A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

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.     

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 and close linkage of two plasma protein loci in dogs

By using a simple method of two-dimensional horizontal electrophoresis, phenotypes of an unidentified plasma protein (PA4) were determined in 967 dogs belonging to 43 different breeds. Two codominant, autosomal alleles (F and S) of PA4 were reported. While many of the breeds of middle and north-eastern Asia (akita inu, Alaskan malamute, chow chow, samoyed, Siberian husky and Tibetan terrier) showed a substantial frequency (0.1 to 0.6) of the S allele, a majority of the European breeds had only the F allele. Evidence was provided that the PA4 locus is closely linked to the plasma pretransferrin 1 locus (PRT1). No recombinant was observed in 45 informative offspring studied. In nearly all breeds, the PA4 S allele was almost always in coupling phase with the PRT1 F allele.     

Genetic relationship between equine apolipoproteins A4 and A1

Genetic polymorphism of equine apolipoprotein (APO) A4 was investigated using two-dimensional electrophoresis in four horse breeds, including Japanese native horses. A linkage relationship between the equine APOA4 and APOA1 structural loci was assumed from the segregation data of these loci in one family line of the Japanese Hokkaido native breed.     

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.     

Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4; synteny and FISH mapping to ECA9

Equine combined immunodeficiency disease (CID) is caused by homozygosity for an autosomal recessive gene. To identify linked markers for the disease, we studied a family segregating for the equine CID gene. A stallion and 19 of his CID-affected offspring were tested for marker segregation at 23 microsatellite DNA loci. His CID-affected offspring inherited only one of his two alleles at the HTG8 and HTG4 loci, namely HTG8–186 and HTG4–124 , respectively. Lod scores for linkage to the CID gene using a Θ of 0·01were 5·34 for HTG8 and 2·37 for HTG4. The apparent genotypes also suggested linkage disequilibrium between the HTG8–186 allele and the gene for CID. The gene for the DNA protein kinase catalytic subunit ( DNA-PK ) was recently suggested as a candidate gene for equine CID. A defect of this gene causes a disease in mice that is similar to equine CID. Therefore, we investigated whether this gene might be associated with the microsatellite markers. Analysis of a somatic cell hybrid panel demonstrated synteny of DNA-PK with HTG4 and HTG8 (Kentucky Synteny Group 3). Fluorescence in situ hybridization (FISH) studies demonstrated that DNA-PK is located on horse chromosome ECA9p12. This work supports the hypothesis of DNA-PK as the probable cause of equine CID.     

The effect of dietary copper on rat plasma apolipoprotein B,E plasma levels,and apolipoprotein gene expression in liver and intestine

The plasma levels of apo B and apo E, and the level of hepatic and intestinal mRNA coding for these apolipoproteins were investigated in weanling male rats pair-fed for 6 wk with a control or copperdeficient diet. Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats. Copper deficiency significantly increased triglyceride levels and decreased cholesterol levels in the liver. No major differences in the levels of hepatic and intestinal apo B and apo E mRNA occurred between control and copper-deficient rats. These data imply that hypertriglyceridemia dn hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in the gene expression at the mRNA level in the liver and intestine of the apolipoproteins studied.     

A previously described serum protein polymorphism in the rat identified as Gc ('vitamin D-binding protein')

The previously published serum protein polymorphisms Gl-1 (Moutier, Toyama & Charrier, 1973) and tf (Bender & Gunther, 1978) are identical and represent genetic variation at the locus of the vitamin D-binding arglobulin, also known as Gc or group-specific component. The identity was established by comparative protein staining, by functional tests with ^;4C-vitamin D₃, by immunological studies with specific anti-Gc sera and by the strain distribution patterns. The Gc polymorphism in the rat may initiate interesting physiological and genetical studies.     

Genetic variation at pig plasma protein locus PLP1 and its assignment to chromosome 5

A new genetic polymorphism of an unidentified plasma protein (PLP1) in pigs was described by using a method of two-dimensional gel electrophoresis and protein staining. Two codominant alleles, with frequencies of 0.83 and 0.17, were found in the Swedish Yorkshire breed. The PLP1 marker was typed in a three-generation pedigree and tested for linkage against a set of 128 markers. The PLP1 locus showed significant LOD score values with three different microsatellite markers (S0092, DAGK and S005), previously assigned to chromosome 5.     

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.     

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.     

Distinctive structure and interfacial activity of the human apolipoprotein A-IV 347S isoprotein

The T347S polymorphism in the human apolipoprotein (apo) A-IV gene is present at high frequencies among all the world''s populations. Carriers of a 347S allele exhibit faster clearance of triglyceride-rich lipoproteins, greater adiposity, and increased risk for developing atherosclerosis, which suggests that this conservative amino acid substitution alters the structure of apo A-IV. Herein we have used spectroscopic and surface chemistry techniques to examine the structure, stability, and interfacial properties of the apo A-IV 347S isoprotein. Circular dichroism spectroscopy revealed that the 347S isoprotein has similar α-helical structure but lower thermodynamic stability than the 347T isoprotein. Fluorescence spectroscopy found that the 347S isoprotein exhibits an enhanced tyrosine emission and reduced tyrosine→tryptophan energy transfer, and second derivative UV absorption spectra noted increased tyrosine exposure, suggesting that the 347S isoprotein adopts a looser tertiary conformation. Surface chemistry studies found that although the 347S isoprotein bound rapidly to the lipid interface, it has a lower interfacial exclusion pressure and lower elastic modulus than the 347T isoprotein. Together, these observations establish that the T347S substitution alters the conformation of apo A-IV and lowers its interfacial activity—changes that could account for the effect of this polymorphism on postprandial lipid metabolism.     

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.     

The effect of apolipoprotein E polymorphism on plasma cholesteryl ester transfer protein activity in type 2 diabetic patients

We studied the relationship between apo E polymorphism and cholesteryl ester transfer protein (CETP) activity in 127 type 2 diabetic patients who did not take lipid lowering drugs. Furthermore, we studied the relationship between apo E and cholesteryl ester transfer protein (CETP) in modulating plasma triglyceride and HDLcholesterol. Apo E genotypes were determined by PCR-RFLP, and CETP activity was measured using an exogenous way. Our results showed that the CETP activity increased significantly in the E2 carrier group compared to E4 carriers and E3/E3 homozygous (84.7 ± 43.9 vs. 62.5 ± 35.9 vs. 52.6 ± 23.6 nmol CE/ml/2h, respectively; p = 0.015). However, there was no association between apo E polymorphism and lipid parameter variations. Even after adjustment for CETP activity, the results remained unchanged, showing that CETP did not step in the relationship between apo E and lipid parameter variations. In conclusion there is an association between apo E polymorphism and CETP activity, and this association did not affect the relationship between apo E polymorphism and triglyceride and HDLcholesterol concentrations. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 931–935. The text was submitted by the authors in English.     

A fourth allele in the plasma esterase-1 (Es-1) system of the domestic fowl

Plasma samples of fowl were analysed by horizontal polyacrylamide gel electrophoresis (pH 9.0). Evidence was presented for the subdivision of an earlier reported esterase-1 allele (Es-1A) into two alleles designated Es-1A1 and Es-1A2. Family data were consistent with the hypothesis that the Es-1 phenotypes were controlled by four codominant, autosomal alleles Es-1C, Es-1A1, Es-1A2 and Es-1B). The White Leghorn samples showed high frequency of Es-1A1 (about 0.7) and also had considerable frequency of Es-1A2 (0.2) and of Es-1B (0.1). The three meat-type breeds studied (White Plymouth Rock, Rhode Island Red and New Hampshire) showed a very high frequency of Es-1B (0.8-1.0).     

Association of polymorphisms in the dopamine D4 receptor gene and the activity-impulsivity endophenotype in dogs

A variable number of tandem repeats (VNTR) polymorphism in exon 3 of the human dopamine D4 receptor gene ( DRD4 ) has been associated with attention deficit hyperactivity disorder (ADHD). Rodents possess no analogous repeat sequence, whereas a similar tandem repeat polymorphism of the DRD4 gene was identified in dogs, horses and chimpanzees. Here, we present a genetic association study of the DRD4 VNTR and the activity-impulsivity dimension of the recently validated dog-ADHD Rating Scale. To avoid false positives arising from population stratification, a single breed of dogs (German shepherd) was studied. Two DRD4 alleles (referred to as 2 and 3a ) were detected in this breed, and genotype frequencies were in Hardy–Weinberg equilibrium. For modelling distinct environmental conditions, 'pet' and 'police' German shepherds were characterized. Police German shepherds possessing at least one 3a allele showed significantly higher scores in the activity-impulsivity dimension of the dog-ADHD Rating Scale than dogs without this allele ( P  = 0.0180). This difference was not significant in pet German shepherds. To the best of our knowledge, this is the first report of an association between a candidate gene and a behaviour trait in dogs, and it reinforces the functional role of DRD4 exon 3 polymorphism.     

Microsatellite diversity, pedigree relatedness and the contributions of founder lineages to thoroughbred horses

The thoroughbred (TB) horse is one of the oldest breeds of domestic animals, with pedigree records spanning three centuries. Because the population is essentially closed, there is concern about loss of genetic variation. Here we report two parallel analyses. In the first, genetic variation in the current population is measured using data from 13 microsatellite loci in 211 horses with relationships calculated based on allele sharing. In the second analysis, pedigree information is used to calculate genetic relationships between animals based on shared ancestry. These two measures of relationship are compared and shown to be closely related. Together, they provide an estimate of the amount of genetic variation which existed in founder animals. This study confirms the narrow genetic base of the breed and provides comprehensive analysis of contributions of founder animals. Seventy-eight percent of alleles in the current population are derived from 30 founders, 27 of these male. Ten founder females account for 72% of maternal lineages, while one founder stallion is responsible for 95% of paternal lineages.     

The Structure of Human Apolipoprotein A-IV as Revealed by Stable Isotope-assisted Cross-linking,Molecular Dynamics,and Small Angle X-ray Scattering

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a “clasp” mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.     

Genetic polymorphism of serum vitamin D-binding protein (GC) in sheep and mouflon

One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by immunoblotting revealed genetic polymorphism of GC protein in sheep (variants F, S, V) and mouflon (variants F and S, apparently identical to F and S of sheep). The frequency of Gcs allele ranged from 0.84 to 1.0 in the 12 breeds of sheep studied. GcV allele was observed only in Tsigai breed with a frequency of 0.017.