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During recent years, there has been an exponential rise in the number of sequences accessible in the public databases. Despite this, a high percentage of partial sequences of cDNA (ESTs) submitted to the databases remain unrecognized (anonymous ESTs). This lack of similarities could be explained by several hypotheses: i) a different part of the transcript is present in the GenBank; ii) the transcript represents a novel gene not yet isolated in other species; iii) alternative splicing of the same gene in different species; iv) inaccurate sequence data; and/or v) the sequence of the transcript has diverged to the extent that it is not recognized as an ortholog. In the present study we selected a sample of 20 ESTs from a pool of 656 anonymous pig small intestine ESTs in order to investigate the possible cause for the lack of similarities with database entries. To test the significant hypotheses we carried out total sequencing of each insert along with zoo-blot and Northern-blot analysis. Extended analyses of the 20 ESTs showed significant matches to seven existing database entries, whereas 13 still did not show significant hits. The results are discussed in the context of the hypothesis listed above.

Three cDNA clones from a porcine small intestine cDNA library, tentatively identified as transcripts of two different cytochrome P450 (CYP) gene subfamilies, were sequenced and mapped. One of the clones was found to comprise an open reading frame of 503 amino acids and showed up to an 80% similarity to genes from the CYP3A subfamily. Translation of the other two cDNA clones revealed high similarity to genes from the CYP2C subfamily. One of these clones was truncated, lacking a part of the 5' end of the coding region, whereas the other encodes a putative pseudogene. The CYP3A gene was mapped cytogenetically by means of a pig/rodent somatic hybrid cell panel to pig chromosome 3, in the region p16-p17 or p11. The CYP2C pseudogene was mapped genetically to pig chromosome 14, using the PiGMaP shared reference pedigrees. The localisations provide information for the human/pig comparative map.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap4A hydrolase (Km = 0.8 microM) being sensitive to inhibition by fluoride (Ki = 24 microM) and adenosine 5'-tetraphosphate (Ki = 10 nM) and yielding ATP and AMP as products. A low Km Ap4A hydrolase (Km = 0.3 microM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of Km for Ap4A and Ki for adenosine 5'-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequences comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P

Frequency distribution and allele size in 20 canine microsatellite loci were analyzed in 33 flat-coated retrievers, 32 dachshunds, 10 red foxes, and 10 Arctic foxes. Overall, the major difference between the two dog breeds was the relative allele frequencies rather than the size ranges of alleles at the individual locus. The average heterozygosity within the two dog breeds was not significantly different. Since the average heterozygosity at several polymorphic loci is a relative measure of heterogeneity within the population, analysis of heterozygosity within microsatellite loci is suggested as a measure for the diversity of populations. Eighty percent (16 of 20) of the canine microsatellite primer pairs amplified corresponding loci in the two fox species. This reflects a very high sequence conservation within the Canidae family relative to findings in, for instance, the Muridae family. This indicates that it will be possible to utilize the well-characterized fox karyotype instead of the dog karyotype as a step towards physical mapping of the dog genome. Analysis of exclusion power and probabilities of genetic identity between unrelated animals by use of the seven most informative loci demonstrated that it will be possible to assemble a panel of microsatellite loci that is effective for parentage analysis in all breeds.

One of the more widely studied simple repeat sequences in the mammalian genome is the (dG-dT) n ·(dC-dA) n dinucleotide repeat sequence. As these repeats are highly polymorphic and fairly evenly distributed in diverse mammalian genomes, they constitute a very powerful tool for genetic mapping in a wide variety of species. So far, the knowledge about repeat sequences in the porcine genome is sparse and only a few areas of this genome have been sequenced. We have isolated and characterized 108 porcine (dG-dT) n ·(dC-dA) n sequences and studied the distribution of these, both by investigating random clones and by performing in situ hybridization. A remarkable correlation between humans and pigs was found with respect to the structure, to the number of repeat blocks, and to the chromosomal distribution.

Investigation of published sequence data from the porcine insulin-like growth factor 1 (IGF1) gene, resulted in the detection of a microsatellite in the first intron of the gene. Polymerase chain reaction (PCR) primers flanking the (CA)19 repeat were constructed. Polymorphism and Mendelian segregation were documented in a three-generation pedigree and allele frequencies were determined in 74 unrelated animals from four different breeds. Seven alleles were encountered. Linkage analysis was performed in a large pedigree established for gene mapping. Linkage between the IGF1 microsatellite and an anonymous microsatellite marker, S0005, was detected. Furthermore, IGF1 and S0005 was found to be linked to the porcine submaxillary gland mucin (MUC) gene, previously assigned to chromosome 5. The results presented here extend the linkage group on pig chromosome 5 and are in accordance with conserved synteny between human chromosome 12, cattle chromosome 5, mouse chromosome 10 and pig chromosome 5.