Jun Kusuda

Molecular cloning of a novel human CC chemokine liver and activation-regulated chemokine (LARC) expressed in liver. Chemotactic activity for lymphocytes and gene localization on chromosome 2

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 iver and ctivation-egulated hemokine. 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 μg/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.

Molecular Cloning of a Novel Human CC Chemokine Secondary Lymphoid-Tissue Chemokine That Is a Potent Chemoattractant for Lymphocytes and Mapped to Chromosome 9p13

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.

Extensive expansion and diversification of the chemokine gene family in zebrafish: Identification of a novel chemokine subfamily CX

BackgroundThe 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.ResultsWe 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.ConclusionThe 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.

Human chemokines fractalkine (SCYD1), MDC (SCYA22) and TARC (SCYA17) are clustered on chromosome 16q13.

Chemokines are a large family of small secreted proteins that regulate migration of white blood cells. Based on the arrangement of the first two of the four conserved cysteine residues, chemokines are classified into four subfamilies, CXC, CC, C and recently identified CX3C. Most of the human genes for the CC chemokines are clustered at chromosome 17q11.2 (Naruse et al., 1996). Previously we identified a novel CC chemokine TARC (thymus and activation-regulated chemokine) (Imai et al., 1996) and localized its gene (SCYA17) at chromosome 16q13 (Nomiyama et al., 1996). Recently, Bazan et al. (1997) and Pan et al. (1997) mapped the CX3C chemokine fractalkine/neurotactin gene (SCYD1) to chromosome 16. More recently, Godiska et al. (1997) reported isolation of a cDNA encoding a novel CC chemokine MDC (macrophage derived chemokine) and mapped its gene (SCYA22) to the same chromosome. It is therefore important to determine the exact relationship of these three chemokine genes on chromosome 16. Materials and methods

Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta

BackgroundCynomolgus macaques (Macaca fascicularis) are widely used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as rhesus macaques (M. mulatta). We isolated 85,721 clones and determined 9407 full-insert sequences from cynomolgus monkey brain, testis, and liver. These sequences were annotated based on homology to human genes and stored in a database, QFbase http://genebank.nibio.go.jp/qfbase/.ResultsWe found that 1024 transcripts did not represent any public human cDNA sequence and examined their expression using M. fascicularis oligonucleotide microarrays. Significant expression was detected for 544 (51%) of the unidentified transcripts. Moreover, we identified 226 genes containing exon alterations in the untranslated regions of the macaque transcripts, despite the highly conserved structure of the coding regions. Considering the polymorphism in the common ancestor of cynomolgus and rhesus macaques and the rate of PCR errors, the divergence time between the two species was estimated to be around 0.9 million years ago.ConclusionTranscript data from Old World monkeys provide a means not only to determine the evolutionary difference between human and non-human primates but also to unveil hidden transcripts in the human genome. Increasing the genomic resources and information of macaque monkeys will greatly contribute to the development of evolutionary biology and biomedical sciences.

Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence

BackgroundIn 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.ResultThe 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.ConclusionsIn 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.

Comparative DNA Sequence Analysis of Mouse and Human CC Chemokine Gene Clusters

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.

Prediction of unidentified human genes on the basis of sequence similarity to novel cDNAs from cynomolgus monkey brain

BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.

Over 80% ofNotI sites are associated with CpG rich islands in the sequenced human DNA

SummaryClones 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.

Genomic structure and chromosome location of RPL27A/Rpl27a, the genes encoding human and mouse ribosomal protein L27A

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.