The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:͞͞mgc.nci.nih.gov).T he gene content of the mammalian genome is a topic of great interest. While draft sequences are now available for the human (1, 2), mouse (www.ensembl.org͞Mus musculus), and rat (http:͞͞hgsc.bcm.tmc.edu͞projects͞rat) genomes, the challenge remains to correctly identify all of the encoded genes. Difficulty in deciphering the anatomy of mammalian genes is due to several factors, including large amounts of intervening (noncoding) sequence, the imperfection of gene-prediction algorithms (3), and the incompleteness of cDNA-sequence resources, many of which consist of gene tags of variable length and quality. Full-length cDNA sequences are extremely useful for determining the genomic structure of genes, especially when analyzed within the context of genomic sequence. To facilitate geneidentification efforts and to catalyze experimental investigation, the National Institutes of Health (NIH) launched the Mammalian Gene Collection (MGC) program (4) with the aim of providing freely accessible, high-quality sequences for validated, complete ORF cDNA clones. In this article, we describe our progress toward the goal of identifying and accurately sequencing at least one full ORF-containing cDNA clone for each human and mouse gene, as well as making these fully sequenced clones available without restriction. Materials and MethodscDNA Library Production. MGC cDNA libraries were prepared from a diverse set of tissues and cell lines, in several different vector systems, by using a variety of methods. Vector maps and details of library construction are available at http:͞͞mgc. nci.nih.gov͞Info͞VectorMaps. The complete sequences for each of the MGC vectors can be found at http:͞͞image.llnl.gov͞ image͞html͞vectors.shtml. The catalog of MGC cDNA libraries can be accessed at http:͞͞mgc.nci.nih.gov.

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

The Raji line of human lymphoblastoid cells, which does not show expression of Epstein-Barr virus, was made resistant to 5-bromodeoxyuridine. Within several weeks after removal of the drug, Epstein-Barr virus particles were detected in the cells. The

Replication of the resident repressed Epstein-Barr virus genome in synchronized nonproducer Raji cells was shown to occur during the early S phase (S-1 period) by hybridization of cell DNA with virusspecific complementary RNA (cRNA). The S-1 period was previously identified as the critical period for virus activation induced by thymidine analogues. The findings reported here and elsewhere are consistent with the proposal that: (i) virus activation is initiated at or near the site of association of the resident viral genome with cell DNA, (ii) replication of the resident virus genome in nonactivated cells is under cell control mechanisms, and (iii) the resident virus genome is physically associated with early replicating cell DNA.Human lymphoblastoid cells latently infected with the Epstein-Barr herpesvirus (EB virus) contain numerous, apparently complete copies of the repressed virus genome (1-4). Virus activation in cultured lymphoblastoid cells occurs spontaneously in producer cells (5), and may be induced in some producer and nonproducer cells by incorporation into DNA of thymidine (dT) analogues (6,7).Previous studies (8) on the mechanism of EB virus activation by 5-iododeoxyuridine (IdU) indicated that activation in synchronized producer or nonproducer cells required incorporation of the drug into DNA during the early S phase (S-i period). It was suggested that the DNA synthesized during the S-1 period contains unique sequences which control activation of the EB viral genome.This report presents findings concerning the time of replication during the cell cycle of the resident repressed EB virus genome in nonproducer Raji cells. The results indicate that replication of the resident EB virus genome occurs during the S-1 period, which corresponds temporally to the previously reported (8) Nucleic Acid Hybridization. Synchronized cells were harvested at intervals after reversal of the dT block and were pelleted at 2000 X g. The cells were resuspended in 0.05 M Tris buffer (pH 9.0) and frozen for subsequent extraction of DNA using previously described procedures (2). The preparation of EB virus-specific complementary RNA (cRNA) and procedures for DNA* cRNA hybridization have been described (2). RESULTSNonproducer Raji cells were selected for these studies since they do not spontaneously undergo productive EB virus replication. On rare occasion, we have observed by immunofluorescence Raji cells positive for EB virus-associated early antigens (EA), but not for late viral structural antigens (VCA) (8). Synthesis of EA occurs prior to productive replication of viral DNA, while VCA synthesis occurs subsequent to productive replication of viral DNA (9) in EB virus producer cells. When Raji cells are exposed to IdU for periods up to several days, only EA is made in the activated cells (8), and attempts to detect productive viral DNA synthesis in IdUactivated Raji cells by sensitive nucleic acid hybridization techniques have been unsuccessful (Nonoyama, unpublished observations). Based on these findings, we assu...

The P3HR-1 line of human lymphoblastoid cells that is Epstein-Barr virus positive was made resistant to 5-bromodeoxyuridine. Epstein-Barr virusassociated antigens, but not virus particles, were produced in P3HR-1(BU) cells maintained on 5-bromodeoxyuridine. However, virus particles did appear within 4 days after removal of the drug. Thymidine kinase activity was limited to P3HR-1(BU) cells producing viral antigen, whereas all control P3HR-1 cells showed thymidine kinase activity regardless of viral antigen synthesis.Cellular DNA in most P3HR-1(BU) cells was made via pathways that did not involve thymidine kinase. In cells having a pathway that involved thymidine kinase, a second DNA of density 1.71 g/cms, corresponding to EpsteinBarr virus, was detected.It was concluded that: (a) a repressed Epstein-Barr virus genome persists in P3HR-1(BU) cells that do not contain thymidine kinase, with activation of the viral genome being accompanied by productive infection and the appearance of enzyme, and (b) thymidine kinase activity in P3HR-1(BU) cells could be used as a marker for viral genome expression.Some human lymphoblastoid cell lines show persistent infection with the human Epstein-Barr herpesvirus (EB virus), which is synthesized in at least a portion of the cell population at any one time. The persistence of EB virus may be due either to a low-grade infection with transmission of infectious virus or to derepression of an integrated viral genome. Cloning experiments with lymphoblastoid cells favor the derepression mechanism (1-3).The studies reported here concern the properties of EB virus-negative cells in a virus-positive cell population made resistant to 5-bromodeoxyuridine (BrdU (v/v); the slides were pressed lightly between the pages of a bibulous paper pad. The cells were scanned by darkfield fluorescence microscopy, and appropriate areas were photographed and their locations were recorded.The coverslips were removed, the cells were washed in water and air dried. Stripping film (Kodak AR-10) was applied and the slides were exposed for 1-3 weeks at 4VC before developing. The cells were stained for 30 see in 0.2% crystal violet in 95% methanol (w/v) and the localized areas were scanned under brightfield microscopy. microscopy)