Takeshi Ariyama

Clonal origin of Philadelphia chromosome negative cells with trisomy 8 appearing during the course of α-interferon therapy for Ph positive chronic myelocytic leukemia

The transient appearance of a Philadelphia chromosome (Ph) negative clone with trisomy 8 was found in the bone marrow cells from a patient with Ph positive CML during the course of alpha-interferon (IFN) therapy. To determine whether this clone was derived from a Ph positive clone or from some other cell lineage, we performed molecular cytogenetic studies on bone marrow cells from the patient by fluorescence in situ hybridization (FISH). No fusion of BCR-ABL could be detected in cells with trisomy 8, clearly indicating that the Ph negative trisomy 8 clone was not derived from the Ph positive CML population.

High resolution ordering of DNA markers by multi-color fluorescent in situ hybridization of prophase chromosomes

To improve resolution for physical ordering of adjacent DNA loci, prophase chromosomes were used for multi-color fluorescent in situ hybridization (FISH). The prophase chromosomes were prepared from cultured lymphocytes by a thymidine synchronization, bromodeoxyuridine release technique and then treating the synchronized cultures with topoisomerase II inhibitors ICRF154 or ICRF193. Almost all mitotic figures exhibited highly elongated prophase chromosomes without significant reduction of the mitotic index. Using multi-color FISH with these prophase chromosomes, we were able to distinguish signals for loci separated by as little as 50 kb, and determine their orientation. Furthermore, using this prophase ordering system, we confirmed the linear order and defined the orientation of seven cosmid markers within a 360-kb region surrounding D10S102, a locus that is closely linked to the disease locus in families segregating an allele causing multiple endocrine neoplasia IIA (MEN2A). This prophase FISH system, by rapidly and precisely providing the linear order of loci that are very close, can expedite construction of fine cytogenetic maps and contribute to positional-cloning studies in which the precise ordering of DNA loci in a target region is critical.

Chromosomal localization of the protein tyrosine phosphatase G1 gene and characterization of the aberrant transcripts in human colon cancer cells

We have recently described the isolation of the human PTPG1 gene which encodes a member of intracellular protein tyrosine phosphatases that may be candidates for tumor suppressor genes. In order to investigate the abnormality of the PTPG1 transcript in various human cancer cell lines, we have analyzed the consensus catalytic region of PTPG1 cDNA, using the reverse transcriptionpolymerase chain reaction. In a colorectal carcinoma cell line, DLD‐1, we found three aberrant transcripts. Sequencing analysis revealed that one had a missense point mutation and the remainders contained 77 bp and 173 bp deletions, respectively. These alterations might directly affect their phosphatase activities. Our findings provide the first evidence for the aberrant transcripts of the protein tyrosine phosphatase in human cancer cells, and suggest that the aberration of PTPG1 gene might be involved in the tumororigenesis. Moreover, the human PTPG1 gene is localized on chromosome 7q11.23, a region with frequent abnormalities implicated in some human cancers.

Human dihydrolipoamide succinyltransferase: cDNA cloning and localization on chromosome 14q24.2-q24.3

We isolated cDNA for dihydrolipoamide succinyltransferase from a human fibroblast cDNA library in lambda gt11. The cDNA revealed that the human dihydrolipoamide succinyltransferase lacked a sequence motif of an E3 and/or E1 binding site. This suggests that the human dihydrolipoamide succinyltransferase possesses a unique structure consisting of two domains in contrast with the dihydrolipoamide acyltransferases of other alpha-keto acid dehydrogenase complexes. In addition, we found that the human dihydrolipoamide succinyltransferase gene is located on chromosome 14 at q24.2-q24.3 and that a sequence related to the dihydrolipoamide succinyltransferase gene is located on chromosome 1 at p31. Interestingly, the gene for the dihydrolipoamide acyltransferase of the branched chain alpha-keto acid dehydrogenase complex is also located on chromosome 1p31 (Zneimer et al. (1991) Genomics 10, 740-747).

Chromosomal localization and cDNA sequence of human BTEB, a GC box binding protein.

Human BTEB cDNA clones have been isolated, sequenced, and the corresponding gene has been assigned to human chromosome 9, region q13, by fluorescent in situ hybridization and DNA blot analysis using DNAs from hybrid cell clones containing a single human chromosome. The cDNA clone encodes a polypeptide of 244 amino acids whose sequence shows a high sequence similarity with the rat BTEB (98% amino acid identity).

Precise ordering of 26 cosmid markers on chromosome region 3p23→p21.3 by two-color FISH on human prophase chromosomes and stretched DNAs

To construct a detailed cytogenetic map of human chromosome region 3p23-->p21.3, we determined the order of 26 cosmid markers (cCI 3 series) previously localized within this region by fluorescence in situ hybridization (FISH). Two-color pairwise FISH analysis of prophase chromosomes provided the order of these markers as follows: pter - 245 (D3S647) - 872 (D3S1018) - 818 (D3S996) - 905 (D3S1022) - 515 (D3S685) - 1195 (D3S1125) - 718 (D3S935) - 911 (D3S1025) - 878 (D3S1020) - 717 (D3S934) - 401 (D3S664) - [708 (D3S926)/524 (D3S686)] - 848 (D3S1011) - 771 (D3S966) - 917 (D3S1029) - 533 (D3S688) - 470 (D3S676) - 940 (D3S1037) - 785 (D3S974) - 810 (D3S988) - 9 (D3S643) - 382 (D3S660) - 769 (D3S965) - 792 (D3S978) - 604 (D3S705) - cen. The two-color signals of 524 (D3S686) and 708 (D3S926) were visualized as an overlapping pattern on prophase chromosomes, and, further, the string signals also overlapped on stretched DNAs, allowing us to determine their precise order as pter - D3S926 - D3S686 - cen. The precise order of 26 DNA markers on 3p23-->p21.3 can provide useful information for the positional cloning of tumor suppressor gene(s) and cancer breakpoint(s) encompassed in this region.

Molecular cloning of the breakpoint of t(11;22) (q23;q11) chromosome translocation in an adult acute myelomonocytic leukaemia.

Southern blot analysis with a cDNA probe of MLL indicated that the breakpoint is in a BamHI 8.3 kb fragment which carries the exon 5–11 of MLL gene in DNA from an adult acute myelomonocytic leukaemia with a t(11;22)(q23;q11) translocation. The structural analysis of the rearranged MLL locus demonstrated that the breakpoint is localized between exon 8 and 9 of MLL locus. The normal counterpart fused to the MLL locus was proved to be derived from chromosome 22q11(AF‐22) by somatic cell hybrids analysis and FISH. By FISH, AF‐22 locus was localized to the region more centromeric to the BCR gene.

Reexamination of chromosomal loci of human muscle actin genes by fluorescence in situ hybridization.

SummaryHuman genes for cardiac (ACTC), skeletal (ACTA), and vascular type (aortic type or α-) smooth (ACTSA) muscle actins have been localized to chromosomes 15q14, 1q42.1, and 10q23.3, respectively, by fluorescence in situ hybridization.

Assignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3.

Human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ; also denoted HPTP zeta or RPTP beta) has a large extracellular region with the N-terminal carbonic anhydrase-like domain and a cytoplasmic region with two tandemly located protein tyrosine phosphatase domains. One of the characteristics of PTPRZ is its preferential expression in the central nervous system. We localized the human PTPRZ gene to chromosome band 7q31.3 by somatic cell hybrid mapping and fluorescence in situ hybridization.