Chromosomal localization of DBL oncogene sequences

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human X rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26-27.2) underwent recombination at its 5' and 3' ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13-q22), respectively. DBL was localized to chromosome Xq27-q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

A key feature in adeno-associated virus (AAV) replication is efficient integration of the viral genome into host cell DNA to establish latency when helper virus is absent. The steps involved in this process remain largely uncharacterized, even though AAV integration was first documented 20 years ago. Using a protein--DNA binding method we isolated AAV--cellular junction DNA sequences. The cellular component hybridized to a single restriction fragment in the virus-free parental cell line, and also co-migrated with AAV-specific sequences in numerous latently infected cell lines. Analysis of somatic cell hybrids indicated that this cellular sequence maps to the distal portion of the q arm of human chromosome 19. In situ hybridization of AAV DNA to chromosomes from latently infected cells confirms the physical location of AAV integrations to be q13.4-ter of chromosome 19. Sequence analysis of several independent integration sites shows breakpoints occurring within a 100 bp cellular region. This non-pathogenic parvovirus thus appears to establish viral latency by integrating its DNA specifically into one chromosomal region. Such specific integration is so far unique among the eukaryotic DNA viruses. The incorporation of site-specific integration into AAV vector schemes should make this vector system attractive for human gene therapy approaches.     

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.     

Assignment of casein kinase 2 alpha sequences to two different human chromosomes

Summary Human casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter and one on 20p13. The existence of two separate chromosomal loci suggests that CK-2 alpha is a member of a gene family. Only the locus on chromosome 11 was confirmed by somatic cell hybrid analysis. The analysis was based on the presence of a CK-2-alpha-specific 20-kb fragment. However, the CK-2 alpha cDNA hybridizes to several additional fragments in total human DNA.     

Chromosomal assignment of human VIP/PHM-27 gene to 6q26----q27 region by spot blot hybridization and in situ hybridization

We determined the chromosomal location of the human VIP/PHM-27 gene in two different ways. First, we performed chromosome sorting with a cell sorter. Sufficient numbers of chromosomes for sorting were obtained by cultivating peripheral blood lymphocytes with interleukin-2. Spot blot hybridization of a molecularly cloned fragment of the gene to the sorted chromosomes revealed that this gene is located on chromosome 6. To define the location more precisely, in situ hybridization of the same fragment to the metaphase spreads was then performed, and human VIP/PHM-27 gene was assigned to a region near the terminus of the long arm of chromosome 6 (q26----q27).     

Chromosomal banding patterns in human large bowel adenomas

Summary The human thyroglobulin gene was mapped by in situ hybridization whereby a ³H-labeled recombinant plasmid DNA containing a fragment of 2.3 kilobases of human thyroglobulin gene was hybridized to human chromosome preparations. A high proportion (25%) of hybridized metaphases exhibited silver grains at the distal portion of the long arm of chromosome 8. Analysis of the grain position at this site indicated that the chromosomal localization of the human thyroglobulin gene was 8q242-8q243.     

Complete nucleotide sequence of the gene for human heparin cofactor II and mapping to chromosomal band 22q11.

Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 lambda phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1749 bp of 5'-flanking sequence, five exons, four introns, and 476 bp of DNA 3' to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5'-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-human somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, we concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and HindIII.     

Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene.

int-2 is one of two cellular genes (int-1 and int-2) currently implicated in the genesis of mammary carcinomas by mouse mammary tumor virus and may constitute a novel cellular proto-oncogene. Using low-stringency hybridization with mouse int-2 probes, we established that homologous genes exist in a variety of mammalian species, including humans, but failed to detect related sequences in other classes and phyla. Recombinant bacteriophage clones and a single cosmid encompassing the human int-2 gene were isolated and characterized by restriction enzyme mapping. A survey of nine primary human breast tumors, three breast tumor cell lines, and three normal individuals revealed no evidence for gross amplification or rearrangement of the int-2 locus. Three distinct restriction fragment length polymorphisms were observed which could prove useful in future linkage studies. By a combination of in situ hybridization of metaphase chromosomes and somatic cell genetics, the human int-2 gene was mapped to chromosome 11, band q13.     

Assignment of the fucosidase pseudogene FUCA1P to chromosome region 2q31----q32

FUCA1P is a pseudogene of the structural fucosidase gene FUCA1. The former has been mapped to human chromosome 2, whereas the latter has been localized to chromosome 1p34----p36. We have further localized FUCA1P to chromosomal band 2q31----q32 by fluorescent in situ hybridization and digital imaging microscopy. This localization was confirmed by linkage analysis between FUCA1P and the COL3A1 gene in 2q24----q32 which gave maximal lod scores of 4.03 at 3% recombination.     

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.     

Isolation of a zinc finger motif (ZNF75) mapping on chromosome Xq26.

We report here the partial characterization of a new human zinc finger (ZNF75) gene of the Kruppel type mapping to the long arm of the X chromosome. A cosmid clone was isolated from a library specific to the Xq24-qter region by hybridization to a degenerate oligonucleotide representing the link between two contigous fingers of the C2H2 type. The sequence of the pertinent cosmid fragments demonstrated five consecutive zinc finger motifs, all pertaining to the Kruppel family. A reading frame starting at least 75 amino acids before the first zinc finger and ending 11 amino acids after the last one was identified; comparison with other ZF genes suggests that this genomic fragment represents the carboxy-terminal exon of the gene. Homology of approximately 55% in the zinc finger region was detected with many zinc finger genes including mouse Zfp-35 and human ZFN7 cDNA clones. Mapping using a panel of sematic cell hybrids and chromosomal in situ hybridization localized the gene to Xq26, in a region not previously known to contain zinc finger genes.     

Refined Localization of the α1-Subunit of the Skeletal Muscle L-Type Voltage-Dependent Calcium Channel (CACNL1A3) to Human Chromosome 1q32 by in Situ Hybridization

We isolated and partially sequenced a cosmid clone containing the human skeletal muscle L-type voltage-dependent calcium channel gene (CACNL1A3). The cosmid clone, which was also found to contain a novel dinucleotide repeat marker for the CACNL1A3 gene, was used for the chromosomal localization of CACNL1A3 by in situ hybridization. Our results refine the localization of CACNL1A3 on the long arm of human chromosome 1 to band q32.     

Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23.

The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism. To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM). The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein. The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand. We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes. High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.     

A highly polymorphic locus in human DNA revealed by probes from cosmid 1-5 maps to chromosome 2q35----37.

The highly polymorphic locus D2S3 is revealed by three single-copy probes from cosmid C1-5. These probes, 1-30, 1-32, and 2-96, collectively reveal seven restriction fragment length polymorphisms. Fifty-three of 56 unrelated individuals (93%) were heterozygous at one or more of the seven loci, making the compound locus a very useful marker for gene mapping. Chromosomal assignment of D2S3 was obtained using a panel of human X hamster and human X mouse somatic cell hybrids. Molecular hybridization of EcoRI-digested DNA from these cell lines with the DNA inserts from subclones 1-30, 1-32, and 2-96 showed that all three probes mapped to the long arm of chromosome 2. Additionally, in situ hybridization of [3H]-labeled probe 2-96 to metaphase chromosome preparations allowed more precise assignment of the locus to the region 2q35----37.     

Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones.

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.