubunit single-stranded DNA-binding protein (SSB), all isolated from HeLa cells, are required (8,9). Replication reactions containing these proteins will hereafter be identified as the monopolymerase system.Proliferating-cell nuclear antigen (PCNA), a known accessory factor for DNA polymerase 6 (Pol-6) (10-12), was shown essential for leading-strand DNA synthesis in crude systems; without PCNA only small-sized DNA products arising from the lagging strand were synthesized (13). In contrast, PCNA had no effect on the monopolymerase system. The reasons for this difference were traced to the effect of multiple protein factors [an elongation inhibitor (El), activators I (Al) and II (All)], which were present in crude fractions but not in the monopolymerase system (14, 15). The El has been characterized as a 120-kDa protein that binds to ends of DNA chains, thus inhibiting enzymes that act at such sites (such as exonuclease III, 5' --3' exonuclease, and DNA ligase).Pol-6 was initially isolated and distinguished from other DNA polymerases by its tightly associated 3' -k 5' exonuclease activity and its PCNA dependency (16,17). Recently another polymerase, also called Pol-6, which does not require PCNA, has been isolated from several mammalian sources (17-20). These different Pol-8 preparations share some common properties, including 3' -+ 5' exonuclea...

DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase a (pol-a), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase 6, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-a extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. 17 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-a, conditions under which pol-a was markedly inhibited. These findings suggest that interactions between T antigen, pol-a-primase, and HSSB position the pol-a-primase complex on the lagging-strand template for RNA primer synthesis.The initiation of replication of simian virus 40 (SV40) DNA includes several discrete steps (1-3). The first step involves the ATP-dependent binding oflarge tumor antigen (T antigen) at the core origin and the formation of double hexamers, followed by multiple structural changes in the core origin (3, 4). In the presence of human single-stranded DNA binding protein (HSSB) and topoisomerase I (topo I), T-antigen hexamers unwind the DNA bidirectionally, each moving in a 3' -+ 5' direction on opposite strands. At some point during these early reactions, the DNA polymerase a (pol-a)-primase complex interacts with T antigen and HSSB, and primase synthesizes small oligoribonucleotides that prime the synthesis of small Okazaki fragments by pol-a. After these initiation steps, synthesis of short DNA chains occurs on the lagging-strand template concomitant with the further unwinding of the duplex. Although pol-a at high concentrations can support leading-strand synthesis, extensive leadingstrand synthesis at low concentrations of pol-a is dependent on proliferating cell nuclear antigen (PCNA), activator 1 (Al), also called RF-C (5), and pol-S. Recent studies suggest that a PCNA-dependent DNA polymerase is also involved in the maturation of short Okazaki DNA chains into mature Okazaki fragments (6,7). After DNA chains are elongated and juxtaposed, they are ligated by the combined action of RNase H, a 5' -> 3' exonuclease, and DNA ligase (8).We have previously reported that oligoribonucleotides were synthesized from duplex circular DNA containing the SV40 origin in the absence of DNA synthesis with a purified system that contained T antigen...

Helicase-related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase-related proteins make up several gene families. In this study, we have identified 134 ORFs that encode helicase-like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase-related proteins. Multiple alignment of the ORF sequences resulted in the 134 ORFs being classified to 11 clusters. Seven out of 21 previously uncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) were identified by systematic gene disruption, to be essential for vegetative growth. Three (YDR332w, YGL064c, and YOL095c) out of the remaining 14 dispensable ORFs exhibited the slow-growth phenotype at 30 degrees C and 37 degrees C. Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growth conditions by Northern analysis and reverse transcription-polymerase chain reaction, indicating that all of the 43 tested ORFs were transcribed. Interestingly, we found that the level of transcript from 34 helicase-like genes was markedly increased by heat shock. This suggests that helicase-like genes may be involved in the biosynthesis of nucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for the repressed synthesis of mRNA and protein.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VI (270 kb) has revealed that it contains 129 predicted or known genes (300 bp or longer). Thirty-seven (28%) of which have been identified previously. Among the 92 novel genes, 39 are highly homologous to previously identified genes. Local sequence motifs were compared to active ARS regions and inactive loci with perfect ARS core sequences to examine the relationship between these motifs and ARS activity. Additional ARS sequences were predominantly observed in 3' flanking sequences of active ARS loci.