Generation and fluorescent in situ hybridization mapping of yeast artificial chromosomes of 1p, 17p, 17q, and 19q from a hybrid cell line by high-density screening of an amplified library.

A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.     

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.     

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.     

Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: Analysis of dinucleotide polymorphisms on native gels

Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p. Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms. Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA. Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19). Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HLA-telomere. A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus.     

Study of numerical aberrations of chromosome 1 by fluorescent in situ hybridization and DNA content by densitometric analysis on (pre)-malignant cervical lesions

Summary In an attempt to determine whether the fluorescent in situ hybridization (FISH) can be used as a rapid approach for the identification of aneuploidy in premalignant cervical smears, a centromeric probe for chromosome 1 was used. The results from the FISH experiments were compared with measurements of the overall DNA content obtained by means of an image analysis system. With progression to neoplasia, a decrease of the frequency of cells with two spots was observed, due to an increasing polysomy of chromosome 1. As far as the DNA content was concerned, an increasing DNA index and 5C-exceeding ratio (fraction of cells with a DNA content higher than 5C) was observed. Classification of the FISH results by a linear discriminant analysis revealed that 67.6% of the cases were classified in agreement with the CIN classification. These data suggest that chromosome 1 may be considered as a marker chromosome for pre-malignant cervical lesions and that the DNA content measurements are complementary to the FISH results.     

Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study—which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent—indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.     

Central serotonin1A receptors: Respective distributions of encoding mRNA, receptor protein and binding sites by in situ hybridization histochemistry, radioimmunohistochemistry and autoradiographic mapping in the rat brain

The regional distribution of the mRNA encoding the 5-HT_1A serotonin receptor (whose selective agonists are potential anxiolytic and antidepressant drugs) was investigated in rat brain sections by in situ hybridization histochemistry using two sets of [³²P]labelled nucleoprobes, a riboprobe of 156 bases and oligoprobes of 30 bases corresponding to highly selective portions within the third intracellular loop and the N terminus domain of the amino acid sequence. These probes allowed the visualization of the 5-HT_1A mRNA mainly in the limbic regions: dentate gyrus and area CA₁ of the hippocampus, amygdala, entorhinal cortex, lateral septum and the dorsal raphe nucleus. These structures were also those which could be labelled by the specific 5-HT_1A radioligand [¹²⁵I]BH-8-MeO-N-PAT and antibodies raised against a synthetic 26 amino acid peptide whose sequence was taken from the most selective portion of the rat 5-HT_1A receptor protein. These data suggest that the 5-HT_1A receptors are not transported to a long distance from their site of synthesis, as it has been already reported for the somato-dendritic 5-HT_1A autoreceptors in the dorsal raphe nucleus. Combined autoradiographic quantification of the 5-HT_1A binding sites (labelled by a selective radioligand such as [¹²⁵I]BH-8-MeO-N-PAT, the 5-HT_1A receptor binding subunit (by radioimmunohistochemistry) and the 5-HT_1A mRNA on adjacent brain sections should be a relevant approach for assessing the molecular mechanisms responsible for the functional alterations of these receptors under various pathological and pharmacological conditions.     

Chromosome mapping of the human RAS-related RAP1A, RAP1B, and RAP2 genes to chromosomes 1p12----p13, 12q14, and 13q34, respectively

Three human cDNAs encoding new RAS-related cDNAs, designated RAP1A, RAP1B, and RAP2, have been isolated previously. The encoded proteins are highly related to RAS in the effector region and share an overall identity with RAS of approximately 50%. Using the complete cDNAs or parts thereof as probes, each RAP gene has been localized on human chromosomes by in situ hybridization. The three genes RAP1A, RAP1B, and RAP2 have been assigned to chromosome bands 1p12----p13, 12q14, and 13q34, respectively.