Polymorphism of pig serum alpha-protease inhibitor-3 (PI3) and assignment of the locus to the Pi1, Po1A, Po1B, Pi2, Igh linkage group

Polymorphism of an alpha-protease inhibitor, PI3, in pig serum samples was detected using 2D agarose gel (pH 5.4)--polyacrylamide gel (pH 9.0) electrophoresis. Evidence was obtained that the five variants observed (A, B1, B2, C and D) are under genetic control by codominant alleles (Pi3A, Pi3B1, Pi3B2, Pi3C and Pi3D) at one autosomal locus. Variants A, B1, B2 and C inhibited chymotrypsin; there was no appreciable inhibition of trypsin and papain. Variant D did not inhibit chymotrypsin, and therefore its classification as a PI3 variant was put in question. PI3 typing was not possible in about 50% of the studied pigs since in those cases the PI3 variants were either too weak or absent. On the basis of backcross matings and haplotyping in complete families for protease inhibitor loci Pi1, Po1A, Pi2 and Pi3 it was proved that the Pi3 locus belongs to the protease inhibitor gene cluster, and the position of the locus in the linkage group was proposed as being Pi1-Po1A-(Po1B)-Pi3-Pi2-(Igh1, Igh2, Igh3, Igh4).     

Parentage testing of dogs using variants of blood proteins: description of five new plasma protein polymorphisms

Plasma samples of 1126 dogs belonging to 21 different European breeds were analysed by two-dimensional agarose gel (pH 5.4 or pH 8.6)--horizontal polyacrylamide gel (pH 9.0) electrophoresis, followed by general-protein staining of gels. Genetic polymorphism was detected for five as yet unidentified proteins designated pretransferrin-1 and -2 (Prt1 and Prt2) and postalbumin-1, -2 and -3 (Pa1, Pa2 and Pa3). Three alleles are reported in the Prt1 and Prt2 systems and two alleles in the Prt2, Pa1 and Pa3 systems. While Prt2 variation was observed only in the cocker spaniel breed, each of the other four proteins showed a high degree of polymorphism in most of the breeds studied. Pa3 fractions were clearly observed only in samples stored at -20 degrees C for more than 2 years. Prt1, Pa1 and Pa2 proteins are additional useful markers for parentage control in dogs. This study corroborated previously published results that dog plasma proteins, in general, show considerably more polymorphism than that reported for haemoglobin and for several blood cell enzymes in this species.     

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electrophoresis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles (Po-2F and Po-2s) at a single locus. The frequency of Po-2F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phosphohexose isomerase locus (Phi). A recombination frequency of 3.2% was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven recombinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd.     

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electropho-resis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles ( Po-2^F and Po-2^S ) at a single locus. The frequency of Po-2^F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phospho-hexose isomerase locus ( Phi ). A recombination frequency of 3.2 % was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven re-combinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd .     

Pig plasma protease inhibitor gene complex: isolation and partial characterization of three inhibitors

1. Pig plasma alpha-protease inhibitors (protease inhibitor-1, PI1; protease inhibitor-2, PI2; postalbumin-1A, PO1A; postalbumin-1B, PO1B), all encoded by one gene complex (gene cluster), were isolated by rivanol-ammonium sulphate fractionation and double-one dimensional IPG-PAGE. The proteins were recovered from the polyacrylamide gel by a combination of electrophoresis and isoelectric focusing. 2. Molecular wt estimated by SDS-PAGE under reducing conditions was 63,000 each for PI1 and PI2 and 60,000 each for PO1A and PO1B. The two main components of a genetic variant of PI2 differed in mol. wt by approx. 1000. 3. PO1A, PO1B and PI2 were shown to be glycoproteins. The major component of both PO1A and PO1B contained about 15% carbohydrate and the two components of PI2 had about 24 per cent and 21 per cent carbohydrate, respectively. 4. Neuraminidase treatment showed that the main component of PO1A had 8 sialic acid residues and fast and slow components of PI2 had respectively 11 and 10 residues. 5. Amino acid compositions of PO1A, PO1B and PI2 were very similar to one another, indicating that the genes for these proteins have evolved by regional duplications of a common ancestral gene. 6. The results (mol. wt, amino acid and carbohydrate compositions) confirm that pig PI2 is homologous to human plasma alpha 1-antichymotrypsin.     

Polymorphic plasma postalbumins of some domestic animals (pig PO2, horse Xk and dog Pa proteins) identified as homologous to human plasma α1B-glycoprotein

Pig, horse and dog plasma proteins, separated by horizontal polyacrylamide gel electrophoresis (pH 9.0) and electrophoretically transferred to nitrocellulose membranes, were tested for cross-reaction with antiserum to human plasma alpha 1B-glycoprotein (alpha 1B). The results showed that one previously reported polymorphic plasma postalbumin in each of these species (pig PO2, horse Xk and dog Pa protein) was homologous to human plasma alpha 1B. In the light of the previously known genetic linkages in these species, this implied: (1) alpha 1B gene is close linked to Phi, Pgd and Hal (halothane sensitivity locus) loci in pigs; and (2) alpha 1B gene is linked to ME1 and Phi loci in horses. This suggested that the alpha 1B gene may also be found to be closely linked to gene(s) controlling susceptibility to malignant hyperthermia in humans and other mammals.     

Pig plasma postalbumin-2 (alpha 1B-glycoprotein): isolation, partial characterization and immunological cross-reactivity with other mammalian sera

1. Components of pig plasma postalbumin-2 (PO2) protein, after rivanol-ammonium sulphate fractionation of plasma, were separated from other proteins by an easy and rapid method of horizontal double-one dimensional IPG-PAGE. The protein was recovered from polyacrylamide gel by combination of electrophoresis and isoelectric focusing. 2. The mol. wt of PO2 was estimated to be 68,000, using SDS-PAGE. 3. Amino acid and carbohydrate compositions of PO2 were very similar to those of human plasma alpha 1B-glycoprotein (alpha 1B), confirming that PO2 is the porcine homologue of human alpha 1B. 4. Neuraminidase treatment resulted in a decrease of electrophoretic migration velocity of all four studied components of PO2. 5. Homologous proteins to pig PO2 (alpha 1B) were observed, not only in human plasma but also in plasma of dog, horse and rabbit, by immunoblotting.     

Extensive genetic polymorphism of four plasma alpha-protease inhibitors in pigs and evidence for tight linkage between the structural loci of these inhibitors

Two-dimensional horizontal gel electrophoresis of pig plasma samples (under non-denaturing conditions) using Immobiline pH gradient gels 4.0-6.0 for the first dimension separation, resulted in clear resolution of the variants of four different alpha-protease inhibitors (protease inhibitor -1 and -2, PI1 and PI2; post-albumin -1A and -1B, PO1A and PO1B). All these variants were readily visualized by general protein staining. About 900 families each of Swedish Landrace (SL) and Yorkshire (SY) breeds were studied. The extensive inheritance data, including the recombinants encountered, indicated that each of these four inhibitors is controlled by a separate, autosomal locus and that the four loci are tightly linked (spread over a distance of 1-1.5 cM) with the order as Pi1-Po1A-Po1B-Pi2. The alleles observed were two of Pi1, 14 of Po1A, 11 of Po1B and 8 of Pi2. About 40 haplotypes were observed in each of the two breeds. The allele frequencies at Po1A, Po1B and Pi2 loci were remarkably different in the two breeds; the alleles at these three loci showed a very strong linkage disequilibrium (0.8-1.0). The females showed much higher recombination frequencies than the males in the Po1A-Pi2 interval, suggesting that gene conversion-like events may be occurring at these loci. This linkage in pigs and similar ones comprising some plasma alpha-protease inhibitor genes in humans and in rodents, reported recently in the literature, indicate evolutionary conservation of a homologous linkage group in these species.     

Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

The serum protease inhibitor linkage group in Belgian pig breeds

Genetic variants of pig serum alpha-protease inhibitors (protease inhibitors-1 and -2, PI1 and PI2; postalbumin-1A and -1B, PO1A and PO1B) were studied by 2-D electrophoresis of serum samples. The inheritance data confirmed the close linkage between the loci of these inhibitors. The order between these loci was indicated as Pi1-Po1A-Po1B-Pi2 and these were spread over a distance of about 1 cM. Very strong linkage disequilibrium was observed between the alleles at these loci. The two breeds studied (Belgian Landrace and Piétrain) showed very different allele and haplotype frequencies. Both breeds showed extensive polymorphism at Po1A and Pi2 loci.     

Plasma alpha 1B-glycoprotein allele frequencies in Finns and Swedish Lapps: evidence for a new alpha 1B allele

A new allele (A1B*5) of human plasma alpha 1B-glycoprotein (alpha 1B) was reported. alpha 1B phenotyping was done by two-dimensional agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis followed by protein staining. The alpha 1B phenotypes 1-1, 1-2, 1-5 and 2-2 were observed in Finns and phenotypes 1-1, 1-2, 1-5 and 2-5 in Swedish Lapps. The respective frequencies of A1B*1, A1B*2 and A1B*5 were 0.9575, 0.0350, 0.0075 in Finns and 0.8922, 0.0653, 0.0425 in Swedish Lapps. The Swedish Lapps showed a higher degree of alpha 1B polymorphism (polymorphism information content = 0.19) than other Caucasian populations that have been studied.     

Preliminary report on two new plasma protein polymorphisms in the goose (Anser anser)

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of plasma proteins in the goose was done. This method resulted in improved separation of proteins in the pre-transferrin and transferrin regions. Preliminary evidence was presented for the existence of two new plasma protein polymorphisms.     

Human alpha 1-antitrypsin genetic polymorphism: PI N subtypes

Three new genetic variants (PI types) of alpha 1-antitrypsin are described. They have been compared to previously described phenotypes by several techniques including narrow pH range isoelectric focusing in ultrathin polyacrylamide gels. In this system, the relevant alpha 1-antitrypsin gel bands, identified by crossed immunoelectrophoresis, focused between PI M2, the most cathodal PI M subtype, and PI P BUD, the most anodal PI P subtype. They were therefore considered to be PI N subtypes. Two of them, PI N GRO and PI N YER, could not be separated by isoelectric focusing, but gave a different pattern in agarose gel electrophoresis. None of the new alleles seemed to be associated with disease. The high resolving power of isoelectric focusing is emphasized with respect to the information it may provide concerning amino acid substitutions, while the use of other techniques proved to be of utmost importance in the differentiation of other variants showing similar isoelectric points.     

Two-dimensional electrophoresis of the plasma proteins of alpacas and llamas: genetic polymorphism of alpha 1B-glycoprotein and three other proteins

Plasma samples of alpacas and llamas were analysed by a simple method of two-dimensional (2-D) agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis, followed by general protein staining of gels. Genetic polymorphism in both species is described for alpha 1B-glycoprotein (alpha 1B) and three other unidentified proteins designated prealbumin (Pr), postalbumin 1 and 2 (Pa1 and Pa2). alpha 1B was identified by cross-reactivity with antisera for human and pig alpha 1B. Altogether, two alleles of Pr, two of Pa1, five of alpha 1B and three of Pa2 are described. Most of the alleles were present in alpacas and llamas. Alpacas showed a high degree of polymorphism at all four loci. Llamas showed considerable polymorphism at only the Pa1 and Pa2 loci. The theoretical probability of exclusion (PE) of an incorrectly assigned parent was estimated to be about 80% in each species by typing for the six polymorphic plasma proteins reported so far in these species. The given method of 2-D electrophoresis revealed no fixed differences in protein mobilities that discriminate between llamas and alpacas.     

Identification of human red cell glutamate-pyruvate transaminase (GPT) phenotypes by isoelectric focusing.

Isoenzymes of human red cell glutamate-pyruvate transaminase (GPT) were resolved by isoelectric focusing (IEF) of hemolysates in polyacrylamide gels at pH 5.0-7.0. The bands of enzyme activity required both alpha-ketoglutarate and L-alanine in the staining mixture for visualization, indicating that the bands were not lactate dehydrogenase or glutamate dehydrogenase. Phenotyping of 41 individuals by IEF, including types GPT 1, 2A, 1-2A, 1-2B, and 2A-2B, agreed with the typing results obtained by electrophoresis in starch gels and in polyacrylamide gels at acid and alkaline pH. Analysis of one kindred demonstrated autosomal codominant transmission of the rare GPT*2B gene through 3 generations. IEF facilitates phenotyping by permitting identification of the GPT types on a single gel with a considerable reduction in time and cost. Although no new variants were found in this investigation, IEF may be more powerful for the recognition of presently undetected variants of GPT.     

Genetic polymorphism of horse serum protein 3 (SP3)

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles (D, F, I, S). Evidence was provided that the F allele can be further divided into two alleles (F1 and F2); the mobilities of F1 and F2 variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Two-dimensional electrophoresis of the plasma proteins of alpacas and llamas: genetic polymorphism of α1B-glycoprotein and three other proteins

Summary. Plasma samples of alpacas and llamas were analysed by a simple method of two-dimensional (2-D) agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis, followed by general protein staining of gels. Genetic polymorphism in both species is described for α 1 B-glycoprotein (α 1 B) and three other unidentified proteins designated prealbumin (Pr), postalbumin 1 and 2 (Pal and Pa2). α 1 B was identified by cross-reactivity with antisera for human and pig α 1 B. Altogether, two alleles of Pr, two of Pa1, five of α 1 B and three of Pa2 are described. Most of the alleles were present in alpacas and llamas. Alpacas showed a high degree of polymorphism at all four loci. Llamas showed considerable polymorphism at only the Pa1 and Pa2 loci. The theoretical probability of exclusion (P e ) of an incorrectly assigned parent was estimated to be about 80% in each species by typing for the six polymorphic plasma proteins reported so far in these species. The given method of 2-D electrophoresis revealed no fixed differences in protein mobilities that discriminate between llamas and alpacas.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.