Assignment of 35 single-copy and 17 repetitive sequence DNA probes to human chromosome 3: high-resolution physical mapping of 7 DNA probes by in situ hybridization

Thirty-five single-copy and 17 repetitive sequence DNA probes specific for human chromosome 3 were isolated from human chromosome 3-derived genomic libraries. Seven DNA clones, including three that are polymorphic for BglII or MspI, were mapped by in situ hybridization. Four probes were mapped to 3p subregions and 3 were mapped to 3q subregions. Three of the DNA sequences map to regions overlapping a segment of chromosome 3 (3p14-23) frequently deleted in small cell lung cancer cells. By Southern blot analysis on a deletion hybrid panel, we previously mapped 6 of these probes to three distinct chromosome 3 subregions. Our in situ data support these assignments and more precisely determine the localization of each clone to the following regions: D3S34 (3p14-21), D3S35 (3p21), D3S39 (3p21), D3S40 (3p12-13), D3S37 (3q21-23), and D3S36 (3q21). Clone pL84c, a low repeat sequence clone (approximately 30 copies), was mapped to the 3q21-29 subregion. These DNA clones mapped by in situ hybridization can provide useful landmarks for the ordering and localization of other clones.     

Chromosomal localization of ceruoplasmin and transferrin genes in laboratory rats,mice and in man by hybridization with specific DNA probes

Fragments of the natural rat ceruloplasmin (Cp) gene and cDNA copies of rat Cp and transferring (Tf) mRNAs highly labelled by nick translation with ¹²⁵I-dCTP were used as specific probes for assignment of these genes to the metaphase chromosomes of rat, mouse and man by in situ hybridization. Both Cp and Tf genes were found to be syntenic in rodents, occupying with high probability the regions 9D and 9F1–3 in mice and 7q11–13 and 7q31–34 in rats respectively. The significant increase in silver grain count over chromosome 15 in rats after hybridization with both the Cp and Tf probes suggests the presence of a related pseudogene cluster on this particular chromosome and thus favours its partial homeology to chromosome 7. The localization of silver grains in metaphase chromosome of man indicates subregional assignment of the Tf gene to 3q21. Use of the rat Cp DNA probe does not indicate synteny of the Cp and Tf genes in man and suggests the existence of a related DNA sequence in 15q11–13. The potential and limitations of the in situ hybridization technique with heterologous DNA probes for gene mapping in mammalian species are discussed.     

The L-isoaspartyl/D-aspartyl protein methyltransferase gene (PCMT1) maps to human chromosome 6q22.3-6q24 and the syntenic region of mouse chromosome 10.

We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) using cDNA probes. We determined that the human gene is present in chromosome 6 by Southern blot analysis of DNA from a panel of mouse-human somatic cell hybrids. In situ hybridization studies allowed us to confirm this identification and further localize the human gene (PCMT1) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we localized the mouse gene (Pcmt-1) to chromosome 10, at a position 8.2 +/- 3.5 cM proximal to the Myb locus. This region of the mouse chromosome is homologous to the human 6q24 region.     

Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3-F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3-F3 region of mouse chromosome 2 and to the q11-q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11-15q13 region.     

用人染色体14q24.3区带探针池直接分离表达顺序

本文报道了从显微切割的人染色体区带直接分离区带专一性表达序列的方法和结果。     

The genes coding for human pro alpha 1(IV) collagen and pro alpha 2(IV) collagen are both located at the end of the long arm of chromosome 13.

We have isolated and characterized a cDNA clone containing DNA sequences coding for the noncollagenous carboxy-terminal domain of human pro alpha 2(IV) collagen. Using this cDNA clone in both Southern blot analysis of DNA isolated from human-mouse somatic-cell hybrids and in situ hybridization of normal human metaphase chromosomes, we have demonstrated that the gene coding for human pro alpha 2(IV) collagen is located at 13q33----34, in the same position on chromosome 13 as the pro alpha 1(IV) collagen gene.     

Cloning of cDNAs for human phosphoribosylpyrophosphate synthetases 1 and 2 and X chromosome localization of PRPS1 and PRPS2 genes

Cloned cDNAs representing the entire, homologous (80%) translated sequences of human phosphoribosylpyrophosphate synthetase (PRS) 1 and PRS 2 cDNAs were utilized as probes to localize the corresponding human PRPS1 and PRPS2 genes, previously reported to be X chromosome linked. PRPS1 and PRPS2 loci mapped to the intervals Xq22-q24 and Xp22.2-p22.3, respectively, using a combination of in situ chromosomal hybridization and human x rodent somatic cell panel genomic DNA hybridization analyses. A PRPS1-related gene or pseudogene (PRPS1L2) was also identified using in situ chromosomal hybridization at 9q33-q34. Human HPRT and PRPS1 loci are not closely linked. Despite marked cDNA and deduced amino acid sequence homology, human PRS 1 and PRS 2 isoforms are encoded by genes widely separated on the X chromosome.     

Molecular gene mapping of human aldolase A (ALDOA) gene to chromosome 16

Summary Mapping of human aldolase A (ALDOA) gene was performed by molecular hybridization techniques using a panel of human-mouse cell hybrids and sorted fractions of human metaphase chromosomes besides in situ hybridization. For the purpose, three kinds of DNA probes derived from the coding region (probe-1), the 3 noncoding region (probe-2), and the coding and 3 noncoding regions (probe-3) of human aldolase A cDNA clone, pHAAL116-3, were selectively employed. The results of RNA and DNA blot analyses indicated that the human ALDOA gene is located on chromosome 16. The in situ hybridization experiment also indicated that the ALDOA gene was localized to 16q22–q24.     

Localization of the human UbB polyubiquitin gene to chromosome band 17p11.1-17p12.

The chromosomal location of the human ubiquitin genes has been evaluated by in situ hybridization. Because of the conservation of the ubiquitin sequence, coding-region probes cannot distinguish between specific ubiquitin genes and reveal ubiquitin sequences in a number of different chromosomal regions. The major sites of hybridization with a coding-region probe include 17p11.1-p12, 12p24.2-q24.32, and 2q21-q24, with weaker hybridization over 1p3, 1q4, 2q3, and 13q. Hybridization with a probe isolated from the UbB gene intron indicated that this gene is located within the region 17p11.1-17p12. This region showed the strongest hybridization with the coding-region probe and is presumably also the location of the duplicated UbB pseudogene.     

The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region

We report the isolation and characterization of the human gene encoding islet amyloid polypeptide (IAPP). Previously characterized cDNA sequences correspond to three exons of which the first is noncoding. A functional promoter region was identified in the 5' flanking DNA; however, this was farther upstream than expected. Northern blot analysis of human insulinoma RNA revealed three IAPP mRNAs of sizes 1.2, 1.8 and 2.1 kb, in agreement with three polyadenylation signals present in the 3' end of the gene. In situ hybridization to metaphase chromosomes resulted in two distinct peaks on chromosome 12, at 12p12-p13 and 12q13-q14. Southern blot analysis of genomic DNA suggested a single IAPP locus but also indicated the presence of additional homologous sequences in human genomic DNA.     

Mapping of the ceruloplasmin gene on human and laboratory mouse chromosomes by direct in situ hybridization

Mapping of ceruloplasmin gene in human and mouse chromosomes was carried out using the cloned fragments of rat chromosomal ceruloplasmin gene and of ceruloplasmin cDNA as specific hybridization probes. DNA probes were nick-translated with 125I-dCTP up to the high specific capacity. The number of silver grains as well as their distribution along the differentially stained chromosomes were analyzed in 120 metaphase plates from bone marrow cells of laboratory mice and in 181 plates from human lymphocyte cultures. The most probable localization of human ceruloplasmin gene is centromeric region q11-13 of chromosome 15(14?). In laboratory mice ceruloplasmin gene is assigned to the euchromatic part of D-disc of chromosome 9. The possible causes for gene synteny in laboratory mouse and in man as well as its evolutionary implication are discussed.     

Reassignment of the human macrophage colony stimulating factor gene to chromosome 1p13-21.

Macrophage colony stimulating factor (CSF-1) is a member of a family of glycoproteins that are necessary for the normal proliferation and differentiation of myeloid progenitor cells. The human CSF-1 gene has previously been assigned to chromosome 5 using somatic cell hybrids, and further localized to 5q33 by in situ hybridization with a 3H labelled cDNA probe. However, the murine macrophage colony stimulating factor gene (csfm) has been localized to a region on mouse chromosome 3 which was previously shown to be syntenic with the proximal region of 1p and not 5q. Using a human genomic DNA clone that contains the CSF-1 gene, we have localized CSF-1 to chromosome 1p13-21 by fluorescence in situ hybridization. The reassignment of the CSF-1 gene argues against its involvement in myeloid disorders with deletions of the long arm of chromosome 5.     

Dual localization of the human gene encoding hnRNP I/PTB protein to chromosomes 19p13.3 and 14q23

A cDNA probe representative of the human hnRNP I/PTB gene was used to perform fluorescence in situ hybridization (FISH) on metaphases of human chromosomes. A new localization was found on band 19p13.3 in addition to the previously reported localization to band 14q23. Identical results were obtained when FISH analysis was repeated with probes covering different parts of the hnRNP I cDNA clone. This supported the notion that most, if not all, of the sequences of the different parts of this clone are present on both chromosomes. Moreover, Southern blot analysis of DNAs from interspecies somatic hybrids containing chromosomes 19 and 14 revealed that the whole hnRNP I cDNA probe generated very similar patterns in each hybrid DNA. These data suggest that two closely related copies of the hnRNP I gene exist in the human genome. Received: 19 January 1996 / Revised: 9 March 1996     

Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.     

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the-A-B-A-B-A-form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant-B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashtion along the DNA strand.     

The human calcitonin gene is located on the short arm of chromosome 11

By molecular hybridization of human calcitonin cDNA probes to DNA from human-rodent hybrid cells containing identified human chromosomes, we have mapped the human calcitonin gene to the short arm of chromosome 11. This location has been confirmed by in situ hybridization, which further localized the calcitonin gene to region 11p13-15. The significance of this region regarding gene linkage and possible markers for inherited cancers is discussed.     

Mapping of AKT3, encoding a member of the Akt/protein kinase B family, to human and rodent chromosomes by fluorescence in situ hybridization

Previously, a rodent cDNA encoding the third member of the Akt/PKB family of serine/threonine kinases was cloned. We have now cloned the human homolog of this cDNA, and we have used this clone to map the AKT3 gene to human chromosome 1q44 by fluorescence in situ hybridization (FISH). We have also mapped the rodent homologs of AKT3 to rat chromosome 13q24-->q26 and mouse chromosome 1H4-6 by FISH.     

Isolation and characterization of DNA sequences from flow-sorted human chromosome 22 libraries

Two flow-sorted chromosome 22 libraries were used to isolate DNA sequences specific for chromosome 22. 45-phage DNAs were probed against human genomic DNA. 12 of them showed unique or low-copy character. Using digested DNA from rodent-human hybrid cell lines, 3 of the 12 recombinants were assigned unique to chromosome 22 and regionally mapped. 1 clone mapped to 22pter-q11, 1 clone to 22q12-qter and 1 clone, for which in situ hybridization was performed, to 22q13.1. 2 low-copy probes, 1 of them displaying polymorphisms in MspI and TaqI digests of individual DNAs, must have similar sequences on 22 and additional chromosomes. Furthermore, a highly repetitive DNA representing a compound locus of some hundred kilobases on chromosome 22 was isolated. These 6 probes may provide useful tools for studying the structure and function of this small chromosome involved in a relatively high number of inherited and acquired diseases.     

Isolation and analysis of DNA markers specific to human chromosome 15.

Chromosome-specific DNA markers provide a powerful approach for studying complex problems in human genetics and offer an opportunity to begin understanding the human genome at the molecular level. The approach described here for isolating and characterizing DNA markers specific to human chromosome 15 involved construction of a partial chromosome-15 phage library from a human/Chinese hamster cell hybrid with a single human chromosome 15. Restriction fragments that identified unique- and low-copy loci on chromosome 15 were isolated from the phage inserts. These fragments were regionally mapped to the chromosome by three methods, including Southern analysis with a mapping panel of cell hybrids, in situ hybridization to metaphase chromosomes, and quantitative hybridization or dosage analysis. A total of 42 restriction fragments of unique- and low-copy sequences were identified in 14 phage. The majority of the fragments that have been characterized so far exhibited the hybridization pattern of a unique locus on chromosome 15. Regional mapping assigned these markers to specific locations on chromosome 15, including q24-25, q21-23, q13-14, q11-12, and q11. RFLP analysis revealed that several markers displayed polymorphisms at frequencies useful for genetic linkage analysis. The markers mapped to the proximal long arm of chromosome 15 are particularly valuable for the molecular analysis of Prader-Willi syndrome, which maps to this region. Polymorphic markers in this region may also be useful for definitively establishing linkage with one form of dyslexia. DNA probes in this chromosomal region should facilitate molecular structural analysis for elucidation of the nature of instability in this region, which is frequently associated with chromosomal aberrations.