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J Ragoussis

Publications and source records attributed to J Ragoussis.

48 records · Page 3Linked to original sources
PubMed1993

Fine mapping of the human MHC class II region within chromosome band 6p21 and evaluation of probe ordering using interphase fluorescence in situ hybridization.

Eight previously well-characterized and mapped probes derived from the human major histocompatibility complex (MHC) class II region were used to investigate the advantages and limitations of fluorescence in situ hybridization (FISH) techniques for fine mapping. The class II region of the MHC was localized within subband 6p21.31 by in situ hybridization on metaphase chromosomes that were banded by immunofluorescence staining with an antibody against 5-bromodeoxyuridine (BrdU). Ordering of probes that were separated by up to 900 kb was achieved by simultaneous hybridization of two or three probes on interphase nuclei. Three-color FISH proved to be an excellent method for ordering of probes within distances of 200-1,000 kb. Under certain conditions, closer probes could be ordered by comparing measured distances between their hybridization signals in interphase nuclei. A linear correlation between measured interphase distances and kilobase distances was observed up to 500 kb. With increasing distances, the measurements become more inaccurate due to chromatin folding.

Chromosome Mapping↗
PubMed1992

Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.

Blotting, Southern↗
PubMed1992

A testis-expressed Zn finger gene (ZNF76) in human 6p21.3 centromeric to the MHC is closely linked to the human homolog of the t-complex gene tcp-11.

A novel testis-expressed Zn finger gene (ZNF76) was identified by screening cDNA libraries with cosmids derived from 6p21. ZNF76 is a member of the GLI-Krüppel family of DNA binding proteins. It is conserved in mouse where transcription in testis is initiated at Day 20 after birth. The mouse tcp-11 gene is located in the distal inversion of the t-complex and is developmentally regulated in the same manner as ZNF76. The human homolog of tcp-11 was isolated to allow a precise chromosomal localization. By using a combination of somatic cell hybrids, radiation hybrids, metaphase and interphase fluorescent in situ hybridization, and pulsed-field gel electrophoresis, we mapped the two genes to the 6p21.2 to 6p21.3 region and linked them to each other within 300 kb of DNA, approximately 2 Mb centromeric to the major histocompatibility complex.

Amino Acid Sequence↗
PubMed1992

Genomic organization of the human folate receptor genes on chromosome 11q13.

There has been interest in the high affinity folate receptor (FOLR) recently because of its high expression in the majority of ovarian tumors. The FOLR genes are part of a family that includes an adult gene, a fetal gene, and one or more pseudogenes, which have been localized to chromosome 11. As a step toward understanding why the adult FOLR gene product is expressed on tumors, we have determined the organization of all the human FOLR-related genes. YAC clones were isolated using the adult FOLR probe. The organization of the locus was determined by PFGE of YAC DNA and by YAC fragmentation. Four FOLR-related genes were found within 140 kb. The adult and fetal genes are not more than 23 kb apart, with the 3' end of the adult gene facing the 5' of the fetal gene. A physical map of over 900 kb of the surrounding region was also constructed. The chromosomal assignment of the FOLR locus was refined to 11q13.3-q13.5 telomeric of the FGF3 locus using fluorescence in situ hybridization.

Adult↗
PubMed1991

Cloning of the HLA class II region in yeast artificial chromosomes.

Yeast artificial chromosomes (YACs) have been applied to clone the entire class II region of the human major histocompatibility complex (MHC), including its flanking regions, in a contig over 1.5 million base pairs (bp) long. The human DNA inserts in the YACs have a size between 60 and 1300 kbp and were isolated from two EcoRI partial digest libraries. The gaps between DRA and DRB, DRB and DQA, and DOB and DPA, which had not been cloned by other means, have been bridged with YAC clones. The contig extends through the 400 kpb of DNA between the DRA and C4 genes, thus linking the class II region with the complement gene cluster in the class III region. The cloning in YACs has been supported by a conventional cosmid walk of 290 kbp in the C4-DRA region. Restriction enzyme sites in the YAC clones were compared to the sites in the cosmid walk, to published cosmid clones, and to the already existing physical maps, leading to a detailed characterization of a region of the human genome over 1500 kbp. The YAC clones will be valuable for functional analysis of the MHC.

Blotting, Southern↗
PubMed1991

Organization of the human HLA-class-II region.

A detailed map of the class-II region of the human MHC has been established by pulsed-field-gel-electrophoresis (PFGE) mapping and cloning in yeast-artificial-chromosome (YAC) vectors. The map revealed CpG islands, which are frequently associated with genes, in the gaps between the known HLA-class-II genes. Using cosmid walking and chromosome jumping, we have cloned 3 of these islands and have identified 5 novel genes, named RINGI-5. This brief report summarizes our present knowledge of these genes.

Chromosome Mapping↗
PubMed1991

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids.

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.

Animals↗
PubMed1989

A physical map including a new class I gene (cda12) of the human major histocompatibility complex (A2/B13 haplotype) derived from a monosomy 6 mutant cell line.

To avoid interpretative problems due to restriction fragment length polymorphisms, the monosomy 6 mutant cell line BM19.7 was employed to establish a molecular map of the human major histocompatibility (HLA) complex in the A2,B13,Bw4,DRw6,DRw52,DQw1,DPw2 haplotype. Results were obtained mainly by field-inversion gel electrophoresis and Southern blotting techniques. The map extends to 4800 kb and includes the HLA complex with a length of 4200 kb. Five HTF islands could be positioned on the map. The class I region has a size of about 2000 kb and includes nonclassical HLA class I genes, some of which must be localized within 200 kb telomeric of HLA-A. A new class I gene, cda12, distinct from HLA-A, HLA-B, or HLA-C, has been localized within 50 kb from HLA-A. The class I region contains a gap of about 500 kb, just telomeric of HLA-C, in which further class I genes could not be detected. The class II region has a size of 1000 kb, which is separated from the class I region by about 1200 kb. The 5' end of the HLA-B gene is situated centromeric, giving an orientation opposite to that of the TNFA and TNFB loci. The estimated length of the HLA complex correlates well with its size determined cytogenetically using mutant cell lines with interstitial deletions.

Cell Line↗
PubMed1987

Assignment of the TCP1 locus to the long arm of human chromosome 6 by in situ hybridization.

TCP1, the human homolog of the Tcp-1 locus in the mouse, which is part of the murine t complex and codes for an abundant testicular germ-cell protein, has been mapped within the human genome by in situ hybridization. Using a cDNA probe for TCP1, pB1.4 hum, we assigned TCP1 to human chromosome region 6q23----qter, with the most likely localization being 6q25----q27.

Animals↗
PubMed1986

Mapping of HLA genes using pulsed-field gradient electrophoresis.

The technique of pulsed-field gradient electrophoresis (PFGE) allows the determination of gene linkage relationships since DNA fragments up to 2 Mb can be separated. PFGE was employed to study linkage of class I, II and III genes belonging to the human major histocompatibility (HLA) complex. The results establish that the class II DO beta and DZ alpha genes are linked with the DP subregion, centromeric to the DQ/DX-DR-C4 chromosomal segment, and allow us to estimate the minimal length of the entire HLA complex.

Chromosome Mapping↗