Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified. These findings assign the human ACHE gene to a single locus on chromosome 7q22 and should assist in establishing linkage between the in vivo amplification of the ACHE gene in ovarian tumors and leukemias and the phenomenon of tumor-related breakage in the long arm of chromosome 7.     

Mapping of human chromosome 22 by in situ hybridization

The second smallest chromosome of the human karyotype, i.e., chromosome 22, is involved in many congenital or acquired structural aberrations. This variety can be taken advantage of to determine the exact linear order, from centromere to telomere, of cloned probes and chromosomal breakpoints. Eleven probes were localized with respect to breakpoints of 11 der(22) of independent cell lines using in situ hybridization on metaphasic spreads. The deduced order of the tested probes and that of the breakpoints are in complete agreement with the published genetic map and the karyotypic analysis, respectively. This approach enables a correlation of the genetic map with the chromosomal banding.     

Assignment of a novel zinc finger gene ZNF191 to human chromosome 18Q12.1 by human/rodent somatic cell hybrid panel and fluorescent in situ hybridization]

A novel human zinc finger gene, ZNF191, was assigned to chromosome 18 by hybridization of human/rodent hybrid cell panel to a full-length cDNA as a probe. Meanwhile, a human genomic DNA lambda/DASH library was screened using this cDNA probe and several positive clones were obtained. Fluorescence in situ hybridization (FISH) was performed by using one of these positive clones, 16-1, as a probe. Thus, the ZNF191 gene was precisely mapped in 18q12. 1. To date, some hereditary diseases and tumors have been found to be associated with this region by analysis of genetic linkage and loss of heterozygosity. Hence, it suggested that the gene ZNF191 can be taken as a candidate gene responsible for those diseases and tumors.     

Mapping of human chromosome 22 with a panel of somatic cell hybrids

The adenylosuccinate lyase (ADSL) which is essential for generating adenylate, maps to the long arm of chromosome 22. By using a Chinese hamster ovary cell line deficient in ADSL activity, we have constructed a set of 17 somatic cell hybrids containing defined regions of human chromosome 22. This panel was extended with six additional hybrids, obtained in other laboratories using various methods of selection. Southern analysis of the hybrids with 38 chromosome 22 probes defined 14 different subregions which could be linearly organized on the long arm of chromosome 22. The order of the probes thus deduced is fully compatible with their previous localization and with the genetic map. The ADSL gene was further sublocalized between the MB and D22S22. This panel, which enables the rapid assignment of chromosome 22 single copy probes to small subregions, will be an important tool in the construction of a detailed physical map of this part of the genome.     

A somatic cell hybrid panel and DNA probes for physical mapping of human chromosome 7p.

To identify by reverse genetics genes on the short arm of human chromosome 7 expected to be involved in the regulation of human craniofacial and limb development, we have set up a human mouse somatic cell hybrid panel that divides 7p into 9 fragments. The breakpoints are defined by deletions or translocations involving one chromosome 7 in the cells of the human cell fusion partners. Particularly densely covered with these cytogenetic anchor points is the proximal area of 7p within and around 7p13. The number of cytogenetic mapping points within proximal 7p could be increased by four, using two diploid human cell lines with small interstitial deletions in this region for dosage studies. We used Southern blots of this panel to assign to 7q or subregions of 7p more than 300 arbitrary DNA probes or genes that provide reference points for physical mapping of 7p. Three reciprocal translocations with one of the breakpoints in 7p13 mark the location of a gene involved in Greig cephalopolysyndactyly syndrome. To define an area in which we could identify candidates for this developmental gene, we established a macrorestriction map using probes flanking the putative gene region. The Greig translocations were found to be located within a 630-kb NotI restriction fragment.     

Regional assignment of 41 human DNA fragments on chromosome 7 by means of a somatic cell hybrid panel

Summary To detect new restriction fragment length polymorphisms that would cover human chromosome 7 with a network of genetic landmarks, a chromosome 7-specific phage gene library was screened for human single-copy fragments. With use of a somatic cell hybrid panel containing defined regions of human chromosome 7, 41 cloned human single-copy sequences were assigned to five regions of this chromosome. Of special importance are the cell hybrid clones GM1059Rag5 and 7851Rag10-1, derived from human cells with interstitial deletions spanning the bands 7q22-q32, within which the cystic fibrosis gene is located. Twelve new probes are described in 7q22-q32, five of which detect a total of six RFLPs.     

Assignment of the developmentally regulated gene NEDD1 to human chromosome 12q22 by fluorescence in situ hybridization

The developmentally regulated mouse gene Nedd 1 encodes a protein showing similarities with the -subunit of heterotrimeric GTP-binding proteins and has growth suppressing activity when overexpressed in various cultured cell types. We have mapped the human homolog (NEDD1) of the mouse gene to chromosome 12q22 by fluorescence in situ hybridization using R-banded human (pro)metaphase chromosomes.     

Direct nonradioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes

Biotinylated DNA from various human-rodent hybrids was hybridized to human lymphocyte spreads after preannealing of the repeated sequences with sonicated total human DNA. Fluorescent labeling was achieved by successive treatments with fluorescein-labeled avidin and biotinylated antiavidin antibody. The use of labeled total DNA from hybrids with known chromosome composition permits the fluorescent staining-("painting") of specific chromosomes, or parts thereof, in human lymphocyte metaphases. Alternatively, the human chromosome content of cell hybrids with unknown chromosome composition is directly assessed from the labeling pattern of human lymphocyte spreads using the total hybrid DNA as probe.     

Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei.

We have used the proximity of probe hybridization sites in interphase chromatin to derive the order of DNA sequences in a 2-3-Mbp region of human chromosome Xq28. The map generated bridges the results of genetic and pulsed-field gel electrophoresis mapping to produce a more complete map of Xq28 than possible with either of these other techniques alone. Two-color fluorescence in situ hybridization (FISH) was used to detect the positions of two or more probes in G1 male interphase nuclei. We show that cosmids that are 50 kbp to 2-3 Mbp apart can be ordered rapidly with two alternative approaches: (1) by comparing the average measured distance between two probes and (2) simply by scoring the order of red and green fluorescent dots after detection of three or more probes with two fluorochromes. The validity of these approaches is demonstrated using five cosmids from a region spanning approximately 800 kbp that includes the factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and color-vision pigment (CV) genes. The cosmid map derived from interphase mapping is consistent with the map determined by restriction-fragment analysis. The two interphase mapping approaches were then used (1) to orient the F8/CV cluster relative to two markers, c1A1 and st14c, which we show by metaphase mapping to be proximal to the F8/CV cluster, (2) to position st14c (DXS52) between c1A1 and F8, and (3) to orient the CV gene cluster relative to G6PD by using two CV-flanking cosmids, 18b41 and fr7. The probe order in Xq28 derived from interphase proximity is cen-c1A1-st14c-5'F8 (p624-p542-p625)-G6PD-18b41-3' green-green-red-fr7-tel. We also show that, to determine their order by using metaphase chromosomes, sequences must be at least 1 Mbp apart, an order of magnitude greater than required in interphase chromatin. The data show that FISH mapping is a simple way to order sequences separated by greater than or equal to 50 kbp for the construction of long-range maps of mammalian genomes.     

A somatic cell hybrid mapping panel for regional assignment of human chromosome 13 DNA sequences

We have constructed somatic cell hybrids containing different overlapping deletions involving human chromosome 13. Cytogenetic characterisation of the breakpoints allowed division of the chromosome into six distinct regions. Molecular characterisation of these hybrids allowed regional assignment of anonymous DNA sequences, cDNAs, and isoenzyme variants and these hybrids should prove valuable in the analysis and isolation of genes and disease loci on chromosome 13.