Yoichi Matsuda

Spatio‐temporally regulated expression of receptor tyrosine kinases, mRor1, mRor2, during mouse development: implications in development and function of the nervous system

Drosophila neurospecific receptor tyrosine kinases (RTKs), Dror and Dnrk, as well as Ror1 and Ror2 RTKs, isolated from human neuroblastoma, have been identified as a structurally related novel family of RTKs (Ror‐family RTKs). Thus far, little is known about the expression and function of mammalian Ror‐family RTKs.

Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation.

Abstract. We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a λgt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif ``IXCXE. The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2–q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication.

Identification and characterization of STK12/Aik2 : a human gene related to aurora of Drosophila and yeast IPL1

Mutations in aurora of Drosophila and related Saccharomyces cerevisiae IPL1 protein kinases are known to cause abnormal chromosome segregation. We earlier isolated a cDNA encoding a novel human protein kinase Aik which shares high amino acid identity with the Aurora/Ipl1 protein kinase family. In the present study, a second human cDNA highly homologous to aurora/IPL1 (Aik2) was identified and the nucleotide sequence was determined (gene symbol STK12). The C-terminal kinase domain of the STK12 encoded protein shares high amino acid sequence identity with those of mouse STK-1 (90%), rat AIM-1 (90%), human Aik (69%), mouse IAK1/Ayk1 (69%), Xenopus pEg2 (68%), Drosophila Aurora (62%), and yeast Ipl1 (45%), whereas the N-terminal domain of the STK12 protein shares little homology with those of Aurora/Ipl1 family members except for AIM-1 and STK-1. Northern blotting analyses revealed that STK12 expression was high in thymus, while low level expression was detected in small intestine, testis, colon, spleen, and brain. The STK12 protein content in HeLa cells is low in S phase, but it accumulates during M phase. STK12 was mapped to human chromosome 17p13.1 by fluorescence in situ hybridization. The chromosome location of STK12 was further defined using a radiation hybrid panel (Stanford G3), that showed a linkage with marker WI-7901 (LOD Score 7.83) located between D17S938 and D17S786.

Molecular cloning and chromosome mapping of rat phospholipase D genes, Pld1a, Pld1b and Pld2

We have previously obtained three partial rat phospholipase D (PLD) cDNA fragments by a reverse transcriptase-polymerase chain reaction (RT-PCR) method using degenerate primers based on two conserved amino acid sequences in PLDs of human and yeast. The entire coding regions of these genes were isolated and sequenced. The longest clone, Pld1a encodes a 1075 amino acid (aa) protein that was highly similar (89% identity) to human PLD1a, especially in four conserved regions present in other PLDs. The nucleotide sequence of the second clone was identical to that of Pld1a except that the clone lacked 114 nucleotides corresponding to 38 aa in the middle. A shorter alternatively spliced form of human PLD1 (PLD1b) lacking the corresponding 38 aa was also identified. Therefore, the second clone (Pld1b) was considered to correspond to the rat counterpart of human PLD1b. The third clone, Pld2 encoding 933 aa was smaller than that of Pld1 and its aa identity to rat Pld1 was 56%. However, it contains four conserved regions and aa sequences of these regions are homologous to those of rat Pld1 and human PLD1. Its entire aa sequence was very similar (96% identity) to the recently cloned mouse PLD, Pld2. Chromosome locations of the Pld1a, Pld1b and Pld2 genes were determined in the rat and mouse by fluorescent in situ hybridization. As expected, both Pld1a and Pld1b clones were hybridized to the same chromosome regions. The Pld1 and Pld2 genes were localized to rat chromosome 2q23.3-->q24 proximal end and the proximal region of mouse Chromosome 3B, and rat chromosome 10q23.3-->q24 proximal end and mouse Chromosome 11B3, respectively. They were mapped in regions where conserved linkage homology has been identified between the two species.

Comparative FISH mapping of mouse and rat homologues of twenty-five human X-linked genes

We constructed a comparative cytogenetic map of 25 functional genes in mouse and rat X chromosomes by direct R-banding fluorescence in situ hybridization. Nineteen and 22 out of the 25 genes, which have been mapped on the human X chromosome, were newly localized to mouse and rat X chromosomes, respectively. Twenty-two additional genes were integrated in the rat-mouse-human comparative map of the X chromosome in this study. Comparison of the gene order indicated the presence of four chromosome segments with conserved linkage homology between mouse and rat X chromosomes, suggesting that a minimum of four chromosomal inversion events occurred between mouse and rat X chromosomes during the evolution of the two species. Four chromosome segments with conserved linkage homology were found between human and rat X chromosomes.

Mouse cdc21 only 0.5 kb upstream from dna-pkcs in a head-to-head organization: an implication of co-evolution of ATM family members and cell cycle regulating genes.

The catalytic subunit of the DNA-dependent protein kinase (DNAPKcs) is a member of the ATM family, which in turn is a branch of the phosphatidylinositol 3-kinase (PI3K) superfamily (Hartley et al. 1995). The ATM family consists of relatively large proteins, all of which have motifs found in PI3Ks’ catalytic domains at their carboxy-terminal region. Many of the family members have been found to be involved in DNA repair, cell-cycle checkpoint control, and cell-cycle transition control (Zakian 1995). Furthermore, the ATM gene itself was demonstrated to be the responsible gene for the genetic disorder ataxia telangiectasia (AT) with a wide spectrum of clinical manifestations including hypersensitivity to ionizing radiation and radiomimetic drugs (Savitsky et al. 1995). The hypersensitivity is due to the dysfunction of radiation-induced checkpoint control in AT cells (Shiloh 1995). DNA-PKcs with Ku components catalyzes double-stranded broken DNA-dependent phosphorylation of proteins (Gottlieb and Jackson 1993), suggesting that DNA-PK is a surveyor of damaged DNA. Although several biochemical and mapping studies suggested that the mouse geneDna-pkcsmight be thescid-responsible gene, the exact nature of the mutation remained unknown. Very recent reports described a candidate mutation of the gene (Blunt et al. 1996; Danska et al. 1996), and we made the definitive identification of the scid mutation inDna-pkcs gene (Araki et al. 1997). The last study proved that the truncation of the DNA-PK csin the carboxy-terminal kinase domain is indeed responsible for the scid mutation. In the following course of studying the mouse promoter region of Dna-pkcs, we obtained several genomic clones that corresponded to the 5 8 end portion of the gene. Nucleotide sequencing of these clones confirmed a homology stretch to the previously determined 5 8 end sequence of the cDNA of Dna-pkcs. The first exon with the putative initial methionine of Dna-pk cs (Araki et al. 1997) was identified, and the successive upstream sequence was also determined (Fig. 1; DDBJ/EMBL/GenBank accession number AB000629). The TFD transcription factor DNA binding site database search with this sequence suggested a variety of ciselemental motifs for transcription factors. In Fig. 1, only basic motifs such as two Sp1 boxes and three CCAAT boxes are indicated. The nucleotide sequence databases search unexpectedly revealed a complete homology stretch to Cdc21(Kimura et al. 1995) in the promoter region. This homologous sequence is also indi-

Genomic organization of Ampd3, heart-type AMPD gene, located in mouse chromosome 7.

AMP deaminase (EC 3.5.4.6., AMPD), an enzyme catalyzing AMP into IMP, plays an important role in purine metabolism, especially in skeletal muscle, since metabolic myopathy associated with AMPD deficiency has been reported (Sabina and Holmes 1995). In higher eukaryotes, several isoenzymes of AMPD are encoded in separate genes (Sabina and Holmes 1995; Morisaki et al. 1990; Mahnke-Zizelman and Sabina 1992). Among three rodent Ampdgenes,Ampd3gene, gene for the heart-type isoform (H-isoform) of AMPD, which is expressed in heart, slow-twitch skeletal muscles and non-muscle tissues (Wang et al. 1997), has not been well studied, whereas Ampd1,encoding the M-isoform, has been extensively characterized (Morisaki et al. 1990). Understanding theAmpd3gene is important since the H-isoform of rodent AMPD also plays an important role in skeletal muscle (Fortuin et al. 1996), and the H-isoform of rodent AMPD, the product of Ampd3,was reported to be immunologically distinct from the E-isoform of human AMPD, the product of AMPD3 gene (Thakkar et al. 1995). Here we report the gene structure and chromosomal localization forAmpd3encoding the H-isoform of mouse AMPD. First, genomic DNA libraries from mouse in lambda FIX II (Stratagene; La Jolla, Calif, USA) were screened with fragments of Ampd3cDNA (Wang et al. 1997) as a probe. Seven independent clones were obtained by screenin g 1 × 10 plaques of genomic DNA libraries. Isolated genomic DNAs were then subcloned into pBluescript II (Stratagene; La Jolla, Calif, USA) and sequenced with automated instrumentation (Applied Biosystems; Foster City Calif, USA). Sequences were verified by multiple runs for both strands. By studying these isolated DNA clones, mouse Ampd3 gene was found to span more than 40 kb (Fig. 1A). Sequencing analysis revealed all of exon-intron boundaries as shown in Fig. 1B. There are 15 exons in total, and the first exon corresponds only to the 58 untranslated region. Exon-intron boundaries are quite well conserved between the human AMPD3 gene and mouse Ampd3 (Fig. 1B), although there is no homology in exon 1 between human and mouse genes. Next, the chromosomal assignment of the mouse Ampd3gene was made by fluorescence in situ hybridization (FISH). Preparation of R-banded chromosomes and FISH were performed as described before (Matsuda et al. 1992, 1996; Matsuda and Chapman 1995). The mouse 1.8-kb Ampd3cDNA (nt + 81 to nt + 1850) fragment inserted in the EcoRI site of pBluescript was labeled by nick translation with biotin 16-dUTP (Boehringer Mannheim; Mannheim, Germany) following the manufacturer’s protocol. Direct R-banding FISH revealed that the Ampd3genes were localized to mouse Chromosome (Chr) 7E2-E3 and rat Chr 1q35-q36, where conserved linkage homology to mouse Chr 7 has been identified (Matsuda et al. 1992; Ronne et al. 1987; Satoh et al. 1989; Somssich and Hameister 1996; Yamada et al. 1994; Fig. 2). Fine linkage mapping of mouse Ampd3 was performed by interspecific backcross analysis with progeny derived from the mating of (C57BL/6 ×Mus spretus ) F1 × M. spretusmice. Recombinant animals for this study were generated by mating males of the feral-derived mouse stocks, Mus spretus,with C57BL/6J females and backcrossing the F 1 females withM. spretusmales (Matsuda et al. 1996). Whole genomic DNAs prepared from kidneys of the backcross mice were digested with the restriction endonuclease, and restriction fragment length variants (RFLVs) were typed by Southern analysis as described previously (Matsuda et al. The nucleotide sequence data reported in this paper have been submitted to EMBL/Genbank/DDBJ and have been assigned the accession numbers, D88984-D88994.

rim2 (recombination-induced mutation 2) is a new allele of pearl and a mouse model of human Hermansky-Pudlak Syndrome (HPS): genetic and physical mapping

A mouse mutation, rim2, is one of a series of spontaneous mutations that arose from the intra-MHC recombinants between Japanese wild mouse-derived wm7 and laboratory MHC haplotypes. This mutation is single recessive and characterized by diluted coat color and hypo-pigmentation of the eyes. We mapped the rim2 gene close to an old coat color mutation, pearl (pe), on Chromosome (Chr) 13 by the high-density linkage analysis. The pearl mutant is known to have abnormalities similar to Hermansky-Pudlak syndrome (HPS), a human hemorrhagic disorder, characterized by albinism and storage pool deficiency (SPD) of dense granules in platelets. A mating cross of C57BL10/Slc-rim2/rim2 and C57BL/6J-pelpe showed no complementation of coat color. Additionally, characteristics similar to SPD were also observed in rim2. Thus, rim2 appeared to be a new allele of the pe locus and serves as a mouse model for human HPS. We have made a YAC contig covering the rim2/pe locus toward positional cloning of the causative gene.