Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

Porteous, David J.; Morten, John; Cranston, Gwen; Fletcher, Judy; Mitchell, A. R.; Heyningen, Veronica van; Fantes, Judy; Boyd, Patricia A.; Hastie, Nicholas D.

doi:10.1128/mcb.6.6.2223

Cited by 43 publications

(18 citation statements)

References 44 publications

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“…The additional bands of HORL9X may be attributed to the presence of the short arm of the X chromosome in this hybrid. The fact that the RFLV patterns of these hybrids can be superimposed indicates that only low levels of polymorphism of the L 1 repeats can exist for individual X chromosomes and supports similar observations for other human chromosomes (Porteous et al 1986;Scambler et al 1986). The discemible Xq-specific bands are illustrated diagrammatically in the figure.…”

Section: Chromosomal Rearrangements Occur In Cmgtsupporting

confidence: 79%

Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.

Pritchard¹,

Goodfellow²

1987

Genes Dev.

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A panel of over 50 hybrid cells containing varying portions of the long arm of the human X chromosome have been obtained by chromosome-mediated gene transfer (CMGT) of human chromosomes to mouse cells deficient in HPRT. This panel is used to investigate the size and integrity of transfected human chromosome fragments and also to examine the effect of including a selectable DNA plasmid in the transfection mix. Chromosomal rearrangements are found to be generated in the chromosome transfer process, and the human X centromeric re#on is detected in the transfected cells at an unusually high frequency. Extensive lengths of X chromosome DNA are transferred intact, suggesting potential uses of CMGT in cloning large genes and loci for which only the chromosomal map position is known.[Key Words: Chromosome transfer; human gene mapping; X chromosome; centromere selection] Received November 6, 1986; revised version received and accepted January 5, 1987.Chromosome-mediated gene transfer (CMGT) can be used to construct hybrid cells that retain fragments of the selected donor chromosome (Mcbride and Ozer 1973}. Chromosome breakage is generally assumed to be random and the chromosome fragment size can vary from 50 kb up to a whole chromosome (Wullems et al. 1976;Olsen et al. 1981). The intermediate size of the transgenome produced by CMGT provides a method for linking and ordering genes over longer distances than the high-resolution mapping techniques of DNA-mediated gene transfer or cosmid cloning and at a more sensitive level than can be achieved by somatic cell hybrids or in situ hybridization. In addition, if fragmentation is random, the frequency with which loci are separated by CMGT could be used as a measure of genetic distance (for review, see Mcbride and Peterson 1980). Despite these advantages CMGT has been restricted in its applications. One major drawback has been the lack of suitable selective methods for isolating the cells containing the required chromosomal fragment. Until recently, only those regions of the genome encoding endogenous selectable loci were amenable to analysis, the best examples being the HPRT locus on the long arm of the human X chromosome (Miller and Ruddle 1978; and the TK locus on the long arm of human chromosome 17 (Klobutcher and Ruddle 1979).In previous experiments, new selection methods were used to extend the number of regions of the genome that could be subjected to analysis by CMGT (Pritchard and Goodfellow 1986). By cotransferring the selectable DNA plasmid, pSV2neo, with human chromosomes to mouse HPRT-cells, it was shown that the frequency of combined uptake of the HPRT chromosomal locus with pSV2neo was close to the frequency of uptake of HPRT alone. This observation demonstrated the feasibility of including an unlinked selectable marker in the transfection mix, followed by preselection for incorporation of that marker as an enrichment step in CMGT experiments. Furthermore, the fact that the neo gene and the HPRT chromosomal locus remained associated after a second chromosome tran...

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Section: Chromosomal Rearrangements Occur In Cmgtsupporting

confidence: 79%

Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.

Pritchard¹,

Goodfellow²

1987

Genes Dev.

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“…1B-8-1/6 is a mouse-human somatic cell hybrid whose sole human component is chromosome 11 (22). The HRASJselected CMGT cell line E67-1 was derived by morphological transformation of mouse C127 cells (23), with mitotic chromosomes isolated from the human bladder carcinoma cell line EJ-18-8D (24), as described (15,16).…”

Section: Methodsmentioning

confidence: 99%

“…Second, DNA sequences that are closely linked on the whole chromosome must be cotransferred intact at high frequency. We chose the HRASI-selected CMGT transformant E67-1 as the most suitable to test this strategy because it contains human DNA equivalent in sequence complexity to less than half of the short arm of chromosome 11, with no evidence for gross molecular scrambling (15). In situ hybridization shows a fragment of human heterochromatin inserted interstitially into a mouse chromosome at a single site (35).…”

Section: Methodsmentioning

confidence: 99%

“…In situ hybridization shows a fragment of human heterochromatin inserted interstitially into a mouse chromosome at a single site (35). Inspection of Table 1 shows that several genes known to be syntenic with HRASJ on the short arm of chromosome 11 have been cotransferred to E67-1 but that the transformant has undergone at least two interstitial deletions (15). However, most important for our purposes, three of the markers immediately flanking the WAGR region-FSHB, CAT, and MICJJ (refs.…”

Section: Methodsmentioning

confidence: 99%

“…However, none of these markers has been found to map as close to WAGR as CAT or FSHB (8). We describe here an alternative, directed approach to the problem of mapping subchromosomal regions (12)(13)(14), by isolation of human DNA recombinants, following HRASJ-selected, chromosome-mediated gene transfer (CMGT) (15,16).…”

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HRAS1-selected chromosome transfer generates markers that colocalize aniridia- and genitourinary dysplasia-associated translocation breakpoints and the Wilms tumor gene within band 11p13.

Porteous¹,

Bickmore²,

Christie³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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We show that chromosome-mediated gene transfer can provide an enriched source of DNA markers for predetermined, subchromosomal regions of the human genome. Forty-four human DNA recombinants isolated from a HRASI-selected chromosome-mediated gene transformant map exclusively to chromosome 11, with several sublocalizing to the Wilms tumor region at llpl3. We present a detailed molecular map of the deletion chromosomes 11 from five WAGR (Wilms tumor/aniridia/genitourinary abnormalities/ mental retardation) syndrome patients, three of which are at the limits of cytogenetic resolution but shown here to be molecularly distinguishable and overlapping. We can define ten distinct regions of the short arm of chromosome 11, five of which subdivide band lipl3. We also map two independent llpl3 translocation breakpoints to within the smallest region of overlap defined by the WAGR deletions. The first comes from a patient with familial aniridia, and the second from a patient with Potter facies and genitourinary dysplasia. The close similarities in map location and affected cell lineage for Wilms tumor and genitourinary dysplasia suggest that they may be alternative manifestations of mutation at the same locus.The association of aniridia, genitourinary abnormalities, and mental retardation with Wilms tumor (WAGR syndrome; genetic locus WAGR) is well established (1, 2). Cytogenetic analysis of independent WAGR syndrome patients has localized the relevant genetic region to band llpl3 (3). We (4) and others (5-8) have utilized available DNA probes and expressed protein markers to start to define the molecular extent of the various deletions. In most studies, only one patient has been examined (5-7) or the deletions have been large and therefore likely to show loss of markers that map some distance from the disease-related genes (8). We now attempt to distinguish the smaller deletions, those which take us beyond the resolving power of cytogenetic analysis towards defining the smallest region of overlap that is within reach of complete molecular analysis. We make use of five independent WAGR deletion hybrids, three of which are at the limit of cytogenetic resolution and involve only part of band llpl3.The short arm of human chromosome 11 (lip) is one of the better mapped regions of the genome (9, 10), but there is still a relative dearth of genetic markers that map within even the larger WAGR syndrome deletions (4, 8). The genes encoding erythrocyte catalase (CAT) and two cell surface markers, MIC4 and MIC11 (synonymous with MIC1), all map to band llpl3 and are closely associated with WAGR but not invariably deleted (4, 8). Most recently (8), the /3 subunit of follicle-stimulating hormone (FSHB) has been shown to map close to the aniridia and Wilms tumor genes. Of the anonymous DNA markers mapped to the short arm of chromosome 11, the majority derive from a somatic cell hybrid considered to retain lip material as the sole human component (11). However, none of these markers has been found to map as close to WAGR as CAT or F...

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Molecular analysis of human monogenic diseases

Davies

Robson

1987

BioEssays

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SummaryOver one hundred genes have been isolated from the human genome and shown to be causally related to specific human genetic diseases. Studies with gene-specific probes have demonstrated that the mutations resulting in aparticular phenotype are highly heterogeneous as a group, ranging from alterations in transcription or RNA processing in the nucleus, through to errors in mRNA translation in the cytoplasm. Even where the gene-speciJic probe is not available, defects have been localized to chromosomal regions by family studies. Recently developed methods for moving along the chromosome from a linked marker to the mutation are resulting in rapid advances in the understanding of many monogenic disorders.

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Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

Cited by 43 publications

References 44 publications

Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.

Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.

HRAS1-selected chromosome transfer generates markers that colocalize aniridia- and genitourinary dysplasia-associated translocation breakpoints and the Wilms tumor gene within band 11p13.

Molecular analysis of human monogenic diseases

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