Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor.

A human genomic DNA library was screened by using conditions of reduced stringency with a bovine cDNA probe coding for the kringle domains in prothrombin in order to isolate the human prothrombin gene. Twelve positives were identified, three of which coded for prothrombin (Degen & Davie, 1987). Phage L5 was characterized in more detail because of its strong hybridization to the cDNA probe and its unique restriction map compared to the gene coding for human prothrombin. The gene in L5 was sequenced and found to code for a kringle-containing protein. A human liver cDNA library was screened by using a genomic probe from the gene in L5. cDNAs were isolated that contained sequence identical with regions in the gene in L5. Comparison of the cDNA with the gene indicated that the gene in L5 was composed of 18 exons separated by 17 intervening sequences and is 4690 bp in length. Exons ranged in size from 36 to 242 bp in length while intervening sequences ranged from 77 to 697 bp in length. The putative protein encoded by the gene in L5 contains four kringle domains followed by a serine protease-like domain. This domain structure is identical with that found in hepatocyte growth factor (HGF), although the two proteins are only about 50% identical. On the basis of the similarity of the protein encoded by L5 and HGF, we propose that the putative L5 protein be tentatively called HGF-like protein until a function is identified. The DNA sequence of the gene and cDNA and its translated amino acid sequence were compared against GenBank and NBRF databases. Sequences homologous to DNF15S1 and DNF15S2, human DNF15S2 lung mRNA, and rat acyl-peptide hydrolase were identified in exon 17 to the 3' end of the characterized sequence for the gene. From our results, it is apparent that the gene coding for human HGF-like protein is located at the DNF15S2 locus on human chromosome 3 (3p21). The gene for acyl-peptide hydrolase is 444 bp downstream of the gene coding for HGF-like protein, but on the complementary strand. The DNF15S2 locus has been proposed to code for one or more tumor suppressor genes since this locus is deleted in DNA from small cell lung carcinoma, other lung cancers, renal cell carcinoma, and von Hippel-Lindau syndrome.     

A partial trisomy 15q due to 15;17 translocation detected by conventional cytogenetic and FISH techniques

We report a case having multiple abnormalities including the simultaneous presence of the heart defect and central nerve system abnormalities, which has been reported in a few cases, and with a partial trisomy 15q. Partial trisomy 15q has been inherited from a balanced translocation carried by his phenotypically normal father, detected by traditional banding and fluorescence in situ hybridization (FISH). Application of FISH using whole chromosome specific library probes, locus specific and repetitive probes allowed us to detect the translocation between chromosomes 15q and 17q. Simultaneous application of probes revealed the position of the translocation. Interestingly, in addition to the chromosomes 15 pericentromeric signals, the use of chromosome 15 beta-satellite III probe demonstrated an extra signal on chromosome 14 in both metaphase, and lighted three signals interphase nuclei which was inherited from his father. This patient is compared with other partial trisomy 15q patients reported in the literature. The results are also discussed in relation to genetic counselling for the possible relation of chromosome abnormality and clinical findings.     

Genomic organization of the human folate receptor genes on chromosome 11q13.

There has been interest in the high affinity folate receptor (FOLR) recently because of its high expression in the majority of ovarian tumors. The FOLR genes are part of a family that includes an adult gene, a fetal gene, and one or more pseudogenes, which have been localized to chromosome 11. As a step toward understanding why the adult FOLR gene product is expressed on tumors, we have determined the organization of all the human FOLR-related genes. YAC clones were isolated using the adult FOLR probe. The organization of the locus was determined by PFGE of YAC DNA and by YAC fragmentation. Four FOLR-related genes were found within 140 kb. The adult and fetal genes are not more than 23 kb apart, with the 3' end of the adult gene facing the 5' of the fetal gene. A physical map of over 900 kb of the surrounding region was also constructed. The chromosomal assignment of the FOLR locus was refined to 11q13.3-q13.5 telomeric of the FGF3 locus using fluorescence in situ hybridization.     

Regional mapping of the human gene encoding the novel pituitary polypeptide 7B2 to chromosome 15q13----q14 by in situ hybridization

Genetic sequences encoding the novel pituitary polypeptide 7B2 were isolated from a human pituitary cDNA library. Hybridization analysis of a panel of human x mouse cell hybrids with a 7B2 cDNA probe indicated that the locus for the human 7B2 gene is probably located on chromosome 15. In situ hybridization analysis of metaphase chromosomes allowed the regional localization of the 7B2 gene to chromosome 15 at q13----q14.     

Application of IGF2-specific identifier probe for cytogenetic study of somatic and sperm cells in horses

The development of molecular techniques with fluorochromes has had an invaluable impact on discovering the nature of chromatin structure. Here, we show the application of a locus specific identifier probe (LSI) for precise and selective visualization of the horse IGF2 gene in the metaphase, interphase nuclei and sperm cells. Our study may be helpful for interpretation of results of interphase fluorescence in situ hybridization (I-FISH). We analyze and discuss the variation in the number and localization of FISH signals in somatic and sperm cells of horse.     

Detection of a single copy gene on a mitotic metaphase chromosome by fluorescence in situ hybridization (FISH) in the sawfly, Athalia rosae (Hymenoptera).

Mitotic metaphase chromosomes of Athalia rosae (Hymenoptera) haploid males were subjected to fluorescence in situ hybridization (FISH) analysis using an rDNA probe and two vitellogenin (Vg) cDNA probes (one representing the 5' half and the other the 3' half of the gene, each about 3 kb long, and together covering the entire coding region). The rDNA probe produced signals in four chromosomes, all in pericentromeric regions (haploid chromosome number = 8), and the Vg probes, either the combined probes or the 3' region alone, produced a twin signal in the middle of a chromosome arm of a single chromosome. Arch.     

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.     

Mosaic loss of 15q11q13 in a patient with hypomelanosis of Ito: is there a role for the P gene?

We report a patient with mental retardation, behavioral disturbances, and pigmentary anomalies, consistent with the phenotype of hypomelanosis of Ito (HMI), and in whom cytogenetic analysis revealed mosaicism for an unbalanced translocation. His karyotype is 45, XY,–7, –15,+der(7)(7;15)t(q34;ql3)/46, XY. He is therefore monosomic for 7q34 to qter and 15pter to q13 in the cells containing the translocation. The human homolog (P) of the p gene (the product of the mouse pink-eyed dilution locus) maps to 15q11q13. Loss of this locus is believed to be associated with abnormalities of pigmentation, such as the hypopigmentation seen in patients with deletions of 15q11q13, and the Prader-Willi and Angelman syndromes. Mutations within the P gene have also been associated with tyrosinase-positive (type II) oculocutaneous albinism. Using fluorescence in situ hybridization, we confirmed that our patient is deleted for one copy of a P gene probe in the cells with the unbalanced translocation, and for loci within the region critical for the Prader-Willi/Angelman syndromes. Although hypomelanosis of Ito is a heterogeneous disorder, we postulate that, in our case and potentially in others, this phenotype may result directly from the loss of specific pigmentation genes.     

Two steroid 15 alpha-hydroxylase genes and a homologous gene family in mice

Steroid 15 alpha-hydroxylase (P45015 alpha) activity is concomitant with the expression of two types of mRNA in the mouse liver. Two discrete genes, designated 15 alpha oh-1 and 15 alpha oh-2, that encode the two mRNAs were recovered from total genomic libraries of the inbred mouse strains 129/J and C57Bl/6J and identified by cDNA hybridization, restriction-site analysis and partial nucleotide sequence. Both genes are approx. 9 kb long and share significant homology, including flanking regions, over a region of at least 30 kb. The two distinct 15 alpha oh genes are members of a larger family of homologous genes and/or pseudogenes of unknown function. The most extensive sequence homology among family members in the 3' portion of the gene with progressively less homology toward the 5' end. The far 5' portions of 15 alpha oh-1 and 15 alpha oh-2 are very similar to one another but there is no observed homology with other genes of the family. The two 15 alpha oh genes and the homologous family have been localized to mouse chromosome 7 by somatic cell hybrid mapping. Analysis of a restriction fragment length polymorphism in recombinant inbred mice shows a close linkage of 15 alpha oh-1 and 15 alpha oh-2 with the Coh locus.     

Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z

We isolated and characterized the gene encoding human transglutaminase (TG)(X) (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG(Z)) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG(X) and TG(Z) showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TG(C),TG(E), and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.     

Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression of a beta tubulin gene of Physarum polycephalum

A beta tubulin gene of Physarum polycephalum has been isolated from a genomic library in the phage EMBL4. Southern-blot hybridization to genomic DNA indicates that the cloned DNA is derived from the betB1 locus of the beta tubulin gene family. A tubulin-specific subfragment of the phage DNA was used as a hybridization probe to construct a restriction map of the betB1 locus. The probe consisted of the almost complete coding region of the 5' half of the tubulin gene, interrupted by one intron. The derived amino acid sequence of this subclone deviates from the protein sequence for Physarum amoebal beta tubulin (amino acids 4-207) in two of 207 amino acids. We used both recA and recBC sbcB bacterial host strains, which have been recommended for cloning of instability-conferring sequences of the Physarum genome, but were unable to subclone the 3' part of the gene from the phage DNA. Primer-extension analysis indicates that the betB gene is expressed in the vegetatively proliferating amoebal and plasmodial stages of the life cycle as well as in differentiating (sporulating) plasmodia.