Horizontal polyacrylamide gradient gel electrophoresis for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle

A simple method of horizontal polyacrylamide gel electrophoresis was described for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle. A step gradient gel of 8, 4, 12 and 14 % acrylamide concentration was used. The method enabled the detection of a new protein polymorphism in the post-transferrin region. Two alleles were observed. The transferrin phenotypes involving D ₁ and D ₂ alleles were clearly separated. The resolution of the post-albumin fractions was also better than described by earlier methods.     

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.     

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.     

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.     

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 post-albumin of cattle and horse plasma identified as vitamin D binding protein (Gc protein)

Cattle and horse plasma samples of known post-albumin types were radiolabelled with ¹⁴C-vitamin D₃. These samples were then analysed by polyacrylamide gel electrophoresis, followed by autoradiography. The patterns observed were identical to those of post-albumin variants. The polymorphic post-albumin protein of cattle and horse was thus identified as the vitamin D binding protein and homologous to the Gc protein of human plasma.     

Simultaneous phenotyping of pig plasma α-protease inhibitors (PI1, PO1A, PO1B, PI2) and four other proteins (PO2, TF, CP, HPX) by a simple method of 2D horizontal electrophoresis

A simple and rapid method of 2D agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis was developed for the simultaneous phenotyping of pig plasma alpha-protease inhibitors (protease inhibitor-1 and -2; postalbumin-1A and -1B), postalbumin-2, transferrin, ceruloplasmin and haemopexin. These eight plasma proteins were clearly visible on gels stained with Coomassie Brilliant Blue G250. The 2D patterns and mobilities of several variants of alpha-protease inhibitors were described. By using two agarose gels and 10 polyacrylamide gels, 120 samples were easily analysed in a day. Since alpha-protease inhibitors show extensive polymorphism and as the gene for postalbumin-2 is closely linked to the halothane sensitivity locus Hal, this method is a useful tool for conducting parentage control and for predicting Hal genotypes of individual pigs.     

Transferrin gene frequencies of feral and domestic goats in New Zealand

Abstract

Blood plasma from three populations of feral goats (Capra hircus L.) and from five domestic breeds was analysed by polyacrylamide gel electrophoresis. The albumin locus was monomorphic in all samples. The macroglobulin locus was variable but lacked resolution on the gel. The transferrin (Tf) locus was polymorphic. The only two Tf alleles observed (Tf A and Tf B) did not vary from those described by other authors. Gene frequencies of the two Tf alleles showed that one feral population could be distinguished from the other two. Gene frequencies of domestic breeds were variable and showed no definitive pattern. Domestic breed type could not be recognised in the feral populations on the basis of transferrin analysis alone.     

Obtaining of pure transferrins D, M and R from equine serum and determination of transferrin level in relation to phenotype

By the method of precipitation with Rivanol (2-ethoxy-6,9-diaminoacridine lactate) and ammonium sulphate followed by chromatography on DEAE cellulose three genetic variants of transferrin were purified from equine serum: D, M and R. Their molecular mass determined in this study was 80 000, and it was identical for all three variants, which differed slightly in their amino acid composition. The protein level was determined in the serum of 535 two-year-old thoroughbred English horses by the method of rocket immunoelectrophoresis using antibodies obtained against three transferrins. The individual variability of the protein level in horses of the same phenotype was fairly high (variability index 9-15%). No differences were observed in the transferrin level related to sex. It was found that the presence of D, F and H alleles was connected with a higher serum transferrin level, while O and R alleles were connected with a lower level.     

Genetic variation in the genus Bufo I. An extreme degree of transferrin and albumin polymorphism in a population of the American toad (Bufo americanus)

A population of Bufo americanus from southwestern Ohio exhibited an extreme degree of transferrin and albumin polymorphism. One hundred and eighty-five individuals were collected from this population, and their transferrin and albumin phenotypes were determined by vertical acrylamide gel electrophoresis. Thirteen transferrin alleles were present in 36 phenotypes, and 11 albumin alleles were present in 29 phenotypes. A deficiency of heterozygotes occurred at both protein loci. The possible mechanisms responsible for the polymorphism and deficiency of heterozygotes are discussed.This work was supported by grants from the following agencies to the Senior author: NSF GB23601, Society of the Sigma Xi, and the American Philosophical Society. Miami University contributed the computer and audiovisual services.     

Transferrin and albumin polymorphisms in buffaloes from Brazil

Starch gel electrophoresis disclosed six transferrin phenotypes, explainable by three alleles (TF A, TF D, TF E), and three albumin phenotypes, determined by two alleles (ALB A, ALB B). Their prevalences suggest that the Brazilian populations have admixed river and swamp buffalo ancestry, the frequency of ALB A being much higher than those found in other regions.     

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.     

Studies of Transferrin Polymorphism in Swedish Cattle Using Agarose Gel Electrophoresis

The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with 59Fe. Six existing phenotypes based on the alleles TfA, TfD and TfE could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencies between the Swedish Red and White and the Swedish Friesian.     

Embryogenetic features of the serum protein composition of chicken blood

Protein composition of the blood serum in chick embryos of two breeds and in their hybrids has been investigated by polyacrylamide gel electrophoresis. Significant changes in the proportion of protein components of pre-albumin, albumin and post-albumin zones during prenatal development of chicks were observed. These changes are alleviated in hybrid embryos. The decrease in protein content of post-albumin zone, which contains foetoproteins, takes place to the end of incubation. This decrease is less significant in hybrid chicks as compared to that in the original hen breeds.     

Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C

In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid.     

Genetic Diversity among Different Clones of the Gynogenetic Silver Crucian Carp, Carassius auratus gibelio, Revealed by Transferrin and Isozyme Markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.     

Frequencies of transferrin types in various breeds of domestic dogs

Plasma transferrin (Tf) types in dogs were studied by a method of horizontal polyacrylamide gel electrophoresis with a 10 % separation gel and a discontinuous buffer system (Tris-citrate-borate, pH 9.0). Samples were analysed from 1127 dogs belonging to 60 different breeds. Tf^B and Tf^c alleles, each in considerably high frequency, were observed in most of the breeds. Tf^A with relatively lower frequency was observed in Beagle, Cocker Spaniel, Doberman Pinscher, German Shepherd, German Shorthaired Pointer, Old Danish Pointer and Poodle. Two rare Tf alleles, Tf^D and the other designated as Tf^E, were observed only in Cocker Spaniel and in Poodle respectively. Basenji (44 samples) and Small Münsterlander (17 samples) showed the highest frequency (0.97) for Tf^C allele while Carelian Bear Dog (19 samples) showed the highest frequency (0.97) for Tf^B allele. In the total material, the frequencies of Tf alleles A, B, C, D and E were found to be 0.0182, 0.5075, 0.4716, 0.0009 and 0.0018 respectively. By using the observed and expected numbers of Tf heterozygotes, the average inbreeding coefficient (F) within breeds was estimated to be 0.14. Six samples of wolf (Canis lupus L.) studied seemed to be of Tf B type. The present study, along with earlier reports, showed that some of the plasma proteins and enzymes (albumin, transferrin, eserine resistant esterase) exhibit considerably more polymorphism than that reported for hemoglobin and some of the red cell and tissue enzymes in the domestic dog.     

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.     

Uterine protein synthesis during the early stages of pregnancy in the rat.

The synthesis of uterine-soluble proteins during early pregnancy in the rat has been examined by means of dual-isotope labelling techniques and subsequent electrophoretic analysis. A protein of similar electrophoretic mobility to the uterine oestrogen-induced protein was observed, and synthesis of this 'presumptive induced protein' was maximal on Day 4 and Day 6 of pregnancy but low on day 5. Pregnancy associated protein synthesis was observed in many regions on polyacrylamide gels, including the beta-lipoprotein, alpha2-macroglobulin, post-transferrin and albumin regions. Synthesis of the post-transferrin species rapidly increased from Day 4 to reach a maximum on Day 6 in the implantation tissue. The temporal pattern of synthesis of post-transferrin protein and and of 'presumptive induced region' suggests involvement in the processes of cell proliferation and decidualization.