An international parentage and identification panel for the domestic cat (Felis catus).

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex 'core' panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.

The fate of foreign DNA in mammalian cells and organisms.

We have investigated the consequences of foreign DNA insertions into the genomes of mammalian cells in transgenic cell lines, in adenovirus type 12 (Ad12)-transformed cells, in Ad12-induced tumor cells or in transgenic mice. We have reported previously on the de novo methylation of integrated foreign genomes and on extensive changes in cellular patterns of DNA methylation upon foreign DNA insertion. These studies have been extended and several independent methods have been applied to document these alterations in cellular DNA methylation and gene expression patterns in transgenic cell lines and in transgenic mice. These data are relevant for the mechanism of (viral) oncogenesis and for the interpretation of data gathered in experiments with transgenic animals.

Integrated viral genomes can be lost from adenovirus type 12-induced hamster tumor cells in a clone-specific, multistep process with retention of the oncogenic phenotype.

In adenovirus type 12 (Ad12)-induced tumor cells, in Ad12-transformed cells and in continuously passaged cell lines from these sources, the viral DNA is integrated in multiple copies, usually at a single chromosomal location. In different tumors or cell lines, the sites of integration of Ad12 DNA are all different. Rare exceptions exist. In most instances, the integrated viral DNA resides very stably in the host cell genomes. However, upon continuous serial passage of such cell lines, the integrated viral DNA can be destabilized and lost. In two instances, i.e. in the Ad12-induced hamster tumor cell lines H1111(1) and CLAC1, we have investigated the loss of integrated viral DNA in detail. After extended serial passage, these two cell lines seemed to be devoid of Ad12 DNA sequences, as detectable by Southern blot hybridization, but continued to induce tumors after reinjection into hamsters. Cells from these two cell lines were now recloned three times, and DNAs from cultures derived from several individual clones were reinvestigated for the presence of several parts of the viral genome by the polymerase chain reaction (PCR). Some of the clones still carried parts of the Ad12 genome. However, several clones were isolated that proved free of all parts of the viral genome, except for minute segments from the right terminus of the Ad12 genome. Apparently, the loss of integrated viral DNA from these cell lines proceeded as a continuous, gradual, multistep process whose pattern could differ from cell clone to cell clone, once destabilization had been initiated. The mechanism of destabilization is not understood. Cell populations of 2 x 10(6) to 3 x 10(7), and as low as 10(2), cells from the clones, that contained only minimal remnants from the right viral DNA terminus, were reinjected into newborn or 13-20 day-old weanling Syrian hamsters (Mesocricetus auratus). Tumors developed within 5-17 days after injection. Tumor cell clones also grew in soft agar. The injection of primary hamster skin fibroblasts never elicited tumor formation. The tumor cells induced by this reinjection proved repeatedly free of Ad12 DNA both by Southern blot hybridization and by PCR, except for those cell and tumor clones that contained small segments of the right terminal E4 region of the Ad12 genome. The tumor cells, however, retained their oncogenic phenotype. The results raise questions about the cell clone-specific excision patterns of integrated foreign DNA from the recipient genome and the possibility of a hit-and-run mechanism of adenoviral oncogenesis.

On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus.

We have previously shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm foreign DNA without homology to the mouse genome, can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice. In spleen, the foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA. We have now extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 microg of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the fluorescent in situ hybridization (FISH) method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH. The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with M 13 DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids. Is maternally ingested foreign DNA a potential mutagen for the developing fetus?

Uptake of foreign DNA from the environment: the gastrointestinal tract and the placenta as portals of entry.

Foreign DNA (deoxyribonucleic acid) is part of our environment. Considerable amounts of foreign DNA of very different origin are ingested daily with food. In a series of experiments we fed the DNA of bacteriophage M13 as test DNA to mice and showed that fragments of this DNA survive the passage through the gastrointestinal (GI) tract in small amounts (1-2%). Food-ingested M13 DNA reaches peripheral white blood cells, the spleen and liver via the intestinal epithelia and cells in the Peyer's patches of the intestinal wall. There is evidence to assume that food-ingested foreign DNA can become covalently linked to mouse-like DNA. When M13 DNA is fed to pregnant mice the test DNA can be detected in cells in various organs of the fetuses and of newborn animals, but never in all cells of the mouse fetus. It is likely that the M13 DNA is transferred by the transplacental route and not via the germ line. The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated.

Integration of foreign DNA and its consequences in mammalian systems.

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.

Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA.

Food-ingested foreign DNA is not completely degraded in the gastrointestinal tract of mice. Phage M13mp18 DNA as a test molecule devoid of homology to mouse DNA was pipette-fed to or added to the food supply of mice. The fate of this foreign DNA in the animals was followed by several methods. In 84 animals, fragments of M13mp18 DNA were detected in the contents of the small intestine, the cecum (until 18 h), the large intestine, or the feces. In 254 animals, M13mp18 DNA fragments of up to 976 bp were found in blood 2-8 h after feeding. In buffer-fed control animals, M13mp18 DNA could not be detected. M13mp18 DNA fragments were traced by PCR in peripheral leukocytes and located by fluorescent in situ hybridization in about 1 of 1000 white cells between 2 and 8 h, and in spleen or liver cells up to 24 h after feeding, but not later. M13mp18 DNA could be traced by fluorescent in situ hybridization in the columnar epithelial cells, in the leukocytes in Peyer's patches of the cecum wall, in liver cells, and in B cells, T cells, and macrophages from spleen. These findings suggest transport of foreign DNA through the intestinal wall and Peyer's patches to peripheral blood leukocytes and into several organs. Upon extended feeding, M13mp18 DNA could be recloned from total spleen DNA into a lambda vector. Among about 2.5 x 10(7) lambda plaques, one plaque was isolated that contained a 1299 nucleotide pair fragment (nt 4736-6034) of sequence-identified M13mp18 DNA. This fragment was covalently linked to an 80 nt DNA segment with 70% homology to the mouse IgE receptor gene. The DNA from another lambda plaque also contained mouse DNA, bacterial DNA, and rearranged lambda DNA. Two additional plaques contained M13mp18 DNA fragments of at least 641 (nt 2660-3300) or 794 (nt 4640-5433) nucleotide pairs. The medical and evolutionary implications of these observations may be considerable.

On the insertion of foreign DNA into mammalian genomes: mechanism and consequences.

We have studied the integration of adenovirus type 12 (Ad12) DNA in transformed and hamster tumor cells over many years. Upon infection of hamster cells with Ad12, viral DNA has been found in association with hamster chromosomes, possibly in part integrated into the host genome. Ad12 DNA integration is not sequence specific. Transcriptionally active sites of the host genome show a preponderance for foreign DNA insertion. We are pursuing the mechanism of Ad12 DNA integrative recombination in a cell-free system prepared from hamster cell nuclear extracts. In a number of Ad12-transformed hamster cell lines or in cell lines carrying foreign DNA, we have located the inserted Ad12 DNA copies on hamster chromosomes by fluorescent in situ hybridization (FISH). Among the consequences of Ad12 DNA integration, we have studied the de novo methylation of the integrated foreign (Ad12) DNA and increases in DNA methylation in several cellular genes and DNA segments in Ad12-transformed and hamster tumor cells. Several lines of evidence argue for the notion that parameters in addition to nucleotide sequence, in particular site of integration and/or the chromatin configuration of the integrated DNA, are important in generating de novo methylation patterns. The de novo methylation of integrated foreign DNA can be interpreted as an old cellular defense mechanism against the activity of foreign genes in an established genome. Pursuing this concept, we have asked for the most likely portal of entry of foreign DNA, supposedly the gastrointestinal tract in most animals. This hypothesis has been tested by feeding mice linearized or circular, double-stranded bacteriophage M13mp18 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice.

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 bp (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M13 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 bp in length were detected. In DNA from blood, M13 DNA fragments of up to 472 bp were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 bp of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)