Why do we still find anonymous ESTs?

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.

Mapping of porcine genes belonging to two different cytochrome P450 subfamilies.

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.

Improvement of the porcine transcription map: localization of 33 genes, of which 24 are orthologous.

From a resource of porcine ESTs, 52 transcripts were selected for regional chromosomal assignments in a somatic cell hybrid panel. Except for six ESTs, the chosen transcripts represented genes where the BLASTX database searches showed high similarity scores (>90%) with a part of the single pass 5' sequence to human, bovine, mouse, or pig entries. PCR primers for hybrid cell analysis of the ESTs were positioned in the 3'UTR of the sequences. Confident regional assignments to pig chromosomes were obtained for 33 of the 52 porcine ESTs. Comparative human mapping data were available for 27 of these. Twenty-four proved to be orthologous genes now placed on the porcine transcription map. The data presented provide further comparative data for 13 autosomes and the X chromosome.

Expansion of the pig comparative map by expressed sequence tags (EST) mapping.

We have used a PCR-based approach for the genetical and physical mapping of 34 transcripts isolated from a porcine small intestine cDNA library. All but one gene were regionally localized by using a somatic pig-rodent cell hybrid panel, and 12 genes were mapped by linkage analysis of single-stranded conformational polymorphisms developed in 3' untranslated regions of transcripts. For 20 of the transcripts, the human homolog has already been mapped. This study thus represents a significant contribution to the pig comparative map. Some important findings were that we could clarify the extent of a previously identified inversion event in a region of conserved synteny between SSC6q and HSA1p, that SSC14q does contain a region homologous to HSA1, a situation not clear from earlier ZOO-FISH studies, and that the homology between SSC17 and HSA20 includes the p-arm of HSA20.

Mapping of 22 expressed sequence tags isolated from a porcine small intestine cDNA library.

Complementary DNA sequences were selected from a resource of tentatively identified clones from a porcine small intestine cDNA library. Forty PCR primer pairs were designed to amplify 101-309 base pairs of the 3' untranslated region of the genes. The PCR conditions were optimized by altering both formamide and magnesium concentrations on samples of pig, mouse, and hamster DNA. Twenty primer pairs that, under stringent conditions, were pig-specific and amplified the expected fragments were chosen for regional assignment in a pig/rodent hybrid cell panel. Furthermore, 22 primer pairs were chosen to amplify DNA from the parental animals of the PiGMaP shared reference families in order to detect possible polymorphisms. Primer pairs that generated polymorphisms were used for genetic mapping. A total of 22 porcine expressed sequence tags (ESTs) were cytogenetically or genetically mapped by this approach. Twelve of the mapped ESTs could be added to the human-porcine comparative map.

Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases.

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.

FISH mapping of seven cDNA sequences in the pig.

Fluorescence in situ hybridization (FISH) technique was applied to localize seven clones derived from a porcine (SSC) intestinal directionally cloned cDNA library. The size of the clones ranged from 1.1 to 1.3 kb. Three of the clones corresponded to histidyl-tRNA synthetase (HARS), immunoglobulin alpha (IGA) and lysozyme (LYZ) and mapped to SSC2q28-q29, 7q2.6 and 5p11 respectively. The available human-pig comparative painting data and sequence homology comparisons assisted in a tentative identification of the other three clones as glutathione-S-transferase (GST), glutathione-S-transferase mu (GSTM1) and immunoglobulin lambda gene cluster (IGL@). These clones mapped to SSC14q21, 5q2.4 and 14q22-q23 respectively. The remaining clone representing an EST mapped to 1p24-p25. These localizations contribute to the transcript map in pig and are significant as comparative markers. Difficulties associated with the mapping of small sequences using FISH are discussed.

Evaluation and characterization of a porcine small intestine cDNA library: analysis of 839 clones.

A porcine small intestine directionally cloned cDNA library was constructed in the vector lambda Zap II. Clones were hybridized with total labeled cDNA such that putative high-copy number transcripts could be differentiated from middle- and low-copy number transcripts prior to selection and characterization by DNA sequencing. More than 2000 non-hybridizing and 242 hybridizing clones were collected. In total, 839 clones were sequenced from the 3' end of the cDNA, and after inter-clone comparison, the unique clones were sequenced from the 5' end of the cDNA. The 5' data were used to query the sequence in databases and resulted in the identification of 630 different gene transcripts, of which 604 are new porcine genes. The identity of 361 transcripts could be identified from sequence comparison studies. The validity of this semi-random selection approach was verified by the identification of a large number of unique transcripts.

Efficient resolution of parentage in dogs by amplification of microsatellites.

Parentage control has been performed for 15 litters from 12 different dog breeds by amplification of microsatellites. As it was possible to include all putative parents in all cases, they were solved by exclusion. Discrimination between parents/non-parents was made after genotyping of 6-9 microsatellite loci. In 12 of the cases all but one of the alleged fathers were excluded while in three cases it was unambiguously shown that superfecundation had taken place. Furthermore, one inclusion case concerning disputed maternity has been investigated. Maternity indices were calculated for 12 loci and probability of maternity was estimated to be 99.99%. These results testify that microsatellites can be applied very efficiently for resolution of parentage in dogs.

The PiGMaP consortium linkage map of the pig (Sus scrofa).

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (approximately 16.5 Morgans) than the genetic map of the homogametic sex (female) (approximately 21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be approximately 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.

Variation of short tandem repeats within and between species belonging to the Canidae family.

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.

Assignment of the gene for porcine insulin-like growth factor 1 (IGF1) to chromosome 5 by linkage mapping.

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.