Ontogeny of erythropoietin receptor mRNA expression in various tissues of the foetal and the neonatal pig.

Erythropoietin receptor (EPOR) mRNA expression in liver, spleen, bone marrow and testes of foetal and neonatal pigs was analysed using a real-time RT-PCR assay. The results showed that early in the foetal life, EPOR expression is greatest in the liver. Later in foetal life, the spleen has the greatest expression of EPOR, whereas at 2 weeks after birth, the main expression of EPOR is found in the bone marrow. These findings contradict our earlier hypothesis that erythropoietin (EPO) acting in a paracrine fashion can account for an extensive erythropoiesis at birth, a point of time when plasma EPO concentrations are low. Results presented in the present paper suggest that the spleen or, alternatively, the bone marrow is able to respond to very low concentrations of circulating EPO around the time of birth. The testes were found to express significant amounts of EPOR. Since EPO mRNA has previously been found in the testes, a paracrine function of EPO may exist in this organ.

Responses of plasma Epo and kidney and liver Epo mRNA to hemorrhage in perinatal pigs.

Despite the fact that pig fetuses in late gestation have extensive erythropoiesis, low blood pO(2) and low hemoglobin concentrations, piglets are born without detectable concentrations of plasma erythropoietin (Epo). In the present study, we have examined the hypothesis that long-term hypoxic stimuli are less efficient than short-term stimuli in stimulating Epo production in perinatal pigs. From fetuses collected by hysterectomy 5 days before term, new-born piglets and piglets 2 and 5 weeks old, blood in amounts corresponding to 2% of body weight was withdrawn from the jugular vein. Twenty-four hours later the animals were killed and their kidney and liver Epo mRNA analysed by a competitive RT-PCR assay. Plasma Epo concentration was estimated by a solid-phase, two-site sequential chemiluminescent enzyme immunometric assay. We found that in nearly fully developed fetuses and in new-born piglets, the concentration of Epo mRNA did not increase upon bleeding. This is in contrast to earlier findings in sheep. In 2- and 5-week-old piglets, bleeding was associated with a 12-15-fold increase in kidney Epo mRNA. In the 2- and 5-week-old piglets, bleeding evoked increased translation of Epo mRNA into the protein hormone. Also in new-born piglets, increased plasma levels of Epo accompanied bleeding, whereas significant changes in gene Epo expression were not observed.

Ontogeny of erythropoietin mRNA expression in liver, kidneys and testes of the foetal and the neonatal pig.

Erythropoietin (EPO) mRNA expression in kidneys, liver and testes of foetal and neonatal pigs was analysed using a competitive RT-PCR assay. The results indicate that in the foetal pig, erythropoietin expression is greatest in the liver, at birth; hepatic and renal expression are nearly identical, and by 5 weeks of age there is mainly renal expression. The dynamics of the renal expression of EPO mRNA in the perinatal period provide a correlate for observations made earlier of plasma EPO concentrations. Early in foetal life (30 days after artificial insemination), the mesonephroi contained large amounts of EPO mRNA. As in the rat, the testes produced EPO mRNA in amounts comparable to the liver on a per gram tissue basis, though much less on a per organ basis.

Genomic organization, comparative analysis, and genetic polymorphisms of the bovine and ovine prion Doppel genes (PRND).

The doppel protein (Dpl) is a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene, PRNP, in human and mouse. This paper describes the isolation and structural organization of the bovine and ovine PRND genes, which are composed of two exons compared with the three of human and mouse. Intergenic distances between PRNP and PRND were covered by means of long-range PCR and found to be 16.8 and 20 kb, in cattle and sheep respectively. The 5' and 3' untranslated regions (UTR) were analyzed to identify transcription regulatory sequences and compared with those from the PRND and PRNP sequences published for other species. Three polymorphisms (R50H, N110H, and R132Q) were revealed in the cattle coding region; two synonymous substitutions (I12I, A26A) were found in sheep. None of the polymorphisms was significantly associated with either Bovine Spongiform Encephalopathy (BSE) in cattle or scrapie in sheep.

The PrP-like protein Doppel gene in sheep and cattle: cDNA sequence and expression.

cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3' untranslated region and the terminal 3' end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.

The porcine erythropoietin gene: cDNA sequence, genomic sequence and expression analyses in piglets.

The porcine erythropoietin (EPO) gene and its cDNA have been cloned and characterized. The cDNA encodes a protein of 194 amino acids. The gene structure and sequence show a high degree of homology to the corresponding human and murine gene. Steroid hormone receptor binding sites are present both in the promoter and in the 3' flanking region of the gene, which also contains an oxygen-sensing sequence. The promoter lacks classical promoter elements such as TATA and CAAT boxes. Expression analyses using a competitive RT-PCR assay showed that the kidneys contain about ten times more erythropoietin mRNA than the liver in five-week-old piglets, thus indicating that the shift from mainly hepatic to mainly renal EPO production has taken place at this age. The testes showed a higher ratio of EPO mRNA to total RNA than the liver. Spleen showed very low levels of expression, while no expression of erythropoietin mRNA was detected in brain tissue, bone marrow, lung, lymph nodes, and ovaries.

Evolution of the neuropeptide Y receptor family: gene and chromosome duplications deduced from the cloning and mapping of the five receptor subtype genes in pig.

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.

The porcine hormone-sensitive lipase gene: sequence, structure, polymorphisms and linkage mapping.

The porcine hormone-sensitive lipase gene and its cDNA have been isolated and sequenced. Several putative regulatory sequences have been detected in the promotor region. The deduced amino acid sequence is 85% identical to the corresponding human, mouse and rat sequence. A search for polymorphisms revealed one intronic and one exonic polymorphism, the latter resulting in a conservative amino acid substitution. Linkage mapping located the LIPE gene close to the calcium release channel (CRC) locus on chromosome 6.

FISH on metaphase and interphase chromosomes demonstrates the physical order of the genes for GPI, CRC, and LIPE in pigs.

The porcine genes for glucose phosphate isomerase (GPI), calcium release channel (CRC), and hormone-sensitive lipase (LIPE) map to the long arm of chromosome 6. Earlier studies, using fluorescence in situ hybridization (FISH), mapped the three loci to the same band, viz., 6q12. To ascertain the relative order of the three genes, we first conducted three double-color FISH experiments, cohybridizing two of the probes at a time. These experiments demonstrated that the gene order was cen-GPI-CRC-LIPE. This order was confirmed by cohybridizing the three probes to both metaphase and interphase chromosomes.

Precise localization of the genes for glucose phosphate isomerase (GPI), calcium release channel (CRC), hormone-sensitive lipase (LIPE), and growth hormone (GH) in pigs, using nonradioactive in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied, using genomic DNA clones, to precisely localize the genes for GPI, CRC, LIPE, and GH on pig chromosomes. The porcine CRC gene was localized to band 6q12 using both genomic and cDNA clones. The GPI and LIPE genes, which are closely linked to the CRC gene, were also mapped to the same band (6q12), using genomic lambda clones. The mapping data are a refinement of earlier findings, wherein radioactive in situ hybridization was used and the assignments included both the short and long arms. Results of the present study clearly exclude the short arm as the location for the three genes. Further, using a genomic cosmid clone, the GH gene was mapped to band 12p14. Compared to the earlier assignments, which included almost the entire short arm of the chromosome due to the use of radioactive in situ hybridization, the present FISH findings provide a band-specific localization for the gene. A modified, simpler version of the posthybridization trypsin/EDTA banding method is also presented.

Localization of the ceruloplasmin (CP) gene to the q32-q33 bands of chromosome 13 in pigs by in situ hybridization.

Ceruloplasmin (CP) is a copper-binding protein in vertebrate plasma. In the present study, the porcine ceruloplasmin gene was localized to the 13q32-q33 bands by in situ hybridization, using a human CP cDNA probe. This confirmed the localization of the porcine linkage group V to chromosome 13. The CP locus is closely linked to the transferrin locus in pigs. Their relative physical order on chromosome 13 is discussed. Comparisons are made with the order of these two loci in other mammalian species.

Characterization of a porcine glucosephosphate isomerase-processed pseudogene at chromosome 1q1.6-1.7.

A porcine glucosephosphate isomerase-processed pseudogene has been isolated and sequenced. The pseudogene has several base substitutions as well as an insertion and deletions, and is 83% homologous to the corresponding cDNA. It contains an intervening sequence of 565 bp, is truncated at the 3' end, and is flanked by direct repeats of seven nucleotides. Fluorescent in situ hybridization to porcine metaphase chromosomes localized the processed pseudogene to Chromosome (Chr) 1q1.6-1.7. A (GT)14(AT)15 microsatellite was detected close to the processed pseudogene.

Genetic recombination between malignant hyperthermia and calcium release channel in skeletal muscle.

Absolute linkage between the gene, on chromosome 19, for the calcium release channel (CRC) of the sarcoplasmic reticulum in skeletal muscle and malignant hyperthermia (MH) has been reported by other workers. In the present study of three Swedish Families informative with respect to this linkage relationship, definite recombinants were found in two families. We conclude that mutations in other genes than the CRC gene can cause the clinical picture of MH. Accordingly, MH appears to be genetically heterogeneous.

Characterization of a porcine variable number tandem repeat sequence specific for the glucosephosphate isomerase locus.

A variable number of tandem repeat from a porcine glucosephosphate isomerase intron has been isolated and sequenced. The repeat has a unit size of 39 bp, is highly conserved and is present in at least 14 copies. Flanking sequences show a sequence periodicity of 53-54 bp and some sequence homology to the 39 bp repeat. A considerable part of the genomic DNA has been lost during subcloning and is considered to be deletion prone or refractory to propagation in E. coli. The tandem repeat is locus specific and detects at least six alleles in BamHI digested porcine DNA. No homology to other tandem repeat sequences has been found.

Localization of the citrate synthase (CS) gene to the p12-p13 bands of chromosome 5 in pigs by in situ hybridization.

Citrate synthase (CS) is a key enzyme of the Krebs tricarboxylic acid cycle. A 1.4 kb porcine CS cDNA probe was used to chromosomally localize the CS gene in pigs by in situ hybridization. Two in situ hybridization experiments were conducted. Although the first experiment indicated a distinct signal on the 5p12-p13 bands, a secondary signal was observed on the 13q24-q32 bands. Hence, a second in situ hybridization experiment was conducted at higher stringency. The results demonstrated a consistent signal on the 5p12-p13 bands, and the signal on chromosome 13 was scattered with no prominent secondary peak. The CS gene was therefore assigned to the p12-p13 bands of chromosome 5 in pigs.

Chromosomal localization of the hormone sensitive lipase (LIPE) and insulin receptor (INSR) genes in pigs.

Using rat hormone sensitive lipase (LIPE) and human insulin receptor (INSR) cDNA probes, the LIPE gene was assigned to chromosome 6p11-q21 and the INSR gene to chromosome 2q11-q21 in pigs by in situ hybridization. In humans, these two genes are located on the q and p arms of chromosome 19, respectively. The present results provide the first in situ hybridization mapping data for porcine chromosome 2.