Genetic polymorphism and close linkage of two alpha 1-protease inhibitors in horse serum.

Two-dimensional electrophoretic analysis of horse serum proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many alpha-globulins. Two groups of alpha 1-globulins, designated Pi1 and Pi2, were found to be protease inhibitors. Preliminary studies indicated that Pi1 and Pi2 proteins differed from each other in molecular weight and in protease inhibiting spectra. Extensive polymorphism was observed for both these proteins. Family data supported the hypothesis that Pi1 and Pi2 types were controlled by autosomal codominant alleles. For both Pi1 and Pi2 systems, most of the homozygous types showed two fractions each while the heterozygous types had 4 fractions. Six Pi1 and five Pi2 alleles were observed in two breeds of Swedish horses. Complete genetic linkage was observed for Pi1 and Pi2 loci as no recombinant type was observed in 40 informative matings studied.     

Two-dimensional gel electrophoresis of sheep plasma proteins: Genetic polymorphism of an ai-protease inhibitor and a post-transferrin

Two-dimensional electrophoretic analysis of sheep plasma proteins was performed by a first-dimension separation in agarose gel (pH 5.0) followed by a second-dimension one in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many α- and β-globulins. Two groups of α₁-globulins, designated Pi-1 and Pi-2, were found to be protease inhibitors. These two inhibitors differed from each other in protease inhibitory spectra. Genetic polymorphism was observed for the Pi-2 protein and another unidentified protein, tentatively designated as post-transferrin (Ptf). Family data supported the hypothesis that Pi-2 and Ptf types were controlled by codominant, autosomal alleles. Three Pi-2 alleles and two Ptf alleles were observed in one population of the Gotland breed of sheep. The analysis of data from 50 informative matings showed no evidence of genetic linkage between the Pi-2, Ptf and transferrin (Tf) loci in the population of sheep studied.     

Polymorphic serum prealbumin (Pa) of pig, identified as an a!-protease inhibitor

Pig serum proteins were analysed by horizontal polyacrylamide gel electrophoresis, with a discontinuous buffer system (pH 9.0). A 12 % acrylamide concentration in the separation gel was used. Each of the two prealbumin (Pa) alleles gave rise to two closely migrating fractions. The polymorhic Pa was identified as an a,-protease inhibitor as the Pa fractions inhibited the esterolytic activity of both bovine trypsin and chymotrypsin. Therefore, it has been proposed that the locus symbol for this prealbumin be changed to Pi-1. The protease inhibitory spectra and electrophoretic mobility of the Pa (Pi-1) fractions suggested that this protein was probably the same as the pig serum a,-protease inhibitor described in some earlier studies and that it corresponds to human serum a,-protease inhibitor (Pi).     

Two-dimensional gel electrophoresis of cattle plasma proteins: Genetic polymorphism of an α1-protease inhibitor

Two-dimensional electrophoretic analysis of cattle plasma proteins was done by a first dimension separation in agarose gel (pH 5.0), followed by a second dimension in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many α- and β-globulins. Three groups of a-globulins, designated Pi-1, Pi-2 and Pi-3, were found to inhibit the estero-lytic activity of bovine trypsin and bovine chymotrypsin. Pi-2 showed appreciable inhibition only for trypsin and genetic polymorphism was observed for this protein. Family data supported the hypothesis that the three Pi-2 types observed were controlled by two codominant, autosomal alleles. The occurrence of a third Pi-2 allele was also postulated in some animals studied. The frequency of the most common allele, Pi-2^s, ranged from 0.5-0.8 in the different breeds of cattle studied (Swedish Red and White, Friesian, Jersey, Charolais and Simmental). The post-transferrins Ptf-1 and Ptf-2 in cattle plasma were shown to be two different genetic systems.     

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

Summary. 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 α-protease inhibitors (protease inhibitor -1 and -2, P11 and P12; postalbumin -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 Pil-Po1A-Po1B-Pi2 . The alleles observed were two of Pil, 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 α-protease inhibitor genes in humans and in rodents, reported recently in the literature, indicate evolutionary conservation of a homologous linkage group in these species.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 2. Protease inhibition

The protease inhibitory spectra of the eight homozygous Thoroughbred Pi types against trypsin, elastase and chymotrypsin have been determined. The α1-protease inhibitor proteins exhibit three classes of inhibitory specificity towards these enzymes. The Pi types F, I, N and U exhibit class I (trypsin, elastase and chymotrypsin) and class II (trypsin and elastase) types of inhibition and fit Juneja et al.s (1979) classification of two separate genetic systems Pi 1 and Pi 2 based on differences in the inhibitory spectra against trypsin and chymotrypsin. The remaining four Pi types are exceptions to Juneja et al.s (1979) classification. Types G, L, S₁ and S₂ possess class I but not class II proteins. A third class of proteins (class III) which exclusively inhibit chymotrypsin was detected in all eight protease inhibitor types. Type G is well represented by class III proteins because two of the three major proteins of the ISO-DALT pattern inhibit only chymotrypsin and is thus an exception to Juneja et al.s (1979) classification.     

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.     

Protease inhibitor system in horses: classification and detection of a new allele

A method of horizontal thin layer polyacrylamide gel electrophoresis at acid pH has been developed for the separation of the prealbumins in equine plasma. Using this method, it has been possible to split the S allele into two, S₁ and S₂, bringing the total number of prealbumin alleles in Thoroughbred horses to eight. The gene frequencies of these eight alleles in Australian Thoroughbreds are presented. All eight prealbumin types exhibit antiprotease activity and therefore, it is suggested that the name prealbumin (Pr) should be abandoned in favour of protease inhibitor (Pi) although at this stage it is not known whether this incorporates the Pi1 and Pi2 described by Juneja et al. (1979).     

The plasma protease inhibitor system (Pi) of Standardbred horses

The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles (PiF, G, I, L, N, S1, S2, U), another 10 alleles, designated PiH, J, K, O, P, Q, R, V, X, Z, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pi1, Pi2 classification of Juneja et al. (1979) were found. Limited family data demonstrated the genetic nature of the 'new' variants and confirmed the allelic inheritance of the 'new' Pi variants.     

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).     

Characterisation of the alpha 1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74-4.43 and have molecular weights ranging from 55 000-72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S1 and S2 which show partial identity. The results do not necessarily support Juneja et al.'s (1979) contention of two closely linked alpha 1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.     

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.     

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.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74–4.43 and have molecular weights ranging from 55 000–72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S₁ and S₂ which show partial identity. The results do not necessarily support. Juneja et al.s (1979) contention of two closely linked α1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.     

A new genetic variant Z2 in the Pi system of horses

A new genetic variant in the horse Pi system, designated Z2, was reported in Polish Arabs by using two-dimensional agarose polyacrylamide gel electrophoresis. The frequencies of Pi alleles F, G, L, L2, N, S, U, W, Z and Z2 were found to be 0.036, 0.005, 0.171, 0.013, 0.008, 0.237, 0.416, 0.003, 0.107 and 0.004 respectively.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

The plasma protease inhibitor system (Pi) of Standardbred horses

The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles ( Pi^{F, g, i, L, N, S1, S2, u)} , another 10 alleles, designated PiH, j, k, ^o, ^p, ^q , ^r, ^v, ^{x, z}, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pil, Pi2 classification of Juneja et al. (1979) were found. Limited family data demonstrated the genetic nature of the 'new' variants and confirmed the allelic inheritance of the 'new' Pi variants.     

Genetic polymorphism of plasma α1B-glycoprotein and transferrin in arctic and silver foxes

Plasma samples of 235 foxes from 38 complete families (14 of arctic foxes, 21 of silver foxes and 3 with arctic x silver fox hybrid offspring) were analysed by one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 followed by general-protein staining of gels. A major postalbumin of fox plasma was identified as alpha 1B-glycoprotein (alpha 1B) by using immunoblotting with antiser m specific to human or pig plasma alpha 1B. Four codominant, autosomal alleles of alpha 1B were found in arctic foxes. Two transferrin (TF) alleles (TfF, TfS) were observed in arctic foxes and two (TfD, Tff) in silver foxes; the TF F type of both of the fox species showed identical electrophoretic mobilities. The arctic foxes showed a high degree of polymorphism for both TF and alpha 1B. The silver foxes showed a scarce polymorphism of TF and were monomorphic for alpha 1B. The arctic fox, silver fox and their hybrids could be clearly differentiated from one another by their plasma protein patterns obtained by the PAGE method.     

Genetic polymorphism of horse serum protein 3 (SP3)

Summary. 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 ( F ₁ and F ₂); 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.     

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.