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Biomedical subjects

J Sumegi

Publications and source records attributed to J Sumegi.

At least 37 records · Page 2Linked to original sources
PubMed1996

The construction of a yeast artificial chromosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) gene in 1q41.

The gene for Usher syndrome type II (USH2A), an autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439. Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb. The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S4 74-EB1-EB2-KB6-AFM144XF2-KB1-K B4-D1S229-D1S490-D1S227-TGFbeta2-D1S439. Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus. The physical distance between the two markers is 1.0 Mb. This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2. A long-range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed.

Base Sequence↗
PubMed1996

Narrowing the genetic interval and yeast artificial chromosome map in the branchio-oto-renal region on chromosome 8q.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial abnormality, hearing loss, and renal anomalies. Recently, the disease gene has been localized to chromosome 8q. Here, we report genetic studies that further refine the disease gene region to a smaller interval and identify several YACs from the critical region. We studied two large, clinically well-characterized BOR families with a set of 13 polymorphic markers spanning the D8S165-D8S275 interval from the chromosome 8q region. Based on multipoint analysis, the highest likelihood for the location of the BOR gene is between markers D8S543 and D8S530, a distance of about 2 cM. YACs that map in the BOR critical region have been identified and characterized by fluorescence in situ hybridization and pulsed-field gel electrophoresis. A YAC contig, based on the STS content map, that covers a minimum of 4 Mb of human DNA in the critical region of BOR is assembled. This lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in BOR syndrome.

Abnormalities, Multiple↗
PubMed1996

Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients.

Usher syndrome type 1b (USH1B) is an autosomal recessive disorder characterized by congenital profound hearing loss, vestibular abnormalities, and retinitis pigmentosa. The disorder has recently been shown to be caused by mutations in the myosin VIIa gene (MYO7A) located on 11q14. In the current study, a panel of 189 genetically independent Usher I cases were screened for the presence of mutations in the N-terminal coding portion of the motor domain of MYO7A by heteroduplex analysis of 14 exons. Twenty-three mutations were found segregating with the disease in 20 families. Of the 23 mutations, 13 were unique, and 2 of the 13 unique mutations (Arg212His and Arg212Cys) accounted for the greatest percentage of observed mutant alleles (8/23, 31%). Six of the 13 mutations caused premature stop codons, 6 caused changes in the amino acid sequence of the myosin VIIa protein, and 1 resulted in a splicing defect. Three patients were homozygotes or compound heterozygotes for mutant alleles; these three cases were Tyr333Stop/Tyr333Stop, Arg212His-Arg302His/Arg212His-Arg302His, and IVS13nt-8c-->g/Glu450Gln. All the other USH1B mutations observed were simple heterozygotes, and it is presumed that the mutation on the other allele is present in the unscreened regions of the gene. None of the mutations reported here were observed in 96 unrelated control samples, although several polymorphisms were detected. These results add three patients to single case reported previously where mutations have been found in both alleles and raises the total number of unique mutations in MYO7A to 16.

Alleles↗
PubMed1995

Mapping of the taurine transporter gene to mouse chromosome 6 and to the short arm of human chromosome 3.

Transport proteins have essential functions in the uptake of neurotransmitters and neuromodulators. We have mapped the gene encoding the taurine transporter, Taut, to the central region of mouse chromosome 6. Analysis of a cross segregating the neurological mutant mnd2 excluded Taut as a candidate gene for this closely linked mutation. To map the human taurine transporter gene, TAUT, a sequence-tagged site (STS) corresponding to the 3' untranslated region of the human cDNA was developed. TAUT was assigned to human chromosome 3 by typing this STS on a panel of somatic cell hybrids. Further analysis of a hybrid panel containing defined deletions of chromosome 3 suggested that TAUT maps to 3p21-p25. These data extend a conserved linkage group on mouse chromosome 6 and human chromosome 3p. Deletion of TAUT might contribute to some phenotypic features of the 3p- syndrome.

Animals↗
PubMed1995

X-linked lymphoproliferative disease: twenty-five years after the discovery.

The X-linked lymphoproliferative disease (XLP), one of six described X-linked immunodeficiencies, stems from a mutation at Xq25 which renders males impotent to mount an effective immune response to the ubiquitous EBV. Purtilo, who first observed this disease in 1969, established a Registry in 1980 to serve as a worldwide resource for the diagnosis, treatment, and research of this condition. Since Purtilo's death in late 1992, the Registry and research unit have not only continued to function as a worldwide consultative service, but have contributed the following. First, the number of affected boys has continued to grow; some 272 among 80 kindreds have been identified. Second, some boys (10%) who inherit the mutated XLP gene are immunologically abnormal even before evidence of EBV exposure. Third, the search for the XLP gene has been narrowed to a small region on Xq25. Its identification is near at hand; once cloned, this gene may well illustrate how the body orchestrates the complex immune response to EBV. Therein lies the justification for the quest for this gene, not only for the benefit of the few surviving boys and those to be born to female carriers, but also for defining its role in defending the body against a ubiquitous DNA virus.

Child↗
PubMed1995

Gene mapping of Usher syndrome type IIa: localization of the gene to a 2.1-cM segment on chromosome 1q41.

Usher syndrome type II is associated with hearing loss and retinitis pigmentosa but not with any vestibular problems. It is known to be genetically heterogeneous, and one locus (termed USH2A) has been linked to chromosome 1q41. In an effort to refine the localization of USH2A, the genetic map of the region between and adjacent to the marker loci previously recognized as flanking USH2A (D1S70 and PPOL) is updated. Analysis of marker data on 68 Usher II families places the USH2A gene into a 2.1-cM region between the markers D1S237 and D1S229. The gene for transforming growth factor beta 2 (TGFB2) and the gene for the homeodomain box (HLX1) are both eliminated as candidates for USH2A, by virtue of their localization outside these flanking markers. The earlier finding of genetic heterogeneity was confirmed in six new families, and the proportion of unlinked Usher II families is estimated at 12.5%. The placement of the USH2A gene into this region will aid in the physical mapping and isolation of the gene itself.

Chromosome Mapping↗
PubMed1993

Alu-PCR approach to isolating NotI-linking clones from the 3p14-p21 region frequently deleted in renal cell carcinoma.

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating NotI sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.

Animals↗
PubMed1991

Intraocular tumor formation of RB reconstituted retinoblastoma cells.

It has been reported that replacement of a functional retinoblastoma (RB) gene in RB defective WERI-27 retinoblastoma cells results in complete loss of their tumorigenic potential in nude mice following s.c. injection. We have repeated the identical studies and found that although tumors did not develop s.c., the RB reconstituted cells, either soon after RB virus infection or after long term cultivation, consistently produced tumors when injected intraocularly. These tumor cells, when reestablished in culture, were found to retain a normal RB protein as determined by direct Western blotting and immunocytochemical staining. The tumors, however, occurred with a longer average latency period and with less frequency compared to those produced by the parental RB defective cells. Our results suggest that reintroduction of the RB gene into WERI-27 cells reduces but does not completely suppress their tumorigenic potential. Since retinoblastoma is an eye tumor it also provides further documentation that the use of an orthotopic injection site can be critical when determining the tumorigenicity of a given cell type.

Animals↗
PubMed1991

Involvement of 3p deletions in sporadic and hereditary forms of renal cell carcinoma.

Deletions of the short arm of chromosome 3 and associated allele losses have been reported in the majority of sporadic renal cell carcinomas (RCC). On the basis of the combined cytogenetic and molecular data, it is reasonable to assume that a putative RCC locus, which contributes to tumor development by its loss, is located telomerically of the D3F15S2 site. Using H3E4, a D3F15S2-specific probe, we have isolated a cDNA clone (cl.4-2), and a sequence comparison revealed that the cDNA clone corresponds to the human acyl-peptide hydrolase gene. The gene is fairly universally expressed, but in RCC biopsies its expression is severely reduced, compared to the normal kidney. Cl.4-2 was used for in situ hybridization on metaphase chromosomes prepared from an Epstein-Barr virus (EBV) transformed lymphoblastoid cell line, derived from a t(3;8) (p14.2;q24.1) carrying member of the RCC family described by Cohen et al. in 1979 (N Engl J Med: 301:592-595). Carriers of this translocation regularly develop RCC by middle age. We now report that D3F15S2 is localized on the telomeric side of the constitutional breakpoint, in 3p21. The region of 3p affected by this familial translocation is thus not identical with the region of 3p most frequently deleted in sporadic RCC.

Alleles↗
PubMed1991

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.

Animals↗
PubMed1991

A new variant 15; 16 translocation in mouse plasmacytoma leads to the juxtaposition of c-myc and immunoglobulin lambda.

Mouse plasmacytomas (MPCs) induced by pristane oil, or by a combination of pristane oil and Abelson virus, carry one of two chromosomal translocations. The typical 12; 15 translocation leads to the juxtaposition of c-myc and immunoglobulin heavy-chain sequences, whereas the 6; 15 translocation links the kappa light-chain locus with the pvt-1 (plasmacytoma variant translocation) locus, located at least 75kb 3' of c-myc [Cory, S., Graham, M., Webb, E., Corcoran, L. & Adams, J. (1985). EMBO J., 4, 675-681]. Unlike the human Burkitt's lymphoma-associated translocation, the lambda/myc juxtaposed variant translocation has not been found previously in MPCs. Using unconventional MPC induction systems in which the tumor precursor cell was induced to proliferate in a secondary host, we have recently identified a 15; 16 translocation in six of the derived MPCs [Wiener, F., Silva, S., Sugiyama, H., Babonits, M. & Klein, G. (1990). Genes Chromosomes Cancer, 2, 36-43]. Chromosome 16 harbors the lambda light-chain gene. To explore whether the 15; 16 translocation represents the lambda/myc juxtaposition, we have mapped the breakpoints on chromosomes 15 and 16 by pulsed-field gel electrophoresis (PFGE). The pvt-1 region was mapped to approximately 220 kb 3' of c-myc. The breakpoint on chromosome 15 in ABPC-Ch-163-10, one of the six 15; 16 translocation-carrying MPCs, was situated approximately 80 kb 3' of c-myc and 140 kb 5' of pvt-1b, the major breakpoint cluster region of the previously analysed 6; 15 variant MPCs. The breakpoint on chromosome 16 was found to cut between the V1 and C3 regions of the lambda locus. Co-migration experiments showed that the C3 and the myc gene were juxtaposed head to tail on the 15; 16 translocation chromosome. On the reciprocal product V1 was juxtaposed to pvt-1.

Animals↗
PubMed1991

Subcellular localization of the retinoblastoma protein.

The subcellular localization of the retinoblastoma (RB) protein has been studied in primate cell lines by immunofluorescence staining using different monoclonal and polyclonal antibodies. The protein appeared as granules of heterogeneous size over the interphase nuclei. Computer assisted digital overlap analysis indicated that it was predominantly localized in euchromatic areas with low DNA density. The largest RB positive grains lined up on the heterochromatin/euchromatin boundary. During mitosis, the RB protein dissociated from the condensing chromosomes. It was dispersed throughout the cytoplasm during metaphase and anaphase, and it reassociated with the decondensing chromatin during telophase. A new monoclonal antibody, designated aRB1C1, was raised against a bacterial TrpE/human retinoblastoma protein. It specifically recognized the nonphosphorylated and differentially phosphorylated forms of the RB protein in immunoprecipitation experiments. A collection of RB expressing cell lines gave a positive staining reaction with the antibody, whereas the RB negative Weri-RB-27 retinoblastoma and OHS osteosarcoma cells failed to react. Wild-type RB complementary DNA was introduced into Weri-RB-27 by retrovirus mediated gene transfer. Similar experiments were performed with the DU145 prostatic carcinoma cell line that expresses a mutant RB protein. Reconstituted cells of both lines expressed the normal size RB protein and gave a positive immunofluorescence reaction with the aRB1C1 and other anti-RB antibodies. The new monoclonal antibody, however, showed cell type dependent differences of the staining pattern compared to other anti-RB antibodies, suggesting differentiation dependent accessibility to its epitope.

Antibodies, Monoclonal↗
PubMed1991

Reintroduction of a normal retinoblastoma gene into retinoblastoma and osteosarcoma cells inhibits the replication associated function of SV40 large T antigen.

The product of the retinoblastoma (Rb) gene can form complexes with the transforming proteins of small DNA tumor viruses, including SV40 large T antigen (Tag), adenovirus E1A, and the human papilloma virus E7. The strong correlation between their ability to transform and their ability to bind Rb protein suggests that these oncoproteins exert their effect through blocking the Rb function. SV40 Tag causes oncogenic cell transformation of rodent cells, and it is also required for viral DNA replication. In this paper, we investigated the effect of the Rb protein on the SV40 replication associated function of Tag. We present evidence suggesting that the complex formation between Rb and Tag interferes with the viral DNA replication. In Y79 retinoblastoma and Saos-2 osteosarcoma cells, which lack functional Rb protein, a SV40 based plasmid vector, pSVEpR4, replicates well. In the same cells reconstituted for Rb expression with an intact Rb gene introduced by retroviral mediated gene transfer, pSVEpR4 replicates to a considerably lower level. The inhibitory effect of Rb protein was surmounted by increasing the intracellular level of Tag. Increasing amounts of Tag in wild-type Rb negative Y79 cells had virtually no effect on SV40 replication. Furthermore, the overexpression of Tag in Rb reconstituted Y79 cells did not alter the growth rate of the cells. These data suggest that Rb protein interacts with Tag and modulates its ability to promote SV40 DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1990

Expression of the RB gene under the control of MuLV-LTR suppresses tumorigenicity of WERI-Rb-27 retinoblastoma cells in immunodefective mice.

Retinoblastomas arise by the loss of the retinoblastoma (RB) gene. The isolation of the RB gene and its expression in RB protein defective tumor cells permits direct tests of the ability of the protein to act as a tumor suppressor. We demonstrate that a functional RB gene introduced into WERI-Rb-27 retinoblastoma cells by retrovirally mediated gene transfer can suppress their tumorigenicity in immunodefective mice.

Animals↗
PubMed1989

Long-range restriction enzyme maps of DNF15S2, D3S2, and c-raf1 loci on the short arm of human chromosome 3.

Physical maps have been constructed around loci DNF15S2, D3S2, and c-raf1 on the short arm of human chromosome 3 using pulsed field gradient gel electrophoresis. The normal restriction pattern has not been altered by a t(3;8)(p14.2;q24.1) characteristic for a hereditary form of renal cell carcinoma, indicating that the breakpoint itself is not included in any of the mapped areas. We have found a CpG island within the DNF15S2 locus, suggesting the presence of a functional gene in the region.

Blotting, Southern↗