Double minutes in the papillary thyroid cancer cell line PTC-1113A.

The cell line PTC-1113A was established from a metastasizing recurrent papillary thyroid cancer. The cell line was growing as monolayer and showed a complex karyotype with chromosome numbers ranging from 30 to 140/metaphase. A proportion of metaphases contained double minutes and/or pulverized chromosomes. Extrachromosomal DNA seemed to originate from a B-group chromosome. A chromosome 4 painting probe hybridized to extrachromosomal material, representing double minutes (dmin) and possibly minutes. In addition, fluorescence in situ hybridization (FISH) with the chromosome 4 library detected a translocation chromosome and a pulverized chromosome originating from chromosome 4. PTC-1113A is, to our knowledge, the single papillary thyroid cancer cell line demonstrating evidence of gene amplification.

Molecular cloning and functional expression of mouse connexin-30,a gap junction gene highly expressed in adult brain and skin.

A new gap junction gene isolated from the mouse genome codes for a connexin protein of 261 amino acids. Because of its theoretical molecular mass of 30.366 kDa, it is named connexin-30. Within the connexin gene family, this protein is most closely related to connexin-26 (77% amino acid sequence identity). The coding region of mouse connexin-30 is uninterrupted by introns and is detected in the mouse genome as a single copy gene that is assigned to mouse chromosome 14 by analysis of mouse x hamster somatic cell hybrids. Abundant amounts of connexin-30 mRNA (two transcripts of 2.0 and 2.3 kilobase pairs) were found after 4 weeks of postnatal development in mouse brain and skin. Microinjection of connexin-30 cRNA into Xenopus oocytes induced formation of functional gap junction channels that gated somewhat asymmetrically in response to transjunctional voltage and at significantly lower voltage (Vo = +38 and -46 mV) than the closely homologous connexin-26 channels (Vo = 89 mV). Heterotypic pairings of connexin-30 with connexin-26 and connexin-32 produced channels with highly asymmetric and rectifying voltage gating, respectively. This suggests that the polarity of voltage gating and the cationic selectivity of connexin-30 are similar to those of its closest homologue, connexin-26.

Homologs of genes and anonymous loci on human chromosome 13 map to mouse chromosomes 8 and 14.

To enhance the comparative map for human Chromosome (Chr) 13, we identified clones for human genes and anonymous loci that cross-hybridized with their mouse homologs and then used linkage crosses for mapping. Of the clones for four genes and twelve anonymous loci tested, cross-hybridization was found for six, COL4A1, COL4A2, D13S26, D13S35, F10, and PCCA. Strong evidence for homology was found for COL4A1, COL4A2, D13S26, D13S35, and F10, but only circumstantial homology evidence was obtained for PCCA. To genetically map these mouse homologs (Cf10, Col4a1, Col4a2, D14H13S26, D8H13S35, and Pcca-rs), we used interspecific and intersubspecific mapping panels. D14H13S26 and Pcca-rs were located on the distal portion of mouse Chr 14 extending by approximately 30 cM the conserved linkage between human Chr 13 and mouse Chr 14, assuming that Pcca-rs is the mouse homolog of PCCA. By contrast, Cf10, Col4a1, Col4a2, and D8H13S35 mapped near the centromere of mouse Chr 8, defining a new conserved linkage. Finally, we identified either a closely linked sequence related to Col4a2, or a recombination hot-spot between Col4a1 and Col4a2 that has been conserved in humans and mice.

Characterization of the mouse loricrin gene: linkage with profilaggrin and the flaky tail and soft coat mutant loci on chromosome 3.

Loricrin is the major component of a specialized structure, termed the cornified cell envelope, that is formed beneath the plasma membrane of stratified squamous epithelial cells and is coexpressed with profilaggrin in terminally differentiating epidermal keratinocytes. Full-length cDNAs for both mouse and human loricrin have been cloned and characterized, as has the human gene. Here we report the isolation and characterization of the mouse loricrin gene. The gene has a simple structure consisting of a single intron of 1091 bp within the 5' noncoding sequence and an uninterrupted open reading frame. Using PCR analyses of DNAs isolated from mouse x Chinese hamster somatic cell hybrids, we have mapped both the loricrin and the profilaggrin genes to chromosome 3. Genetic linkage analysis has shown that mouse loricin and profilaggrin lie within 1.5 +/- 1.1 centimorgans of each other. We have further shown that both genes map in the vicinity of the flaky tail (ft) and soft coat (soc) loci. These mouse mutants exhibit a number of changes in their integument, suggesting that abnormalities in these genes may contribute to the mutant phenotype.

Chromosomal aberrations in two adrenocortical tumors, one with a rearrangement at 11p15.

Adrenocortical tumors are detected with increasing frequency, but symptomatic cases with excessive hormone production are rare. We investigated cytogenetically one benign aldosterone-producing tumor (Conn Syndrome)(case 1) and one malignant cortisol-producing tumor (Cushing Syndrome)(case 2). Radioimmunoassay of cell culture supernatant of case 2 detected cortisol secretion during 2 months in culture. Flow cytometry of spill-out cells from case 2 showed a bimodal pattern (DNA Index 1.0, 1.4). Case 1 revealed a marker chromosome in 4/25 cells analyzed; the marker was a long acrocentric partially derived from chromosome 2,der(2q). In case 2, a cytogenetic harvest was achieved after prolonged culture time (6 weeks) and a marker chromosome, add(11)(p15), was detected in 16/22 cells. A breakpoint of 11p13, as well as loss of heterozygosity of alleles on 11p15, has been reported in the literature for other malignant adrenocortical cancers.

Chromosomal assignments of mouse genes for connexin 50 and connexin 33 by somatic cell hybridization.

The 12 known connexin genes coding for the subunit proteins of the gap junction channels and related in nucleotide sequence are widely dispersed in the mouse genome. By using a series of mouse X Chinese hamster somatic cell hybrids and molecular probes of murine connexin genes, we have assigned the connexin 50 gene to mouse chromosome 3. The connexin 33 gene has been mapped to the X chromosome, thus confirming the previous chromosomal assignment of this gene based on interspecific back-cross mapping.

Cloning, genomic organization, and chromosomal localization of the Scya5 gene encoding the murine chemokine RANTES.

RANTES is a member of the C-C subfamily of chemokines that functions as a proinflammatory chemoattractant for CD4+ T cells, monocytes, and eosinophils, and as an activator of basophils to release histamine. Like other members of the chemokine superfamily, RANTES has been implicated in a number of chronic inflammatory and autoimmune processes based on its function and its pattern of regulation. To begin study of the transcriptional regulation of RANTES, we have determined the genomic organization of the gene encoding the small inducible cytokine A5 (Scya5) and performed an initial analysis of its promoter elements. The Scya5 gene is located on chromosome 11. By Southern blot, it is a single-copy gene approximately 4.5 kb long composed of 3 exons. This chromosomal localization and pattern of genomic organization is conserved among the other C-C subfamily of chemokines. Primer extension analysis was used to identify the transcriptional initiation site that is located 27 bp downstream of a typical TATAA box. Sequence analysis of 1040 bp 5' to the start site of the Scya5 gene revealed a number of regulatory motifs that are also shared among the chemokine family including a PU.1 box, a NF-kappa B, and an IFN regulatory factor-1 response element. This region of genomic DNA was also cloned into a luciferase reporter vector. Transfection of this reporter construct into murine proximal tubular cells reveals that TNF-alpha can induce a transcriptional activation of the gene, as would be predicted from the rise in mRNA transcripts encoding RANTES in cells stimulated with TNF-alpha.

Characterization of the mouse haptoglobin gene.

Plasmids containing mouse cDNA encoding haptoglobin, a major plasma protein that binds free hemoglobin, have been isolated and characterized. The amino acid sequence predicted by the mouse haptoglobin cDNA was 80% identical to human haptoglobin and 90% identical to rat haptoglobin sequence. The mouse haptoglobin probe was used to demonstrate a single haptoglobin gene in the genome of C57BL6 mice mapped to chromosome 8. Sequence analysis of the mouse Hp gene promoter revealed two unique features: the presence of a second TATA box with a 48-bp trinucleotide repeat immediately upstream. The enhancer element and the sequences shown to be required for cytokine and hormonal regulation of the rat Hp gene are highly conserved in mouse. Interestingly, the single nucleotide variation G to A, which completely inactivates the IL-6 responsive element A in the rat Hp gene, is identical in mouse. This suggests that the presence of an inactive IL-6-responsive element A in Hp genes is common in rodents.

Chromosomal aberrations in two sporadic gastrinomas.

Results of cell culture and cytogenetic analysis (standard and fluorescent in situ hybridization, FISH) of two sporadic gastrinomas are reported. Maintenance of hormonal activity was assessed by detection of gastrin levels during the first 3 months in culture. Case 1 showed clonal aberrations consisting of two marker chromosomes: marker 1 is a large metacentric chromosome and marker 2 is a small acrocentric chromosome. Case 2 showed a constitutional polymorphism with chromosome 15p+ and a clone in the tumor cell culture with trisomy for chromosome 3. To our knowledge, this is the first cytogenetic report of sporadic gastrinomas (Zollinger-Ellison syndrome).

Assignment of the mouse tartrate-resistant acid phosphatase gene (Acp5) to chromosome 9.

Tartrate-resistant acid phosphatase is a marker enzyme for osteoclasts, the multinucleated cell responsible for bone resorption. Interspecific somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map the gene encoding tartrate-resistant acid phosphatase (Acp5) to mouse Chromosome 9. Acp5 is therefore a member of a syntenic family of genes that map to human chromosome 19p13.1-p13.3 and mouse Chromosome 9.

Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos.

We have isolated two mouse genes, Mox-1 and Mox-2 that, by sequence, genomic structure and expression pattern, define a novel homeobox gene family probably involved in mesodermal regionalization and somitic differentiation. Mox-1 is genetically linked to the keratin and Hox-2 genes of chromosome 11, while Mox-2 maps to chromosome 12. At primitive streak stages (approximately 7.0 days post coitum), Mox-1 is expressed in mesoderm lying posterior of the future primordial head and heart. It is not expressed in neural tissue, ectoderm, or endoderm. Mox-1 expression may therefore define an extensive 'posterior' domain of embryonic mesoderm before, or at the earliest stages of, patterning of the mesoderm and neuroectoderm by the Hox cluster genes. Between 7.5 and 9.5 days post coitum, Mox-1 is expressed in presomitic mesoderm, epithelial and differentiating somites (dermatome, myotome and sclerotome) and in lateral plate mesoderm. In the body of midgestation embryos, Mox-1 signal is restricted to loose undifferentiated mesenchyme. Mox-1 signal is also prominent over the mesenchyme of the heart cushions and truncus arteriosus, which arises from epithelial-mesenchymal transformation and over a limited number of craniofacial foci of neural crest-derived mesenchyme that are associated with muscle attachment sites. The expression profile of Mox-2 is similar to, but different from, that of Mox-1. For example, Mox-2 is apparently not expressed before somites form, is then expressed over the entire epithelial somite, but during somitic differentiation, Mox-2 signal rapidly becomes restricted to sclerotomal derivatives. The expression patterns of these genes suggest regulatory roles for Mox-1 and Mox-2 in the initial anterior-posterior regionalization of vertebrate embryonic mesoderm and, in addition, in somite specification and differentiation.

Significance of trisomy 7 in thyroid tumors.

Standard cytogenetic studies of a multifocal metastasizing papillary thyroid carcinoma revealed two clonal chromosome aberrations: rearranged 10q and trisomy 7. Trisomy 7 seemed to be restricted to tumor nodule A, whereas era (10q) was detected in tumor nodule B and in a metastatic lymph node. We applied fluorescent in situ hybridization to ask whether trisomy 7 was a feature of the original tumor nodule or an in vitro phenomenon changing quantitatively during early passages and to see whether trisomy 7 was restricted to tumor nodule A. We used the biotinylated chromosome 7 alpha-satellite probe D7Z1 on freshly dropped slides from metaphase harvests from tumor nodule A,B, and the lymph node and on touch preparations from the frozen specimen of tumor nodule A. Trisomy 7 was present in the original tumor nodule (6% of cells), as well as in early passages (P1-3) from both tumor nodules and the metastatic lymph node with a frequency of 10.7-13.2%. The detection of trisomy 7 as a stable component in short-term cell culture and its presence in the original tumor material indicates that this common numerical aberration is an in vivo phenomenon.

Two gap junction genes, connexin 31.1 and 30.3, are closely linked on mouse chromosome 4 and preferentially expressed in skin.

Two new gap junction genes isolated from the mouse genome code for connexin homologues of 271 and 266 amino acids, designated here Cx31.1 and Cx30.3, respectively. The two open reading frames, oriented in the same direction, are only 3.4 kb apart on mouse chromosome 4. Within the connexin family, these two proteins are most closely related to one another (70% amino acid sequence identity) and to Cx31 (65 and 68% identity, respectively). Comparison of the Cx31.1 mouse gene with a Cx31.1 cDNA showed a similar genomic organization to that found with other members of the connexin gene family, i.e. the coding and 3'-untranslated regions are contained within a single exon, which is preceded by an intron, less than 25 bases upstream of the ATG start codon. Northern blot hybridization revealed highly tissue-specific coexpression of the 1.6-kb Cx31.1 mRNA and two Cx30.3 transcripts of 1.9- and 3.2-kb size, predominantly in skin and two related mouse keratinocyte cell lines. Minor levels of Cx31.1 mRNA were detected in testis. Microinjection of Cx30.3, but not Cx31.1 cRNA, into Xenopus oocyte pairs induced formation of functional gap junction channels with unique voltage-gated parameters compared to other connexins expressed similarly.

Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization.

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.

Chromosomal assignments of mouse connexin genes, coding for gap junctional proteins, by somatic cell hybridization.

The connexin genes Cx31 and Cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by Southern blot hybridization of specific gene probes to DNA from mouse x Chinese hamster somatic cell hybrids. In addition, our results confirm the recent assignment of mouse connexin genes Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 to mouse chromosomes 14, X, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. Our assignment of the Cx31 gene to mouse chromosome 4 locates the fourth connexin gene on this mouse chromosome to which the genes for Cx31.1, Cx37, and Cx30.3 have previously been assigned. Interestingly three of them (coding for Cx31, Cx31.1, and Cx30.3) are preferentially expressed in skin. Possibly some of the connexin genes clustered on mouse chromosome 4 may be regulated coordinately.

Assignment of tyrosine-specific T-cell phosphatase to conserved syntenic groups on human chromosome 18 and mouse chromosome 18.

Phosphorylation of proteins on tyrosine is crucially involved in signal transduction and mitogenesis and is regulated by both kinases and phosphatases. Recently, a number of soluble and transmembrane receptor-linked protein tyrosine phosphatases (PTPase) have been characterized. Among these is a 48.4-kDa PTPase encoded by a cDNA isolated from a T-lymphocyte library by low-stringency screening with probes derived from placental PTPase 1B. A human T-cell PTPase (PTPT) cDNA and somatic cell hybrids were used to assign a PTPT gene to conserved syntentic groups on human chromosome 18 and on mouse chromosome 18. Two unlinked sequences, one on human chromosome 1, were also detected.