Forensic animal DNA typing: Allele nomenclature and standardization of 14 feline STR markers.

Since the domestic cat (Felis catus) has become one of the most popular pets and owners usually develop a close relationship to their cats, it is necessary to take traces of cats into account for forensic casework. For this purpose feline short tandem (STR) repeat markers have been investigated in several earlier studies, but no detailed description of sequence data, allelic variations or a repeat-based nomenclature is available. The aim of the study was to provide a suggestion for the allele nomenclature of 14 cat STR markers according to the recommendations of the International Society for Forensic Genetics (ISFG) for human DNA typing and to present a standardized system for a secure DNA typing of samples. Samples of 122 unrelated cats from a local animal shelter and private owners in Germany were used to generate a population database with allele frequencies and to analyze the tandemly repeated sequence variations within the alleles of each STR marker. These markers could be grouped into two STR classes: simple repeat STRs and complex STRs (some with the supplement highly complex), consisting of di- and tetranucleotide repeat motifs. After analyzing the repeat structure and elaborating a repeat based nomenclature, allelic ladders of common and rarely occurring alleles for each marker were designed to enable accurate typing of alleles that differ in fragment length and to facilitate data exchange.

Validation of two canine STR multiplex-assays following the ISFG recommendations for non-human DNA analysis.

To gain general acceptance forensic DNA testing in animals needs to improve standardization of analysis methods and data interpretation. Recently, the International Society of Forensic Genetics (ISFG) took particular care of this topic by publishing recommendations for forensic non-human DNA analysis following the successful example of human DNA analysis in order to provide a basis for harmonization of the still existing inter-laboratory variability. By following these recommendations we demonstrate the performance of two short tandem repeat (STR) multiplexes for forensic identity testing of canine biological material. Thirteen STRs and two sex-specific markers were selected and validated according to the ISFG guidelines. Population genetic parameters were calculated based on 295 dog samples collected in Austria (124) and Germany (171). A repeat-based nomenclature of the mainly tetrameric STRs and corresponding allelic ladders are presented. All 146 different alleles included in the ladders were sequenced for correct allele calling. Additionally, a canine cell line (DH82-D3167) was evaluated as standard reference material.

Expression of genes related to the human erbB, erbA, pdgf and pdgf-r in tumors of different etiology in Xiphophorus.

The melanoma determining Tu locus of the teleost Xiphophorus contains an accessory gene, x-erbB*a, which is closely related to the EGF receptor gene family, and is probably oncogenic. x-erbB*a exists in allelic forms that are specific for distinct Tu-loci, and shows high homology to a non-allelic non-oncogenic counterpart x-erbB*i which is transcribed into mRNA of 4.6 kb in non-tumorous and tumorous tissues of fish harboring and lacking Tu. Expression of a 4.0-kb mRNA in tumors (melanoma and fibrosarcoma) of different etiology is strictly correlated with the inheritance of X. maculatus x-erbB*a alleles; transcripts of 8.0 kb were detected in melanoma and carcinoma of fish harboring a certain x-erbB*a of X. variatus. The expression of the putative x-erbB*a transcripts parallels the stage of malignancy of the tumor. The expression of the xiphophorine EGF receptor gene (x-erbB) was detected in almost all tumors, is strongly enhanced in carcinoma, and is positively correlated with the degree of malignancy of melanoma and fibrosarcoma. Some tumors show expression of erbA-related genes. The PDGF receptor mRNA is expressed in all tumors analyzed and shows enhanced expression in malignant tumors of neurogenic, epithelial and mesenchymal origin. Expression of x-pdgf was observed in several cases of melanoma, but more frequently in carcinoma and fibrosarcoma. We conclude that x-erbB*a might be involved in initiation of tumors of different cellular origin and etiology in fish harboring Tu, as well as in the determination of the malignancy of the tumor. Furthermore, we assume that x-erbB*i, x-erbB, x-pdgf and x-pdgf-r play a role in secondary events in tumorigenesis by, e.g., conferring a selective growth advantage to the tumor cells.

[Attempts at analyzing the initiation of the initial processes of carcinogenesis based on the Xiphophorus melanoma model]. / Ansätze zur Analyse der Initiation von Initialprozessen der Krebsbildung am Melanom-Modell von Xiphophorus.

Certain backcross hybrids (BC8-22) of a spotted X. maculatus (platyfish) and a non-spotted X. helleri (swordtail; recurrent parent) are highly sensitive to mutagenic carcinogens and, after a latent period of 8 to 12 months, develop melanoma of unicellular origin that is genealogically related to the spots of the platyfish. Sensitivity to the carcinogen or susceptibility to melanoma, respectively, are inherited in a Mendelian fashion and can be assigned to a "tumor gene-complex" (Tu-complex) consisting probably of almost 20 genes. The Tu-complex is located at the end of an autosome or sex chromosome, and is largely deregulated by crossing conditioned replacement of platyfish chromosome carrying regulatory genes (tumor suppressor genes, oncostatic genes, antioncogenes) for the Tu-complex by swordtail chromosomes lacking them. The melanoma-free condition of these BC-hybrids depends upon the skin-specific regulatory gene Bs (body side) that requires impairment in a pigment cell precursor for the outgrowth of melanoma. Structural mutations involving different breakpoints indicate that the signal for melanoma formation comes from a particular region of the Tu-complex where an accessory v-erb B related oncogene (x-erb B*a; 85% homology to the human EGF receptor gene) is located. Northern blot analyses of the melanoma cell line showed an about 20-fold overexpression of x-erbB*a. Both the inositol lipid turnover [(3H)inositol incorporated into phosphoinositides], and the xiphophorine pp60x-src kinase activity that are assumed to be causally involved in tumor formation showed a remarkable elevation in the melanoma as compared to the normal tissue (brain) of the tumorous and non-tumourous (with or without the Tu-complex) segregants. Other BC hybrids carrying the Tu-complex but lacking the linked regulatory gene develop melanoma "spontaneously". This kind of melanoma occurs early in the course of life, is of multicellular origin, and is inherited as a Mendelian character. In contrast to the BC hybrids requiring somatic mutation for melanoma formation, both inositol, lipid turnover and x-src activity are remarkable enhanced in both melanoma and normal tissues. A mutant of the laller BC hybrids carrying in addition of the Tu-complex the homozygous oncostatic gene g (g/g, "golden") that arrests pigment cell differentiation in the stem cell stage is incapable to develop melanoma spontaneously. Nevertheless it shows the elevation of inositol lipid turnover and x-src activity in its always healthy tissues. Following treatment with tumor promoters such as TPA and steroid hormones pigment cell differentiation recovers and melanoma of multicellular origin develops within 4 to 8 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Search for genes critical for the early and/or late events in carcinogenesis: studies in Xiphophorus (Pisces, Teleostei).

Southern blot analyses of the xiphophorine genome with probes specific for 15 viral and cellular oncogenes revealed that only three v-erbB related EcoRI fragments comprising 4.9 kb of a certain X, 11.5 kb of another X, and 6.7 kb of both a Y and a Z chromosome are inherited in parallel with the Tu complex and melanoma formation. They are accessory in the genome, and are highly homologous with each other and with an ubiquitous autosomal 7.5-kb fragment. The latter one is probably linked to the indispensable Tu complex that is postulated to be present in all individuals of Xiphophorus irrespective of whether they possess or lack the capacity to form melanoma in interspecific hybrids. Three restriction fragments, the X-chromosomal 4.9-kb, the Y-chromosomal 6.7-kb and the ubiquitous Tu-nonlinked 5.5-kb EcoRI fragments were cloned and sequenced. The X- and the Y-chromosomal fragments show perfect identity in the regions of the putative exons C and D of the EGF receptor gene and minor but significant differences to the putative exon C (exon D not identified) of the Tu-nonlinked fragment of 5.5 kb, indicating that at least two different types of x-erb B genes coding for slightly different EGF-receptors exist in the fish. Northern blot analyses revealed expression of the Tu-linked x-erbB genes (x-gfrB genes) in both transformed and nontransformed tissue, suggesting their essential role in regulation of normal cell proliferation and in carcinogenesis. We conclude that the indispensable x-egfrB genes remain unchanged and strictly regulated, while the sex chromosomal accessory x-egfrB genes possibly undergo dramatic changes in structure and/or function (e.g., unscheduled expression, ectopic expression, point mutations, truncation) leading to activation of the oncogenic potential of these genes, which in turn could induce several cellular events involved in the switch from the normal to the transformed state of the cell. In contrast, none of the x-erbA restriction fragments could be assigned to the Tu-complex or to any regulatory gene (R or S). These results, however, do not exclude the existence of a structural and/or functional relation between x-erbA genes and R and S genes. We therefore analyzed x-erbA genes by cloning, sequencing, and expression studies.(ABSTRACT TRUNCATED AT 400 WORDS)

v-erbB related sequences in Xiphophorus that map to melanoma determining Mendelian loci and overexpress in a melanoma cell line.

Certain genotypes of Xiphophorus harbour an accessory Mendelian gene which following loss, impairment, or insufficiency of its regulatory genes in the germ line, mediates the hereditary capacity to develop neoplasia spontaneously or following induction with initiating and promoting carcinogens. Together with its linked regulatory genes it forms a 'tumor gene-complex' (Tu-complex). We concentrated on accessory sex chromosomal Tu-complexes that are responsible for sex chromosome-linked melanoma formation. Southern analyses of the xiphophorine genome with 15 authentic oncogene probes revealed so far that only three v-erbB related EcoRI fragments comprising 4.9 kb of a certain X-, 11.5 kb of another X-, and 6.7 kb of both a Y- and Z-chromosome are inherited in parallel with the Tu-complex and melanoma formation. They are accessory in the genome, and are highly homologous with each other. The sequence of the X-chromosomal 4.9 kb fragment shows minor but significant differences to that of the invariably present autosomal xiphophorine erbB (x-erbB) fragment of 5.5 kb, indicating that at least two different x-erbB genes coding for different EGF receptors can exist in the fish. Northern analyses showed expression of both genes in a fibroblast cell line, and overexpression of the sex chromosomal x-erbB in a melanoma cell line. The co-segregation of the hereditary trait of melanoma with the sex chromosomal x-erbB fragments, suggests that the accessory x-erbB gene may be responsible for the switch from the normal to the neoplastic state of the pigment cells.