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.     

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 in the blood of llamas, Llama glama, and alpacas, Llama pacos

Blood samples of llamas and alpacas were typed using haemolytic, electrophoretic and isoelectric focusing procedures to assay polymorphism at 13 loci. Blood group variation was assessed using six antibody specificities produced by allo- and heteroimmunizations. Two red cell factors (A and B) behave as autosomal, codominant alleles at a closed A locus. The other four factors (C, D, E and F) behave as autosomal, dominant traits. Biochemical variation was found for red cell enzymes catalase, phosphogluconate dehydrogenase, glucose phosphate isomerase and for plasma proteins transferrin and post-albumin. No variants were found for haemoglobin, phosphoglucomutase and albumin. Estimates of probability of exclusion were 0.883 for llamas and 0.681 for alpacas, which are adequate initial levels of efficacy for purposes of parentage verification. Preliminary estimate of Nei's genetic distance measure (D) suggests that llamas and alpacas are more likely related as subspecies than as separate species.     

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.     

Two-dimensional electrophoresis of horse serum proteins: genetic polymorphism of ceruloplasmin and two other serum proteins

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum proteins revealed genetic polymorphism of ceruloplasmin (Cp) and two unidentified serum proteins tentatively designated serum protein 1 (SP1) and serum protein 2 (SP2). Family data were consistent with the hypothesis that the observed Cp and SP1 phenotypes were each controlled by two co-dominant, autosornal alleles. The three common SP2 phenotypes were shown to be controlled by two codominant, autosomal alleles. Population data and limited family data indicated the occurrence of two additional SP2 alleles. Altogether more than 600 horses representing 13 different breeds were typed for Cp, SP1 and SP2, and allele frequency estimates were calculated. SP2 was highly polymorphic in all breeds studied whereas SP1 and Cp showed quite low degrees of polymorphism. SP1 polymorphism was observed in seven breeds while Cp polymorphism was observed only in the Icelandic toelter horse breed.     

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.     

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.     

Low genic variation in male-reproductive-tract proteins of Drosophila melanogaster and D. simulans

We report results, using two-dimensional gel electrophoresis (2DE), of natural population surveys of allelic variation in approximately 300 male-reproductive-tract polypeptides in both Drosophila melanogaster and its sibling species, D. simulans. Despite our efforts to maximize operational sensitivity of our 2DE gels to polymorphism, variation estimates in both species were low (proportion of polymorphic loci [P] = 9%, and average heterozygosity [H] = 1%-3%), compared with those by one-dimensional gel electrophoresis (1DE) (P = 29%-55%; H = 8%-19%) in the same populations. However, H of polymorphic loci was very similar for 2DE and 1DE proteins; and for 17 of a total of 54 polymorphic proteins, 2DE detected three or four distinct alleles. The results suggest that the differing levels of variability widely seen with 1DE and 2DE are real and reflect differing intensities of functional constraint between different classes of structural loci. However, the alternative possibility remains that 2DE has a greater between-locus unevenness of variant detection sensitivity than does 1DE.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.     

Genetic control of protein synthesis in white lupine (Lupinus albus L.) seeds

Using polyacrylamide gel electrophoresis in the glycine-acetic acid system (pH 3.2), variants of proteins of white-lupine seeds were revealed. The study of conglutin polymorphism in the culture of the autogamous population F_→∞ (var. Dega) revealed two loci, Con A and Con B, which control protein synthesis. The loci were situated in the same linkage group within a distance of 11.48 ± 3.4% of recombination. Natural selection in favor of genotypes that contain Con A1 and Con B2 alleles is proposed. It is established that conglutins A and B (CON A and CON B) contain cysteine residues, which form intermolecular disulfide bonds between peptides.     

Genetic polymorphism and close linkage of two α1-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 α-globulins. Two groups of aj-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.     

猪几个血清蛋白质位点的等位基因频率在选育中的变化趋势

采用淀粉凝胶电泳测定了杜洛克猪的转铁蛋白(Tf)、前白蛋白(Pa)、血液结合素(Hpx)、铜兰蛋白(Cp)和淀粉酶(Am)多态性,统计和计算了表现型数和等位基因频率,并同1968年测得的基因频率进行了比较,揭示了杜洛克猪几个血清蛋白质位点的等位基因频率在长期选育中的变化趋势,即随着人们有目的地进行选育,这些血清蛋白质位点的等位基因数减少,基因频率向某个等位基因集中,形成优势等位基因。目前的优势等位基因是Tf^B、Pa^A、Hpx^3、Cp^B、Am^8。此外,将杜洛克猪的基因频率同湖南部分地方猪进行了比较,计算了它们的平均杂合度、遗传相似系数和遗传距离系数,并做了聚类分析。     

Genetic control of PI and GC variants in the American Mink

Genetic polymorphism of the serum α-protease inhibitor (PI) and group-specific component (GC) in minks was revealed using one-dimensional polyacrylamide gel electrophoresis and immunoblotting. Two codominant alleles were identified at each of the two loci. The data ruled out the possibility of any linkage between the PI, GC and the coat colour gene Crystal ( Cr ).     

Polymorphic plasma postalbumin (Po) of llamas and alpacas identified as Gc protein

Immunoblotting with antiserum specific to human Gc protein was used to identify Gc protein as the previously reported polymorphic plasma postalbumin (Po) of llamas and alpacas. This is the first report of Gc polymorphism in camelid species. One Gc variant appeared to be identical in llamas, alpacas, dromedaries and bactrian camels.     

Polymorphic plasma postalbumin (Po) of llamas and alpacas identified as Gc protein

Summary. Immunoblotting with antiserum specific to human Gc protein was used to identify Gc protein as the previously reported polymorphic plasma postalbumin (Po) of llamas and alpacas. This is the first report of Gc polymorphism in camelid species. One Gc variant appeared to be identical in llamas, alpacas, dromedaries and bactrian camels.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum.

Horizontal polyacrylamide gel electrophoreses, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, GcF and GcS, and three Pa alleles, Pa D, Pa F and Pa S, were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. GcF and PaF, ranged from 0.72-0.93 and from 0.58-0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the GcS fraction and thus were found unsuitable for Gc typing.     

Genetics of four plasma protein loci in Equus przewalskii: new alleles at the prealbumin, postalbumin and transferrin loci

This paper reports genetic variation at the prealbumin ( Pr ), postalbumin ( Pa ) and transferrin ( Tf ) loci in Equus przewalskii found using thin layer isoelectric focusing and an amphoteric separator. The method resolves all three loci plus serum esterase ( Es ) on a single gel, and typing of all four loci is readily achieved. In addition to the esterase alleles previously reported by Fisher & Scott (1979), five alleles were found at the Pr locus. three at the Pa locus and six at the Tf locus. Analysis of several mating types confirms inheritance is autosomal and codominant for all four loci.     

Genetics of four plasma protein loci in Equus przewalskii: new alleles at the prealbumin, postalbumin and transferrin loci

This paper reports genetic variation at the prealbumin (Pr), postalbumin (Pa) and transferrin (Tf) loci in Equus przewalskii found using thin layer isoelectric focusing and an amphoteric separator. The method resolves all three loci plus serum esterase (Es) on a single gel, and typing of all four loci is readily achieved. In addition to the esterase alleles previously reported by Fisher & Scott (1979), five alleles were found at the Pr locus, three at the Pa locus and six at the Tf locus. Analysis of several mating types confirms inheritance is autosomal and codominant for all four loci.     

Separation of allelic variants by two-dimensional electrophoresis.

The resolving power of two-dimensional (2-D) gel electrophoresis has been tested using 17 allele products at five loci. Standard O'Farrell gels could separate 13 of these isozymes. The addition of a second pH gradient made it possible to separate all but one of the variant proteins. These results indicate that 2-D gel electrophoresis can resolve more than 90% of variants originally detected by one-dimensional (1-D) electrophoresis. The implications of these results for the low estimates of average heterozygosity obtained in surveys using 2-D gel electrophoresis are discussed.     

Human salivary proline-rich (Pr) proteins: A posttranslational derivation of the phenotypes

The acidic proline-rich proteins (Pr) showing genetic polymorphism were purified from human parotid salivas by gel filtration and ion exchange chromatography. Molecular weight determinations, amino acid composition analyses, and polypeptide mapping experiments indicate that the Pr 3 protein is a fragment of the Pr 1 protein. Studies of a parotid saliva factor capable of converting Pr 1 to Pr 3 and Pr 2 to Pr 4 indicate that Pr 3 and Pr 4 are generated from Pr 1 and Pr 2, respectively. Evidence suggests that the converting factor is a protease capable of posttranslationally cleaving Pr 1 and Pr 2, the primary or derived products of alleles Pr1 and Pr2.