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.

Effect of ethanol metabolism on initiation of protein synthesis in rat hepatocytes.

Rat liver parenchymal cells were incubated in the presence and absence of ethanol (80 mM). Polysomes were isolated and analysed on sucrose gradients. Ethanol was shown to inhibit the incorporation of 14C-valine into proteins, result in a shift in the distribution of polysomes towards smaller sizes, inhibit the formation of 40S initiation complexes, and diminish the concentration of glucose-6-phosphate in the hepatocytes. Addition of 4-methylpyrazole (0.5 mM) partially prevented the inhibition of protein synthesis and completely restored the polysomal distribution. It is concluded that ethanol inhibits protein synthesis partly by a mechanism linked to ethanol metabolism. This effect takes place at the level of initiation and may be mediated by a reduced gluconeogenesis.

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.

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.

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.

Localization of the calcium release channel gene in cattle and horse by in situ hybridization: evidence of a conserved synteny with glucose phosphate isomerase.

In situ hybridization techniques were used to localize regionally the calcium release channel (CRC) gene on cattle and horse chromosomes, using a porcine CRC cDNA probe. In cattle, the hybridization signal peaked on the 18q23-q26 bands and in horse on the 10pter region. Previous studies have shown that the glucose phosphate isomerase (GPI) gene localizes at the same site in both species, indicating that the two loci are syntenic. As CRC and GPI are syntenic in human, pig and mouse, the present results in cattle and horse represent another example of synteny conservation in the evolution of mammalian chromosomes.

Isolation and sequencing of porcine lipoprotein lipase cDNA and its use in multiallelic restriction fragment length polymorphism detection.

Porcine lipoprotein lipase (LPL) cDNA has been cloned and sequenced. The deduced amino acid sequence shows a high degree of identity to LPL from other species, and contains the Ser/His/Asp triade characteristic of serine proteases and esterases. A repetitive element is present in the 3'-untranslated region of the cDNA. A partial cDNA covering the coding region of LPL detects three restriction fragment length polymorphisms with HindIII. This represents the first marker assigned to porcine chromosome 14.

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.

Chromosomal localization of the glucose phosphate isomerase (GPI) gene in cattle, sheep and goat by in situ hybridization--chromosomal banding homology versus molecular conservation in Bovidae.

A porcine genomic glucose phosphate isomerase (GPI) DNA probe was used for in situ hybridization with metaphase chromosomes in cattle, sheep and goat. The probe gave distinct signals on the q22----proximal part of the q24 segment of chromosome 18, 14 and 18 in cattle, sheep and goat, respectively, indicating the location of GPI gene. The three species belong to the family Bovidae and have high resemblance in chromosome banding patterns. The localization of the GPI locus to the same site on chromosomes with almost similar banding patterns suggests high degree of homology at these sites in the three species. Correlation between banding homologies and possible similarities at the molecular level is discussed.

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.

A partial cDNA clone for porcine glucosephosphate isomerase: isolation, characterization and use in detection of restriction fragment length polymorphisms.

A cDNA library for porcine skeletal muscle was established in the vector pBR322. The library was screened with an oligonucleotide probe coding for a hexapeptide from glucosephosphate isomerase (Gpi). A positive clone with an insert of about 450 bp and restriction sites for PstI, BamHI and PvuII was isolated. A 362-bp PstI fragment was sequenced and shown to contain the codons for the hexapeptide as well as the remaining 29 amino acids of this Gpi peptide. The PstI fragment was used to probe pig genomic DNA. The restriction enzymes PvuII and SacI detected a set of polymorphisms with five bands, behaving as a set of insertion/deletion polymorphisms.

DNA sequence of the skeletal muscle calcium release channel cDNA and verification of the Arg615----Cys615 mutation, associated with porcine malignant hyperthermia, in Norwegian landrace pigs.

Porcine calcium release channel (CRC) cDNA from skeletal muscle has been cloned and sequenced. The deduced amino acid sequence showed 97% identity to the corresponding rabbit and human sequences. Using oligonucleotide primers based on the nucleotide sequence, CRC cDNA fragments from seven pigs representing HALNN, HALNn and HALnn genotypes have been amplified. Sequencing and restriction digestion of the amplified cDNA confirm that the reported C----T mutation, which gives rise to Arg615----Cys615 change in the calcium release channel, is associated with the halothane sensitive allele in Norwegian Landrace pigs. The mutation may alter the reactivity of a neighbouring serine residue which is potentially phosphorylated.

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.

Assignment of the bovine immunoglobulin gamma heavy chain (IGHG) gene to chromosome 21q24 by in situ hybridization.

By the use of in situ hybridization, the bovine immunoglobulin gamma heavy chain (IGHG) gene was assigned to chromosome 21q24. Fifteen percent of the total grains were scored on chromosome 21, with about 65 percent of these grains located on the q24 band. The present results confirm a previous synteny study and provide the precise chromosomal localization of this gene in the cattle genome.

Multiple restriction fragment length polymorphisms in the porcine calcium release channel gene (CRC): assignment to the halothane (HAL) linkage group.

Two cDNA probes for the porcine calcium release channel gene (CRC) were used in restriction fragment length polymorphism (RFLP) analysis in an attempt to develop genetic markers linked to the malignant hyperthermia (stress susceptibility) gene (HAL). Three TaqI RFLPs, denoted CRC1-CRC3, each composed of two alleles, were detected. RFLPs were also detected with MspI and PvuII, but the MspI RFLP correlated completely with CRC3 in this material and the PvuII RFLP could not be scored reliably due to a minute size difference between the two allelic fragments. The autosomal codominant inheritance of these RFLP loci was confirmed by family analyses. Significant evidence for genetic linkage between the CRC1/CRC3 loci and the A1BG locus in the HAL linkage group confirmed a previous assignment of the CRC gene to chromosome 6 in the pig.

Isolation, characterization and chromosomal assignment of a partial cDNA for porcine 6-phosphogluconate dehydrogenase.

A partial cDNA for 6-phosphogluconate dehydrogenase (PGD, EC 1.1.1.44) was isolated from a porcine liver cDNA library using a rat PGD cDNA. The identity of the PGD cDNA was confirmed by DNA sequencing and comparison of the amino acid sequence with the corresponding ovine sequence. The PGD cDNA was assigned to 6q2.5-2.7 by in situ hybridization.