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Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.

Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.

The genetic basis of the phenotypic difference between human and chimpanzee is one of the most actively pursued issues in current genomics. Although the genomic divergence between the two species has been described, the transcriptomic divergence has not been well documented. Thus, we newly sequenced and analyzed chimpanzee full-length cDNAs (FLcDNAs) representing 87 protein-coding genes. The number of nucleotide substitutions and sites of insertions/deletions (indels) was counted as a measure of sequence divergence between the chimpanzee FLcDNAs and the human genome onto which the FLcDNAs were mapped. Difference in transcription start/termination sites (TSSs/TTSs) and alternative splicing (AS) exons was also counted as a measure of structural divergence between the chimpanzee FLcDNAs and their orthologous human transcripts (NCBI RefSeq). As a result, we found that transposons (Alu) and repetitive segments caused large indels, which strikingly increased the average amount of sequence divergence up to more than 2% in the 3'-UTRs. Moreover, 20 out of the 87 transcripts contained more than 10% structural divergence in length. In particular, two-thirds of the structural divergence was found in the 3'-UTRs, and variable transcription start sites were conspicuous in the 5'-UTRs. As both transcriptional and translational efficiency were supposed to be related to 5'- and 3'-UTR sequences, these results lead to the idea that the difference in gene regulation can be a major cause of the difference in phenotype between human and chimpanzee.

Coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin (Ig) superfamily and a component of epithelial tight junction. CAR also functions as a primary receptor for coxsackievirus B and adenovirus (Ad) infection. In this study, we report the identification of a novel protein, CAR-like soluble protein (CLSP), which is closely related to CAR. Mouse CLSP (mCLSP) was composed of 390 amino acids, including three Ig domains, and showed strong homology to the IgV domain of CAR. Interestingly, mCLSP lacks a transmembrane domain, indicating that this is a soluble protein. mCLSP mRNA was detected primarily in the brain and ovary. When mCLSP cDNA was introduced into SK HEP-1 cells, which were known to be CAR positive and easily infected with Ad vector, the infection with Ad vector was severely inhibited. On the other hand, mCLSP promoted the infection with Ad vector in CAR-negative NIH3T3 cells. Furthermore, recombinant CLSP directly bound to Ad and inhibited the Ad vector-mediated transduction in SK HEP-1 cells. Computational analysis for a genome database showed that the CLSP gene is rodent-specific, and that human and bovine lack this gene. These results suggest that CLSP may play a role in the antiviral defense of the host in rodent animals.

Finding genetic novelties that may contribute to human-specific physiology and diseases is a key issue of current biomedical studies. TMEM30C is a gene containing two transmembrane (TM) domains and homologous to the yeast CDC50 family, which is related to polarized cell division. It is conserved among mammals along with two other paralogs, TMEM30A and TMEM30B. We found that TMEM30C is expressed specifically in the testis of mammals, in contrast to the relatively wide expression distributions of the other paralogs. While macaques expressed two alternative splicing isoforms which include one or two TM domains, humans and chimpanzees predominantly expressed truncated transcripts because of the mutations in the splicing and/or poly(A) signal sites. The major transcript in humans harbored non-stop ORF (open reading frame) while the chimpanzee counterpart encoded a protein with one TM domain. The difference was due to the 1-bp indel upstream of the poly(A) signal site. In addition, both the hominoids expressed minor transcripts encoding short proteins with one TM domain. Phylogenetic analysis has showed the acceleration of amino acid substitution after the human and chimpanzee divergence, which may have been caused by a recent relaxation in functional constraints or positive selection on TMEM30C. Elucidating the precise reproductive function of TMEM30C in mammals will be important to the foundation of divergence in higher primates at a molecular level.

Chemokines are a rapidly evolving cytokine gene family. Because of various genome rearrangements after divergence of primates and rodents, humans and mice have different sets of chemokine genes, with humans having members outnumbering those of mice. Here, we report the occurrence of lineage-specific chemokine gene generation or inactivation events within primates. By using human chemokine sequences as queries, we isolated a novel cynomolgus macaque CXC chemokine cDNA. The encoded chemokine, termed CXCL1L (from CXCL1-like) showed the highest similarity to human CXCL1. A highly homologous gene was also found in the rhesus macaque genome. By comparing the genome organization of the major CXC chemokine clusters among the primates, we found that one copy of the duplicated CXCL1 genes turned into a pseudogene in the hominids, whereas the gene in macaques has been maintained as a functionally active CXCL1L. In addition, cynomolgus macaque was found to contain an additional CXC chemokine highly homologous to CXCL3, termed CXCL3L (from CXCL3-like). These results demonstrate the birth-and-death process of a new gene in association with gene duplication within the primates.

The Borg (binder of Rho GTPases) family proteins interact with CDC42 and TC10 in a guanosine triphosphate (GTP)-dependent manner. We have isolated a full-length cDNA of the mouse Borg4 gene, which is a member of this family. Sequence analysis revealed that this gene encoded a putative 349-amino acid protein. By reverse transcription — coupled polymerase chain reaction (RT-PCR) analysis, we observed that Borg4 was expressed ubiquitously in adult tissues. Additionally, we determined the entire cDNA sequence of the putative human Borg4 orthologue. By fluorescence in situ hybridization, mouse Borg4 and the putative human orthologue have been assigned to mouse chromosome 11E and human chromosome 17q24–25, which has been described as syntenic to the mouse region.

Casein kinase 1 gamma1(CK1 gamma1) is known to be involved in the growth and morphogenesis of eukaryotic cells. We have isolated two types of cDNA for human casein kinase 1 gamma1 (hCK1 gamma1). One of them (hCK1 gamma1S) was found to encode a polypeptide consisting of 393 amino acids, which is highly homologous with already reported rat CK1 gamma1 (rCK1 gamma1). The other type of cDNA (hCK1 gamma1L) encodes a polypeptide consisting of 422 amino acids, which is quite identical in the kinase domain, but different in the C-terminal sequence from hCK1 gamma1S. Namely, hCK1 gamma1L has a characteristic sequence of 50 amino acids at the C-terminal end and this motif was shown to be shared by the casein kinase gamma2 and gamma3 from rat and human, suggesting that it is a signature sequence of the gamma-isoforms. In this sense, newly isolated hCK1 gamma1L might be the original form of CK1 gamma1 subspecies rather than rCK1 gamma1 and hCK1 gamma1S. RT-PCR analysis revealed that hCK1 gamma1S mRNA is predominantly present in the testis, whereas the abundance of hCK1 gamma1L mRNA was nearly the same in the twelve tissues examined. These results suggest that novel hCK1 gamma1L may have a unique functional role different from that of hCK1 gamma1S and rCK1 gamma1. The human hCK1 gamma1 gene (CSNK1G1) was mapped to chromosome 15q22.1--q22.31 by fluorescence in situ hybridization.

The intron-containing genes encoding human and mouse ribosomal protein (r-protein) L27A were cloned and sequenced. The human r-protein L27A gene (RPL27A) shared an identical exon/intron structure with the mouse r-protein 27A gene (Rpl27a). The translational start codon ATG was separated from the main reading frame by the first intron sequence in both genes. An approximately 200-bp sequence upstream of the translational start site of both genes displayed remarkable similarity, and contained the putative promoters lacking canonical TATA, but harbored Sp1 binding sites and a short stretch of pyrimidine cluster, similar to other r-protein genes. Transcriptional regulatory elements, Box-A and GABP, found in the promoters of some other r-protein genes were also conserved in both genes. These structural features were included in the typical CpG island identified in the 5′-end sequences, suggesting that RPL27A/Rpl27a cloned here are authentic and transcriptionally active. Fluorescence in situ hybridization (FISH) analysis localized the mouse intron-containing Rpl27a to chromosome 7E2–F1 syntenic to human chromosome 11p15, where human RPL27A was located.

A sequence homologous to the transaldolase gene (TALDO) was identified in a polymorphic cosmid DNA mapped on human chromosome 11p15 by exon trapping with pSPL3. Analysis of lambda clones contiguous to the cosmid clone showed that the related gene (TALDOR) consists of 8 exons spanning approximately 19 kb from the translation start site to the polyadenylation signal. The exon sequence of TALDOR was almost identical with that of TALDO localized on 1p33-34.1, but its exons corresponding to exons 4 and 5 of TALDO were found to be split by 4 introns in TALDOR. To examine the evolutionary conservation of two genes for transaldolase, we have isolated the cDNA for its mouse homolog and determined the nucleotide sequence covering the complete coding region. Fluorescence in situ hybridization using the cDNA as a probe showed that the mouse transaldolase gene (Taldo) is localized on chromosome 7 F3-F4 as a single copy gene. This chromosomal region is known to be syntenic to human chromosome 11p15 rather than to 1p33-p34.1, suggesting that TALDOR is the ancestral form. The existence of TALDOR implies a duplication of the mammalian transaldolase gene after divergence of rodent and primate.

By searching the expressed sequence tag (EST) data base, we identified partial cDNA sequences encoding a novel human CC chemokine. We determined the complete cDNA sequence that encodes a highly basic polypeptide of a total 98 amino acids with 20 to 30% identity to other human CC chemokines. We termed this novel chemokine from EBI1-Ligand Chemokine as ELC (see below). The ELC mRNA was most strongly expressed in the thymus and lymph nodes. Recombinant ELC protein was expressed as a fusion protein with the Flag tag (ELC-Flag). For receptor-binding assays, recombinant ELC protein fused with the secreted form of alkaline phosphatase (SEAP) was used. By stably expressing five CC chemokine receptors (CCR1 to 5) and five orphan receptors, ELC-SEAP was found to bind specifically to an orphan receptor EBI1. Only ELC-Flag, but not MCP-1, MCP-2, MCP-3, eotaxin, MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed and secreted), thymus and activation-regulated chemokine (TARC), or liver and activation-regulated chemokine (LARC), competed with ELC-SEAP for EBI1. ELC-Flag-induced transient calcium mobilization and chemotactic responses in EBI1-transfected cells. ELC-Flag also induced chemotaxis in HUT78 cells expressing endogenous EBI1 at high levels. By somatic hybrid and radiation hybrid analyses, the gene for ELC (SCYA19) was mapped to chromosome 9p13 instead of chromosome 17q11.2 where the genes for CC chemokines are clustered. Taken together, ELC is a highly specific ligand for EBI1, which is known to be expressed in activated B and T lymphocytes and strongly up-regulated in B cells infected with Epstein-Barr virus and T cells infected with herpesvirus 6 or 7. ELC and EBI1 may thus play roles in migration and homing of normal lymphocytes, as well as in pathophysiology of lymphocytes infected with these herpesviruses. We propose EBI1 to be designated as CCR7.

Transaldolase catalyzes the transfer of a C3 fragment corresponding to dihydroxyacetone from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, forming erythrose 4-phosphate and fructose 6-phosphate in the pentose phosphate pathway. The pathway provides mainly D-ribose 5-phosphate for nucleic acid synthesis and NADPH for lipid biosynthesis. 10 refs., 1 fig.

Partial overlapping cDNA sequences likely to encode a novel human CC chemokine were identified from the GenBank Expressed Sequence Tag data base. Using these sequences, we isolated full-length cDNA encoding a protein of 96 amino acid residues with 20-28% identity to other CC chemokines. By Northern blot, this chemokine was mainly expressed in liver among various tissues and strongly induced in several human cell lines by phorbol myristate acetate. We thus designated this chemokine as LARC from Liver and Activation-Regulated Chemokine. We mapped the LARC gene close to the chromosomal marker D2S159 at chromosome 2q33-q37 by somatic cell and radiation hybrid mappings and isolated two yeast artificial chromosome clones containing the LARC gene from this region. To prepare LARC, we subcloned the cDNA into a baculovirus vector and expressed it in insect cells. The secreted protein started at Ala-27 and was significantly chemotactic for lymphocytes. At a concentration of 1 microg/ml, it also showed a weak chemotactic activity for granulocytes. Unlike other CC chemokines, however, LARC was not chemotactic for monocytic THP-1 cells or blood monocytes. LARC tagged with secreted alkaline phosphatase-(His)6 bound specifically to lymphocytes, the binding being competed only by LARC and not by other CC or CXC chemokines. Scatchard analysis revealed a single class of receptors for LARC on lymphocytes with a Kd of 0.4 nM and 2100 sites/cell. Collectively, LARC is a novel CC chemokine, which may represent a new group of CC chemokines localized on chromosome 2.

We screened potential promoter regions from NotI-linking cosmid clones mapped on human chromosome Xq28 region with our constructed trapping vector and isolated six fragments containing transcription activity. Using one of the obtained fragments as a probe, a novel gene was isolated by screening a human skeletal muscle cDNA library. The isolated cDNA, termed HXC-26, contained an open reading frame of 975 nucleotides encoding 325 amino acids (38,848 Da). The HXC-26 gene was composed of 13 exons that span approximately 8 kb. Several potential GC boxes were found in the putative promoter region, but no typical TATA box. The HXC-26 gene associated with a CpG island was located adjacent to the rab GDP dissociation inhibitor (GDI) gene.

我々はニワトリ肝細胞の初代培養法について,炭酸ガスインキュベーターを使用せずに通常の空気インキュベーターを用いる簡便な方法を確立した。これは肝細胞を通常のコラゲナーゼ潅流法で調製し,通常の空気インキュベーター中で,30mM Hepes-NaOHでpH7.8に調整した5%血清(ニワトリあるいは仔牛血清)-L-15培地を用いて蒔込み,3時間後に無血清培地に交換して長期間培養するものである。ニワトリ肝細胞はこの弱アルカリ性の無血清培地で接着•伸展し,10日間程度容易に維持できた。このような条件で培養した肝細胞は肝特異的機能の1つであるアルブミン合成能を,特に培養後期(8日目以降)に強く発現することが確認され,肝特異的機能の1つであるアルブミン生合成能を発現している肝細胞実験系として有用であることが示された。

Background Consolidating transcriptome data of non-human primates is essential to annotate primate genome sequences, and will facilitate research using non-human primates in the genomic era. Macaca fascicularis is a macaque monkey that is commonly used for biomedical and ecological research. Findings We constructed cDNA libraries of Macaca fascicularis, derived from tissues obtained from bone marrow, liver, pancreas, spleen, and thymus of a young male, and kidney of a young female. In total, 5'-end sequences of 56,856 clones were determined. Including the previously established cDNA libraries from brain and testis, we have isolated 112,587 cDNAs of Macaca fascicularis, which correspond to 56% of the curated human reference genes. Conclusion These sequences were deposited in the public sequence database as well as in-house macaque genome database http://genebank.nibio.go.jp/qfbase/. These data will become valuable resources for identifying functional parts of the genome of macaque monkeys in future studies.

Intracellullar trafficking of lipids is fundamental to membrane biogenesis. For the synthesis of sphingomyelin, ceramide is transported from the endoplasmic reticulum to the Golgi apparatus by the ceramide transfer protein CERT. CERT is phosphorylated by protein kinase D at S132 and subsequently multiple times in a serine-repeat motif, resulting in its inactivation. However, the kinase involved in the multiple phosphorylation remains unclear. Here, we identify the gamma2 isoform of casein kinase I (CKIgamma2) as a kinase whose overexpression confers sphingomyelin-directed toxin-resistance to Chinese hamster ovary cells. In a transformant stably expressing CKIgamma2, CERT was hyperphosphorylated, and the intracellular trafficking of ceramide was retarded, thereby reducing de novo sphingomyelin synthesis. The reduction in the synthesis of sphingomyelin caused by CKIgamma2 was reversed by the expression of CERT mutants that are not hyperphosphorylated. Furthermore, CKIgamma2 directly phosphorylated CERT in vitro. Among three gamma isoforms, only knockdown of gamma2 isoform caused drastic changes in the ratio of hypo- to hyperphosphorylated form of CERT in HeLa cells. These results indicate that CKIgamma2 hyperphosphorylates the serine-repeat motif of CERT, thereby inactivating CERT and down-regulating the synthesis of sphingomyelin.

SummarySeveral genetic mosaics for larval body marking of the silkworm,Bombyx mori, have been induced by X-ray irradiation. It is hypothesized that the occasional loss of chromosomal fragments carrying the genes for body marking during development may give rise to this type of mosaicism. Using pulsed field gel electrophoresis (PFGE), we find that a DNA molecule of about 2·5 megabases (Mb) is present in one type of mosaic (mottled striped strains pSm788 and pSm872), and not in any other strain. This DNA fragment hybridizes strongly with some chorion genes which are less than 6·9 cM away from thepslocus, and hence it corresponds to a chromosomal fragment containing genes for both striped marking (ps) and the chorion. In the non-mottled psstrain, the phenotype before X-ray irradiation, no band was detected either on a PFGE gel or after hybridization with the chorion probe. These results suggest that the mottled psstrains carry short chromosome fragments which are lost differentially during cell divisions.

Background The chemokine family plays important roles in cell migration and activation. In humans, at least 44 members are known. Based on the arrangement of the four conserved cysteine residues, chemokines are now classified into four subfamilies, CXC, CC, XC and CX3C. Given that zebrafish is an important experimental model and teleost fishes constitute an evolutionarily diverse group that forms half the vertebrate species, it would be useful to compare the zebrafish chemokine system with those of mammals. Prior to this study, however, only incomplete lists of the zebrafish chemokine genes were reported. Results We systematically searched chemokine genes in the zebrafish genome and EST databases, and identified more than 100 chemokine genes. These genes were CXC, CC and XC subfamily members, while no CX3C gene was identified. We also searched chemokine genes in pufferfish fugu and Tetraodon, and found only 18 chemokine genes in each species. The majority of the identified chemokine genes are unique to zebrafish or teleost fishes. However, several groups of chemokines are moderately similar to human chemokines, and some chemokines are orthologous to human homeostatic chemokines CXCL12 and CXCL14. Zebrafish also possesses a novel species-specific subfamily consisting of five members, which we term the CX subfamily. The CX chemokines lack one of the two N-terminus conserved cysteine residues but retain the third and the fourth ones. (Note that the XC subfamily only retains the second and fourth of the signature cysteines residues.) Phylogenetic analysis and genome organization of the chemokine genes showed that successive tandem duplication events generated the CX genes from the CC subfamily. Recombinant CXL-chr24a, one of the CX subfamily members on chromosome 24, showed marked chemotactic activity for carp leukocytes. The mRNA was expressed mainly during a certain period of the embryogenesis, suggesting its role in the zebrafish development. Conclusion The phylogenic and genomic organization analyses suggest that a substantial number of chemokine genes in zebrafish were generated by zebrafish-specific tandem duplication events. During such duplications, a novel chemokine subfamily termed CX was generated in zebrafish. Only two human chemokines CXCL12 and CXCL14 have the orthologous chemokines in zebrafish. The diversification observed in the numbers and sequences of chemokines in the fish may reflect the adaptation of the individual species to their respective biological environment.

The CC chemokines are a closely related subfamily of the chemokine superfamily. Most of the CC chemokine genes form a cluster on chromosome 11 in mice and chromosome 17 in humans. To date, 11 and 16 functional genes have been localized within the mouse and human clusters, respectively. Notably, some of the genes within these clusters appear to have no counterparts between the two species, and the orthologous relationships of some of the genes are difficult to establish solely on the basis of amino acid similarity. In this study, we have taken a comparative genomic approach to reveal some of the features that may be involved in the dynamic evolution of these gene clusters. We sequenced a 122-kb region containing five chemokine genes of the mouse CC cluster. This mouse sequence was combined with those determined by the Mouse Genome Sequencing Project, and the entire sequence of the mouse CC cluster was compared with that of the corresponding cluster in the human genome by percent identity plot and dot-plot analyses. Although no additional chemokine genes have been found in these clusters, our analysis has revealed that numerous gene rearrangements have occurred even after the diversification of rodents and primates, resulting in several species-specific chemokine genes and pseudogenes. In addition, phylogenetic analysis and comparison of the genomic sequences unambiguously identified the orthologous relationships of some of the chemokine genes in the mouse and human CC gene clusters.

Background In order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues. Result The full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl. We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames) to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously. Conclusions In this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies.

It is possible to assess positive selection by using the ratio of Ka (nonsynonymous substitutions per plausible nonsynonymous sites) to Ks (synonymous substitutions per plausible synonymous sites). We have searched candidate genes positively selected during primate evolution by using 5′-end sequences of 21,302 clones derived from cynomolgus monkey (Macaca fascicularis) brain cDNA libraries. Among these candidates, 10 genes that had not been shown by previous studies to undergo positive selection exhibited a Ka/Ks ratio > 1. Of the 10 candidate genes we found, 5 were included in the mitochondrial respiratory enzyme complexes, suggesting that these nuclear-encoded genes coevolved with mitochondrial-encoded genes, which have high mutation rates. The products of other candidate genes consisted of a cell-surface protein, a member of the lipocalin family, a nuclear transcription factor, and hypothetical proteins.

Twenty-nine novel human genes were predicted with putative coding regions sharing an open reading frame with the cynomolgus monkey, and the expression of 21 pairs of genes was confirmed by reverse transcription-coupled polymerase chain reaction., Background The complete assignment of the protein-coding regions of the human genome is a major challenge for genome biology today. We have already isolated many hitherto unknown full-length cDNAs as orthologs of unidentified human genes from cDNA libraries of the cynomolgus monkey (Macaca fascicularis) brain (parietal lobe and cerebellum). In this study, we used cDNA libraries of three other parts of the brain (frontal lobe, temporal lobe and medulla oblongata) to isolate novel full-length cDNAs. Results The entire sequences of novel cDNAs of the cynomolgus monkey were determined, and the orthologous human cDNA sequences were predicted from the human genome sequence. We predicted 29 novel human genes with putative coding regions sharing an open reading frame with the cynomolgus monkey, and we confirmed the expression of 21 pairs of genes by the reverse transcription-coupled polymerase chain reaction method. The hypothetical proteins were also functionally annotated by computer analysis. Conclusions The 29 new genes had not been discovered in recent explorations for novel genes in humans, and the ab initio method failed to predict all exons. Thus, monkey cDNA is a valuable resource for the preparation of a complete human gene catalog, which will facilitate post-genomic studies.

An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37 degrees C. Moreover, the cells showed a sensitive response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (theta-GST) and albumin genes.

A clone of immature cDNA for human casein kinase I gamma 2 (CSNK1G2) was isolated by screening the human testis cDNA library with a PCR-amplified probe (about 400 bp) representing the kinase domain of rat casein kinase I gamma 2 (CKI gamma 2). Comparison of the entire sequence with that of rat CKI gamma 2 showed that the cDNA contained the complete coding sequence of CKI gamma 2 as well as an intron-like sequence of 1006 bp, part of which was homologous to the Alu sequence. To obtain an insertion-free CSNK1G2 cDNA, PCR cloning was performed based on the above sequence. The amplified 1687-bp fragment was subcloned and sequenced. The predicted amino acid sequence consisted of 416 residues, 94% of which were identical to that of the rat homologue. Although there are two Src homology 3 (SH3) domain-binding motifs (Pro-X-X-Pro consensus), Pro-Lys-Val-Pro and Pro-Ser-Glu-Pro in the C-terminal region of rat CKI gamma 2, only the latter was conserved in the human counterpart. This finding suggests that the latter motif is important for binding to the signal transduction adaptor protein Nck (NCK). The human CSNK1G2 gene was mapped to chromosome 19p13.3 by fluorescence in situ hybridization and PCR analysis of the human/rodent hybrid cell panel.

By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine. We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids. The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21–33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2. Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes. From these results, we designated this novel CC chemokine as SLC fromsecondary lymphoid-tissuechemokine. SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor. Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nm) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC. By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligandchemokine (SCYA19) also exists. Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13.

Nomiyama, Hisayuki ; Imai, Toshio ; Kusuda, Jun ; Miura, Retsu ; Callen, David F. ; Yoshie, Osamu

To study liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37 degrees C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L15-medium )pH 7.) and 37 degrees C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L12 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.

When cDNA encoding rat rasGTPase-activating protein (rat GAP1m) was used as a probe, two partial cDNA clones of a human counterpart of rat GAP1m were isolated from a cDNA library derived from growth-arrested normal human ectocervical epithelial cells. One clone was found to be a cDNA of premature mRNA with two introns. A complete cDNA of human GAP1m was constructed by a series of reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from human epidermoid carcinoma A431 cells. Human GAP1m shows 87.7% nucleotide identity to rat GAP1m in open reading frame and encodes an 850-amino acid protein that shows 89.2% identity to rat GAP1m. A 100-kDa protein was detected in A431 cells by Western blotting with anti-rat GAP1m antibody. The human GAP1m gene was mapped to chromosome 3q24-q26.

This report describes the localization of two ADP ribosylation factor (ARF) genes, ARF1 and ARF3, to human chromosomes 1q42 and 12q13, respectively, using fluorescence in situ hybridization and PCR screening with a hybrid cell panel. ARFs are a family of guanine nucleotide-binding proteins which are responsible for the vesicular transport of newly synthesized proteins. 14 refs., 1 fig., 1 tab.

A cDNA clone coding for human casein kinase I (CK1) has been isolated and sequenced. The insert of 1911 bp contained an open reading frame of 415 amino acids. The entire amino acid sequence of human CK1 was 97% homologous to that of rat CK1 delta, and their sequences in the kinase domain (284 amino acid residues) were completely identical, predicting that the obtained cDNA is for a human homolog of the CK1 delta isoform (CSNKID). The considerable similarity in the amino acid sequence of the kinase domain of human CK1 delta to the Saccharomyces cerevisiae CK1, HRR25 (66%), and to the Saccharomyces pombe CK1, HHP1 (78%), which are involved in the repair of DNA strand break, supports the speculation that human CK1 delta might also act in DNA metabolism through excision and recombinational repair. The human CK1 delta gene was mapped to chromosome 17q25.2-q25.3 by fluorescence in situ hybridization and polymerase chain reaction analysis of the human/rodent hybrid cell panels.

The human genome is a mosaic of isochores, long DNA segments which are compositionally homogeneous and which can be partitioned into five families, L1, L2, H1, H2 and H3, characterized by increasing GC levels and by increasing gene concentrations. Previous investigations showed that in situ hybridization with a DNA fraction derived from the GC-richest and gene-richest isochores of the H3 family produced the highest concentration of signals on 25 R(everse) bands that include the 22 most thermaldenaturation-resistant T(elomeric) bands, a subset of R bands. Using an improved protocol for in situ hybridization and cloned H3 isochore DNA, we have now shown (i) that the number of bands which are characterized by strong hybridization signals, and which are here called T or H3+, is 28; (ii) that 31 additional R bands, here called T or H3+ bands, also contain H3 isochores, although at a lower concentration than H3∗ bands; and (iii) that the remaining R bands (about 140 out of 200, at a resolution of 400 bands), here called R″ or H3- bands, do not contain any detectable H3 isochores. H3+ and H3+ bands contain all the generichest isochores of the human genome. The existence of three distinct sets of R bands is further supported (i) by the different compositional features of genes located in them; (ii) by the very low gene density of chromosomes 13 and 18, in which all R bands are H3- bands; (iii) by the compositional map of a H3∗ band, Xq28; (iv) by the overwhelming presence of GC-rich and GC-poor long (>50 kb) DNA sequences in H3+/H3∗ and in H3-/G bands, respectively; and (v) by the large degree of coincidence of H3+ and H3∗ bands with CpG island-positive bands. These observations have implications for our understanding of the causes of chromosome banding and provide a classification of chromosomal bands that is related to GC level (and to gene concentration).

In order to determine the chromosomal locations of an autoimmune antigen, the coilin gene and its pseudogene, we amplified the segments of the two genes by the polymerase chain reaction (PCR) and screened a panel of somatic cell hybrids for the presence of the gene products. The results indicate that the human coilin gene and its pseudogene can be assigned to chromosome 17 and chromosome 14, respectively. Further analysis of cell hybrids bearing chromosome 17 with various deletions localized the coilin gene to the region q21-q23.

Clones containing DNA segments linked to NotI sites are not only useful for ordering the NotI fragments fractionated by pulsed field gel, but also valuable in the search of unknown genes, because they often contain the CpG rich islands and genes related with them. To know the probability of association of NotI sites with CpG rich islands, we screened 5,188 sequences accumulated in DNA data base for the presence of NotI site and examined the distribution of CpGs around them. The sequential calculation of G + C content and frequency of CpG occurrence at each nucleotide position identified the CpG rich domains close to NotI sites in 77 sequences, which corresponds to 84% of total number of candidate sequences. This frequency is consistent well with the prediction that 89% of NotI sites in mammalian genome are likely to be present in CpG rich islands and would stress the importance of cloning of NotI linking sequences for direct isolation of desired genes. Furthermore, 63 islands newly identified in this study should provide a clue for understanding the transcriptional regulation of associated genes.

Background The complete assignment of the protein-coding regions of the human genome is a major challenge for genome biology today. We have already isolated many hitherto unknown full-length cDNAs as orthologs of unidentified human genes from cDNA libraries of the cynomolgus monkey (Macaca fascicularis) brain (parietal lobe and cerebellum). In this study, we used cDNA libraries of three other parts of the brain (frontal lobe, temporal lobe and medulla oblongata) to isolate novel full-length cDNAs.

A physical map of the cloned ribosomal DNA of tobacco chloroplasts was constructed by cleavage with several restriction endonucleases and hybridization of the restriction fragments with labeled 16S and 23S rRNAs. The location of the 16S and 23S rRNA genes was determined by electron microscopic analysis of the R-loop containing molecules. No intervening sequences were detected in these genes. The 16S and 23S rRNA genes are separated by a 1.3×106 daltons spacer and coded for by the same DNA strand. The direction of transcription is from gene 16S to gene 23S.

Ovarian imaginal discs prepared from fifth-instar larvae of the silkworm, Bombyx mori were treated with graded concentrations of glycerol, cooled at a rate of 1°C/min to −35°C and preserved in liquid nitrogen for 2 days or more and then rapidly thawed (500°C/min). The frozen and thawed ovaries were transplanted into fifth-instar female larvae, in which more than 20% of the ovaries developed to produce mature eggs with a chorion according to the state of host development. By parthenogenetic activation, the mature eggs started embryogenesis and hatched to produce larvae. About 50% hatching occurred in the eggs developed in a C 108 × Cambodge host, and about 10% in a C 108 × Aojuku host. The hatched larvae completed post-embryonic development as did the normal larvae.

The fibroin gene of the wild silkworm Bombyx mandarina was identified through hybridization with the fibroin DNA of the domesticated species, B. mori. Using their cross homology, we isolated the clone carrying the fibroin gene from a B. mandarina genomic library in λ Charon 30. A resultant recombinant phage contained the DNA fragment corresponding to the 5′-end region of the B. mori fibroin gene. After subcloning the DNA fragment into pUC12, its DNA sequence was partially determined. Comparison of the nucleotide sequence of the 5′-end region from the two species showed that the B. mandarina fibroin gene is highly homologous to that of B. mori but the extent of homology varies in different functional domains within this region. In particular, the promoters and enhancer-like element sequences are identical in the two species, indicating that these regions are essential for fibroin gene expression irrespective of species. In contrast, there is an appreciable amount of base change in the region far upstream from the enhancer-like element sequence and in the intron-region. These results support the view that the B. mandarina which exists in nature at present is a possible ancestor of the domesticated silkworm, B. mori.

Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x106 and 2.8x106 daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.

The levels of guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase in the larval and pupal tissues of Bombyx mori were estimated. This activity was highest in the fat body of the female pupa. The enzyme showed a significant variation in activity during development of adult in female. Male silkworm gave less significant results. The cGMP-dependent kinase partially purified from the pupa could be activated by a high concentration of adenosine 3′,5′-monophosphate (cAMP) as reported for cGMP-dependent protein kinases from other sources. The nature of the enzyme thus activated and that of the enzyme activated by a low concentration of cGMP were found to be similar in several aspects. This indicates that the intrinsic activity of protein kinase from the silkworm pupa is independent of the kind of cyclic nucleotide as an activator.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the `two out of three' and `U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

1. 1. A blue-colored, red-fluorescent protein was purified from the digestive juice of silkworm, Bombyx mori L. 2. 2. The chromoprotein and its chromophore show well-defined absorption peaks at 278 and 605 mμ, and 495 and 575 mμ respectively. 3. 3. The chromoprotein and chromophore were negative to Gmelin, Ehrlich, diazo- and Carr-Price tests but were positive to the chromium oxide-chlorine-benzidine test for pyrrole. 4. 4. Chemical and spectral results obtained, together with the previous findings mentioned in the text, suggest that this chromoprotein is a biliprotein, probably formed in the midgut of the silkworm from the chlorophyll lipoprotein complex of mulberry leaves.