Chromosome localization and cDNA sequence of murine and human genes forras p21 GTPase activating protein (GAP)

The cDNA coding for mouse and human rasp21 GTPase-activating protein (GAP) was isolated; the deduced amino acid sequences share over 96% homology with that previously determined for bovine brain GAP. Both the mouse and human GAP cDNAs were used as probes for the chromosomal localization of this gene. The locus designations for the gene encoding GAP in human and mouse are RASAand Rasa (for ras-activating protein), respectively. By somatic cell hybrid analysis and in situ chromosomal hybridization, we have assigned the RASAgene to human chromosome band 5q13.3. In addition, with somatic cell genetics and linkage analysis in recombinant inbred mouse strains, the murine Rasagene was localized to the distal end of mouse chromosome 13. These assignments place the gene encoding GAP in a known conserved syntenic region.     

Analysis of chromosomal copy number changes and oncoprotein expression in primary central nervous system lymphomas: frequent loss of chromosome arm 6q

Primary central nervous system lymphoma (PCNSL) is a rare disease. A small number of cytogenetic studies of PCNSL have been conducted and several reports have been published on associated molecular and protein expression data. We combined these approaches in a series of eight PCNSL cases, analyzing the chromosomal abnormalities using comparative genomic hybridization (CGH), testing for Epstein Barr virus (EBV) involvement by in situ hybridization for EBER, assessing expression of p53, Bcl-2, Bcl-6 and CD10 by means of immunohistochemistry, and screening for mutations of the TP53 gene by DGGE. TP53 gene mutations and EBV expression were not detected. Most of the cases showed p53, Bcl-6 and Bcl-2 protein expression. CGH revealed DNA copy number changes in all eight cases. The most frequent changes were gains of chromosome 12 (63%), chromosome 18 (50%) and 20q (38%), and loss of chromosome arm 6q (75%). No correlation between protein expression and chromosomal abnormalities was found in these eight cases. Although gains of chromosome 12, 18 and 20q and loss of 6q have also been reported in systemic diffuse large B-cell lymphomas, the frequency of 6q deletion is clearly higher in PCNSL. This creates a similarity to primary lymphomas of the testes that also frequently have deletions of 6q. This suggests that suppressor genes located on chromosome 6q may play a role in the development of lymphomas at immunoprivileged sites, like the CNS.     

Chromosome loss is responsible for segregation at theHPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT)locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRTlocus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the HPRTlocus to the X chromosome in Chinese hamster, and in fact allow mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

Expression, Genomic Structure and Mapping of the Thymus Specific Protease Prss16: A Candidate Gene for Insulin Dependent Diabetes Mellitus Susceptibility

PRSS16 is a serine protease specifically expressed by epithelial cells in the thymic cortex. The human gene is encoded on 6p21.3-p22 where recent linkage analysis has identified an association with insulin dependent diabetes mellitus (IDDM) susceptibility independent of HLA-DR3. To further investigate its potential role in autoimmunity, we characterized the mouse orthologue, Prss16. The genomic structure of Prss16 shows conservation with the human gene in size, number of exons and chromosomal location. Mapping of Prss16 places it on mouse chromosome 13 centromeric of thesatin locus. This region is comparable to the PRSS16 region on human chromosome 6 and has also been linked to quantitative trait locus for IDDM in the nonobese diabetic mouse. Similar to the human gene, Prss16 expression is highly specific in the mouse with expression limited to the cortical thymic epithelium. Notably, embryonic expression coincides with population of the thymic anlage with T-cell precursors and initiation of T-cell development. We also show that NOD and New Zealand Black mice, which have a disrupted thymic architecture and autoimmune phenotype, have lower levels of Prss16 expression compared to C57BL/6 mice. These findings support the role of Prss16 in T-cell development and susceptibility to autoimmunity in the mouse.     

Generation and characterization of irradiation hybrids of human chromosome 4

In recent years investigators have attempted to develop more rapid and precise methods to isolate specific chromosomal DNA regions. In this paper we demonstrate a modification of the method first developed by Goss and Harris for generation of irradiation hybrids. The gene encoding the dominant selectable marker for resistance to neomycin was introduced into human chromosome 4 using retroviral insertion into human fibroblasts. Transfer of these chromosomes via microcells into the mouse cell line NIH3T6 produced a somatic cell line containing chromosome 4 as the only human chromosome. Irradiation of this cell line followed by fusion with the hamster cell line CHTG49 generated hybrids containing only small portions of chromosome 4p on a hamster background. The use of selection produced stable hybrids that retained chromosome 4 fragments over long periods of tissue culture passage. To obtain new polymorphic markers for Huntington's disease, one of these hybrids was used to isolate new genomic fragments. We identified 41 single-copy fragments, of which 27 have been mapped to specific regions of chromosome 4; 52% of these fragments map to the region of chromosome 4 containing theHD gene.     

Construction of microcell hybrid panel containing differentNeo gene insertions in mouse chromosome 17 used for chromosome-mediated gene transfer

A panel of four microcell hybrids representing different sites of insertion of the exogeneous neogene into mouse chromosome 17 has been constructed. These constructions were based on a cotransfer of mouse chromosome 17 and neomycin resistance generated in a stepwise procedure involving (1) random insertion of the neogene into a primary cell hybrid containing mouse chromosome 17 in a hamster cell background, (2) microcell-mediated chromosome transfer (MMCT) to segregate mouse and hamster chromosomes, and (3) identification of the mouse chromosome containing cells using a novel cell dotting procedure for mass screening at the cell colony level by molecular hybridization. Using this panel of four microcell hybrids for chromosome mediated gene transfer (CMGT), we obtained one transformant containing a chromosome fragment derived from the t-complex region located on mouse chromosome 17. It is concluded that the specific chromosome based procedure used here to generate CMGT transfectants may provide a general means to produce large numbers of transfectants containing megabase fragments covering, in principle, all regions of a given chromosome.     

Recurrent chromosomal aberrations in peripheral T-cell lymphoma

Histological, immunological, and cytogenetic analysis of the same neoplastic tissues have been performed on seven patients with peripheral T-cell lymphomas (PTCL). Clonal chromosomal abnormalities in five cases are reported. The most common chromosomal aberration, observed in four patients, is a rearrangement of chromosome 14 with a breakpoint in q11.2. Aberrations of chromosome 8 also occurred in four patients, three of whom had an extra 8q. The data indicate that breakpoints of malignant diseases affecting similar cell types might cluster to specific chromosomal regions, which can be helpful in recognition and classification of PTCL.     

An extended panel of hamster-human hybrids for chromosome 2q

A hamster-human hybrid containing only the q arm of chromosome 2 has been used to construct a panel of hybrids bearing reduced regions of chromosome 2 using the technique of irradiation fusion gene transfer. The human chromosome 2 carried theEcogpt gene and all hybrids were selected using this marker. The integratedEcogpt gene was localized to the region 2q33-34, resulting in the selective retention of this region in the hybrids. These data were combined with another previously constructed panel of hybrids containing regions of 2q, which were enriched for the region 2q36-37. The combined hybrid panel is useful for the mapping of new markers to defined regions of chromosome 2 and for the cloning of genes located on 2q by a positional strategy.     

Localization of the human homolog of the yeast cell division control 27 gene (CDC27) proximal to ITGB3 on human chromosome 17q21.3

The human homolog of theSaccharomyces cerevisiae cell division control 27 gene (CDC27) was mapped to human chromosome 17q12-q21 using a panel of human/rodent somatic cell hybrids and localized distal to the breast cancer susceptibility gene,BRCA1, using a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of humanCDC27 on human chromosome 17 is between the marker D17S409 and the beta 3 subunit of integrin (ITGB3). Further confirmation of this localization comes from the sequence tagged site (STS) mapping of humanCDC27 to the same yeast artificial chromosomes (YACs) positive forITGB3. The estimated distance between ITGB3 and humanCDC27 is less than 600 kb.     

Subchromosomal localization of a gene (XRCC5) involved in double strand break repair to the region 2q34-36

We have previously shown that human chromosome 2 can complement both the radiation sensitivity and the defect in double strand break rejoining characteristic of ionizing radiation (IR) group 5 mutants. A number of human-hamster hybrids containing segments of human chromosome 2 were obtained by microcell transfer into two group 5 mutants. In most, but not all, of these hybrids, the repair defect was complemented by the human chromosomal DNA. Two complementing microcell hybrids were irradiated and fused to XR-V15B, an IR group 5 mutant, to generate further hybrids bearing smaller regions of chromosome 2. All hybrids were examined for complementation of the repair defect. The region of chromosome 2 present was determined using PCR with primers specific for various human genes located on chromosome 2. A complementing hybrid bearing only a small region of chromosome 2 was finally generated. From this analysis we deduced that theXRCC5 gene was tightly linked to the marker,TNP1, which is located in the region 2q35.     

Cloning and identification of a DNA fragment coding for the sup1 gene of Saccharomyces cerevisiae

Summary A plasmid, pYsup1-1, containing a DNA fragment able to suppress the recessive mutant phenotype of the suppressor locus sup1 (allele sup1-ts36) of Saccharomyces cerevisiae was isolated from a bank of yeast chromosomal DNA cloned in cosmid p3030. The complementing gene was localized on a 2.6 kb DNA fragment by further subcloning. Evidence is presented that the cloned DNA segment codes for the sup1 structural gene (chromosome IIR).     

Assignment of retinoblastoma susceptibility gene to mouse chromosome 14

A human cDNA probe was used to screen a panel of mouse-Chinese hamster somatic cell hybrids to determine the chromosomal location of the retinoblastoma susceptibility gene (Rb-1)in mouse. The Rb-1gene mapped to mouse chromosome 14. Thus, the retinoblastoma susceptibility gene is syntenic with esterase 10 (the mouse homolog of human esterase D). The chromosomal assignment of the mouse Rb-1gene was further confirmed by using the same probe to study mouse-rat microcell hybrids. Since the human retinoblastoma susceptibility gene (RB1)along with the gene for esterase D is on chromosome 13q14, these data indicate this linkage group is conserved in man and mouse.     

Human cyclin B1 gene (CCNB1) assigned to chromosome 5 (q13-qter)

Cyclins play an important role in cell cycle regulation. At least five classes of cyclins have been identified—A, B, C, D, and E. B cyclins are generally of two types in most organisms—B1 and B2. We have mapped the gene for human cyclin B1 (CCNB1) to human chromosome 5 (region q13-qter) by Southern blot analysis of human × Chinese hamster somatic cell hybrid panels. Many more cyclin B-related sequences have been identified in the mouse (Cycb-1 to Cycb-10) and have been mapped to chromosomes 4, 5, 7, 8, 13, 14, 15, and 17. Based on our mapping of humanCCNB1 and known evolutionary conservation of chromosomal regions, we propose that the homologous cyclin B1 locus,Cycb-4, on mouse chromosome 13 is a functional gene.     

High resolution copy number analysis of IRF4 translocation‐positive diffuse large B‐cell and follicular lymphomas

Translocations affecting chromosome subband 6p25.3 containing the IRF4 gene have been recently described as characteristic alterations in a molecularly distinct subset of germinal center B‐cell‐derived lymphomas. Secondary changes have yet only been described in few of these lymphomas. Here, we performed array‐comparative genomic hybridization and molecular inversion probe microarray analyses on DNA from 12 formalin‐fixed paraffin‐embedded and two fresh‐frozen IRF4 translocation‐positive lymphomas, which together with the previously published data on nine cases allowed the extension of copy number analyses to a total of 23 of these lymphomas. All except one case carried chromosomal imbalances, most frequently gains in Xq28, 11q22.3‐qter, and 7q32.1‐qter and losses in 6q13‐16.1, 15q14‐22.31, and 17p. No recurrent copy‐neutral losses of heterozygosity were observed. TP53 point mutations were detected in three of six cases with loss of 17p. Overall this study unravels a recurrent pattern of secondary genetic alterations in IRF4 translocation‐positive lymphomas. © 2012 Wiley Periodicals, Inc.     

Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse

Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, we have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. GenesDPDE1, DPDE2, DPDE3, andDPDE4 that share sequence homology with theDrosophila dunce gene were assigned to human chromosomes 19 (DPDE1 andDPDE2), 5q12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.     

Organization of the HPRT gene and related sequences in the human genome

Comparative Southern hybridization of cDNA probes to DNA from cells carrying either one or four X chromosomes has been used to distinguish sequences derived from the functional locus for hypoxanthine-guanine phosphoribosyltransferase (HPRT) on the X chromosome from four independent HPRT-like autosomal sequences in the human genome. Subfragments of cDNA were then used to orient fragments from the HPRTlocus with respect to the mRNA sequence. The chromosomal origin of each of the autosomal sequences was determined by Southern analysis using DNA from a panel of human-Chinese hamster somatic cell hybrids. Two of the HPRT-like sequences were localized to chromosome 11, the third to chromosome 3, and the fourth to the region between p13 and q11 on chromosome 5. Three of these four autosomal sequences were isolated from genomic recombinant libraries and subcloned fragments from each were used as probes to study restriction fragment length polymorphisms (RFLP) at these loci. A RFLP for MspIwas found at the HPRT-like locus on chromosome 5 with a 1.3-kb major allele (frequency=0.8) and a 3.6-kb minor allele (frequency=0.2).     

The human galactosyltransferase gene is on chromosome 9 at band p13

The structural gene for galactosyltransferase (glycoprotein 4-B-galactosyltransferase, EC 2.4.1.38) was localized to human chromosome 9 band p13 by chromosome in situ hybridization using a cloned bovine galactosyltransferase cDNA probe. This chromosomal location is at the same position to which galactose-1-phosphate uridyltransferase, an enzyme which provides the nucleotide sugar substrate (UDP-galactose) for galactosyltransferase, has been mapped.     

FISH mapping of theα-S2 casein gene on river buffalo and cattle chromosomes identifies a nomenclature discrepancy in the bovine karyotype

A mixture of five genomic clones spanning the-S2 casein gene (CSN1S2) were mapped to river buffalo (Bubalus bubalis L.) and cattle (Bos taurus L.) chromosomes by fluorescencein situ hybridization (FISH) and R-banding. Clear probe hybridization signals were detected on river buffalo chromosome 7q, band 32, and the homologous cattle chromosome. These mapping data allow the indirect assignment of the entire cattle U15 syntenic group to river buffalo chromosome 7. The assignment of U15 to river buffalo chromosome 7 is discussed in the light of chromosomal nomenclature discrepancies involving the homologous cattle chromosome.accepted for publication by D. Ward     

T-cell lymphoma developing in Hodgkin's disease: Evidence for two clones

A case of peripheral T-cell lymphoma (PTL) occurring in a patient with Hodgkin's disease (HD) in relapse is described. The second neoplasm developed 25 months after the diagnosis of HD. Cytogenetic analysis on the lymph node biopsy at the time of diagnosis of PTL revealed the co-existence of two distinct, abnormal cell clones. The first clone was characterized by a reciprocal translocation t(5;7)(q13;q35) involving 7q35, namely the TCR-β gene, as expected in T-cell lymphomas. The second cell clone carried trisomies for chromosomes 2, 5, 7, and 14. By immunophenotypic and molccular analysis as well as by in situ hybridization, it was possible to prove that the malignant T-cells and the Reed-Sternberg cells corresponded to different cell clones, one carrying the structural chromosome abnormalities and one carrying the numerical chromosome anomalies. These results indicate that the present case represented a true composite lymphoma.