Genetic variation in Przewalski's horses, with special focus on the last wild caught mare, 231 Orlitza III

In our continuing efforts to document genetic diversity in Przewalski's horses and relatedness with domestic horses, we report genetic variation at 22 loci of blood group and protein polymorphisms and 29 loci of DNA (microsatellite) polymorphisms. The loci have been assigned by linkage or synteny mapping to 20 autosomes and the X chromosome of the domestic horse (plus four loci unassigned to a chromosome). With cumulative data from tests of 568 Przewalski's horses using blood, hair or tooth samples, no species-defining markers were identified, however a few markers were present in the wild species but not in domestic horses. Inheritance patterns and linkage relationships reported in domestic horses appeared to be conserved in Przewalski's horses. A derived type for the last wild caught mare 231 Orlitza III provided evidence for markers apparently not found in (or not currently available by descent from) the other species founders that were captured at the end of the nineteenth century. This information has been critical to the development of parentage analyses in the studbook population of Przewalski's horses at Askania Nova, at one time the largest herd of captive animals and the source of stock for reintroduction efforts. Some horses in the study showed genetic incompatibilities with their sire or dam, contradicting published studbook information. In many cases alternative parentage could be assigned from living animals. To assist in identification of correct parentage, DNA marker types for deceased horses were established from archived materials (teeth) or derived from offspring. Genetic markers were present in pedigreed animals whose origin could not be accounted for from founders. Genetic distance analysis of erythrocyte protein, electrophoretic and microsatellite markers in Przewlaski's horses and ten breeds of domestic horse place the Przewalski's horse as an outgroup to domestic horses, introgression events from domestic horses not withstanding.     

Horse domestication and conservation genetics of Przewalski's horse inferred from sex chromosomal and autosomal sequences

Despite their ability to interbreed and produce fertile offspring,there is continued disagreement about the genetic relationshipof the domestic horse (Equus caballus) to its endangered wildrelative, Przewalski's horse (Equus przewalskii). Analyses havediffered as to whether or not Przewalski's horse is placed phylogeneticallyas a separate sister group to domestic horses. Because Przewalski'shorse and domestic horse are so closely related, genetic datacan also be used to infer domestication-specific differencesbetween the two. To investigate the genetic relationship ofPrzewalski's horse to the domestic horse and to address whetherevolution of the domestic horse is driven by males or females,five homologous introns (a total of 3 kb) were sequenced onthe X and Y chromosomes in two Przewalski's horses and threebreeds of domestic horses: Arabian horse, Mongolian domestichorse, and Dartmoor pony. Five autosomal introns (a total of6 kb) were sequenced for these horses as well. The sequencesof sex chromosomal and autosomal introns were used to determinenucleotide diversity and the forces driving evolution in thesespecies. As a result, X chromosomal and autosomal data do notplace Przewalski's horses in a separate clade within phylogenetictrees for horses, suggesting a close relationship between domesticand Przewalski's horses. It was also found that there was alack of nucleotide diversity on the Y chromosome and highernucleotide diversity than expected on the X chromosome in domestichorses as compared with the Y chromosome and autosomes. Thissupports the hypothesis that very few male horses along withnumerous female horses founded the various domestic horse breeds.Patterns of nucleotide diversity among different types of chromosomeswere distinct for Przewalski's in contrast to domestic horses,supporting unique evolutionary histories of the two species.     

Major histocompatibility complex variation in the endangered Przewalski's horse.

The major histocompatibility complex (MHC) is a fundamental part of the vertebrate immune system, and the high variability in many MHC genes is thought to play an essential role in recognition of parasites. The Przewalski's horse is extinct in the wild and all the living individuals descend from 13 founders, most of whom were captured around the turn of the century. One of the primary genetic concerns in endangered species is whether they have ample adaptive variation to respond to novel selective factors. In examining 14 Przewalski's horses that are broadly representative of the living animals, we found six different class II DRB major histocompatibility sequences. The sequences showed extensive nonsynonymous variation, concentrated in the putative antigen-binding sites, and little synonymous variation. Individuals had from two to four sequences as determined by single-stranded conformation polymorphism (SSCP) analysis. On the basis of the SSCP data, phylogenetic analysis of the nucleotide sequences, and segregation in a family group, we conclude that four of these sequences are from one gene (although one sequence codes for a nonfunctional allele because it contains a stop codon) and two other sequences are from another gene. The position of the stop codon is at the same amino-acid position as in a closely related sequence from the domestic horse. Because other organisms have extensive variation at homologous loci, the Przewalski's horse may have quite low variation in this important adaptive region.     

Bistable properties of the hock joint of horses (Equus spp.)

The hock (ankle) joint of horses is bistable: if placed in an intermediate position it springs either to full extension or to strong flexion. We have performed experiments to measure the torques involved and to elucidate the mechanism. The torques were much smaller in hocks of Przewalski's horse ( E. ferus ) and zebra ( E. grevyi ) than in those of domestic horses. We suggest that the bistability of the hock is too slight to have much importance for wild horses but may have been enhanced in the breeding of domestic horses by selection for elegance of gait. The elbow joint is also bistable but the stifle (knee) and carpal joints are not.     

Mouse hemoglobin during gestation. Absence of fetal hemoglobin

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.     

Equine piroplasmoses at the reintroduction site of the Przewalski's horse (Equus ferus przewalskii) in Mongolia

Piroplasmosis has been identified as a possible cause of mortality in reintroduced Przewalski's horses (Equus ferus przewalskii) in the Dsungarian Gobi (Mongolia). A cross-sectional and a longitudinal study were conducted in a representative sample (n = 141) of the resident domestic horse population and in 23 Przewalski's horses to assess the prevalence of Theileria equi and Babesia caballi. Piroplasms were detected in blood by light microscopy in 6.7% (95% confidence interval [CI]: 3.6-12.2%) of the domestic horse samples. Antibody prevalence was 88.6% (95% CI: 82.4-92.9%) for T. equi and 75.2% (95% CI: 67.4-81.6%) for B. caballi. Antibody prevalence did not change over time, but antibody prevalence for both piroplasms were significantly lower in animals less than 1 yr of age. For both piroplasms, the prevalence of presumably maternal antibodies (falling titers) in foals was 100%. Only one of 16 foals seroconverted against T. equi during the study period, despite that piroplasms were found in two other individuals. The incidence density (ID) of T. equi in foals was therefore 0.0012 seroconversions per horse day (95% CI: 0.00029-0.0057). In contrast, yearlings had an ID of 0.0080 (95% CI: 0.0049-0.010) for T. equi and 0.0064 (95% CI: 0.0036-0.0093) for B. caballi, and in seven individuals piroplasms were detected. The seroprevalence of both piroplasms rose from 20% in spring to 100% in autumn. Comparison of domestic and Przewalski's horses resulted in a standardized prevalence ratio (SPR) of 0.98 (95% CI: 0.80-1.24, not significant) for B. caballi; in contrast, the prevalence of T. equi in Przewalski's horses was significantly lower than expected (SPR = 0.51, 95% CI: 0.50-0.64).     

Ligand-dependent Bohr effect of Chrionomus hemoglobins.

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation. For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2-liganded and CO-liganded states of hemoglobin III. Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi; here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively. On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine-E11, obstructing the movement of the heme-iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved. The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.     

Studies of rhesus monkey (Macaca mulatta) hemoglobin.

One hemaglobin was found in the adult rhesus monkey (Macaca mulatta) by the technique of isoelectric focusing. In addition to the adult hemoglobin, one fetal hemaglobin was observed in neonates of the same species. The alpha and non-alpha globins of rhesus monkey hemoglobins showed similar electrophoretic mobilities as those of human hemoglobin A by cellulose acetate membrane electrophoresis. It appeared that the rhesus monkey hemoglobin system is a good model system for the study of human hemoglobin development.     

Isoelectric focusing purity criteria and 1H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata

Three major monomeric hemoglobins have been isolated from the erythrocytes of Glycera dibranchiata. Their importance to structure-function studies of heme proteins lies in the fact that they have been shown to possess an exceptional amino acid substitution. In these proteins, the E-7 position is occupied by leucine rather than the more common distal histidine. This substitution alters the polarity of the heme ligand binding environment compared to myoglobin. Due to this, the G. dibranchiata monomer hemoglobins are attracting much attention. However, until now no purity criterion has been developed. Here we demonstrate that, for all of the Glycera monomer hemoglobins, multiple line patterns are shown on high-voltage isoelectric focusing (IEF) gels. Most of these lines are shown to be a consequence of heme-related phenomena and can be understood on the basis of changes in oxidation and ligation state of the heme iron. The multiple line pattern does not indicate significant impurities in the monomer hemoglobin preparations. Similar behavior is also demonstrated for horse heart myoglobin. The multiple line patterns on IEF gels disappear when gels of the apoproteins alone are focused. Single bands occur in this case for all of the monomer hemoglobins except component II, which displays two bands, one major and one minor. The minor band is found to be a modified apoprotein form. It is sensitive to apoprotein handling prior to focusing and depends upon whether the IEF gel is prefocused or not. From this analysis, IEF is shown to be a valuable purity criterion, and the purity of our monomer hemoglobin component II preparation is 97% one globin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A reinvestigation of hemoglobin alterations in ground squirrels while in various hibernation activity states

Characterization of the hemoglobins of winter-hibernating, winter-active and summer-active Arctic ground squirrels (Citellus undulatus) by citrate agar electrophoresis and isoelectric focusing (IEF), pH 5.5-8.5, showed no differences in hemoglobin electrophoretic patterns. Previous studies showing alterations in hemoglobins were most likely the result of artifacts due to the use of whole blood. The Arctic ground squirrel's hemoglobin amino terminal sequence was determined for each activity state and was identical in all cases.     

A Koelliker hemoglobin in chick erythrocytes

1. Adult chicken hemoglobins were analysed by ion exchange chromatography and isoelectric focusing and a minor hemoglobin fraction (HbK) was isolated. 2. Analysis of the constituent chains shows that HbK differs from the two major hemoglobins HbA and HbD in the alpha globin. 3. The amino acid composition, the tryptic peptide maps, the results of carboxypeptidase digestion and the functional properties show that the HbK alpha globin is quite similar to that of HbA except that the C-terminal amino acid Arg 141 is lacking. 4. HbK must then be considered a Koelliker-type hemoglobin.     

Multiple hemoglobins of the cutthroat trout, Salmo clarki

Nine hemoglobins were purified from blood of Salmo clarki by ion-exchange chromatography and preparative isoelectric focusing. The subunit structures of eight of the purified hemoglobins were studied by electrophoresis of globins in the presence of urea. Six are alpha 2 beta 2 tetramers while two appear to be heterotetramers of the type alpha alpha' beta 2 and alpha alpha' beta beta'. The effects of pH, nucleotides, and temperature on the oxygen equilibria of the purified hemoglobins were studied. Five hemoglobins with isoelectric points from 9.1 to 7.1 and one minor hemoglobin with an isoelectric point of 5.9 appear to have essentially identical oxygen binding properties. All have similar oxygen equilibria which are independent of pH and temperature and not affected by saturating amounts of ATP. Another minor hemoglobin with an isoelectric point below 5.9 has similar oxygen equilibria except for a possible pH dependence. Two hemoglobins, with isoelectric points of 6.5 and 6.4, have oxygen binding properties which are strongly pH and temperature dependent. Addition of ATP or GTP causes a large decrease in the oxygen affinity without affecting the cooperativity of oxygen binding. The effect of GTP is slightly greater than that of ATP. No significant differences were observed in the oxygen equilibria of these two hemoglobins. The red blood cells of S. clarki were found to contain large amounts of both ATP and GTP, with an ATP:GTP ratio of 3:1. Both nucleotides may be important modulators of hemoglobin oxygen affinity in S. clarki, in contrast to the situation in S. gairdneri, in which red blood cell GTP concentrations are considerably lower. The presence of six or possibly seven hemoglobins with identical oxygen binding properties in S. clarki suggests that, to a large extent, the physiological role of multiple hemoglobins in this species involves phenomena not directly related to the oxygen binding properties of the hemoglobins.     

Kinetic studies on the five principal components of normal adult human hemoglobin.

The five principal components of human hemoglobin (Ala, Alb, Alc, Ao, and A2) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A0 and A2 are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A0 is greatly reduced in Ala and Alc and is essentially absent in Alb. The general effect of p-mercuribenzoate bindind on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin Ala is anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1l') showed the greatest variation among the five hemoglobins, with A0 having the smallest constant, and Alb the largest. For the native hemoglobins, 1l' for Alb was more than twice that for A0; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH form 7 to 8 increases 1l' for all hemoglobins, but Ala is anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A0, the fastest, and Ala, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for Ala and is 4.4 times that for A0, and 5.6 times that for Alc, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for Ala and Alb, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN-minus and N3-minus binding to the five methemoglobins.     

Suppression of the antibody response to type III pneumococcal polysaccharide with antigen coupled to syngeneic lymphoid cells

Nine allophenic mice of the type C57BL/10Sn … A were analyzed quantitatively, at weekly intervals over a period of 6 weeks, for the relative parental contributions to their red blood cell and white blood cell populations. It was found that four of the mice showed a significant change (termed “chimeric drift”) in the parental composition of their peripheral white blood cells, as determined by cytotoxicity testing. Six of the mice analyzed showed chimeric drift in their red blood cell population, as determined by hemoglobin analysis on isoelectric focusing gels. The isoelectric points of the hemoglobins of six inbred strains of mice were determined as an outgrowth of this study. Chimeric drift was observed in the direction of either parental cell type, and was found to be independent of the coat color, age, or sex of the mice.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Turnover of amino-terminal acetylated and non-acetylated hemoglobins in vivo

The significance of amino terminal acetylation in the turnover of hemoglobin was analyzed by measuring the synthesis and degradation of hemoglobins A and B in domestic cat blood. The two hemoglobins occur as mixtures in cat blood and are structurally very similar except for β-chain amino terminal acetylation found in HbB which is chiefly responsible for their difference in isoelectric pH. The hemoglobins were labeled by administering radioactive amino acids to anemic animals and their specific radioactivities were monitored at intervals for several days thereafter until the average life span of the erythrocytes was exceeded. The results showed that the turnover of hemoglobin is unrelated to isoelectric pH or to amino-terminal acetylation of the protein.     

Fixed nucleotide differences on the Y chromosome indicate clear divergence between Equus przewalskii and Equus caballus

The phylogenetic relationship between Equus przewalskii and E. caballus is often a matter of debate. Although these taxa have different chromosome numbers, they do not form monophyletic clades in a phylogenetic tree based on mtDNA sequences. Here we report sequence variation from five newly identified Y chromosome regions of the horse. Two fixed nucleotide differences on the Y chromosome clearly display Przewalski's horse and domestic horse as sister taxa. At both positions the Przewalski's horse haplotype shows the ancestral state, in common with the members of the zebra/ass lineage. We discuss the factors that may have led to the differences in mtDNA and Y-chromosomal observations.     

Genetic differentiation between breeds of horses by polymorphic blood protein loci]

The estimation of genetic differentiation between 27 horses breeds originated in USSR, based on serum proteins polymorphism (loci Tf, Al, Es) is shown. Genetical variability among aborigine breeds is higher then among cultural ones. The erosion of gene pool of Przewalski's Horse is explained by special history of this population and a few horses in analyzing group. Genetic distances reflect the directions and intensity of breeding. High genetical distances between Przewalski's Horse, Shetland Pony and other horses obtained could be explained by overcoming the "bottle neck" of selections in breeding process. Results of investigation shown that 9 aborigine breeds of USSR are clustered in a special group, differed from foreign horse breeds, because their gene pool was quite unique.     

Genetic studies of blood markers in Przewalski's horses

Ninety-six Przewalski's horses (Equus przewalskii) were blood typed using systems of inherited blood variants known to be highly effective for parentage testing of domestic horses (E. caballus). Sixteen red cell antigenic factors detected using sera prepared by alloimmunization of domestic horses were shown to be inherited in six systems (A, C, D, P, Q, and U) and in the same patterns as domestic horses. Family data confirmed autosomal, codominant inheritance at five loci of serum protein variants (Al, Tf, Xk, Pi, and Es) and three loci of red cell proteins (PGM, PHI, and Hb). One serum protein locus (Gc) and two red cell protein loci (PGD and CA) appeared to be monomorphic. Despite the narrow genetic base and high inbreeding coefficients of captive Przewalski's horses, average heterozygosity calculated over 18 loci was estimated to be 0.320 +/- 0.05, which was similar to that found in five breeds of domestic horses.     

Hemoglobin alterations of the 13-lined ground squirrel while in various activity states.

1. Characterization of fetal, winter-hibernating, winter-active, summer-active and summer-induced hibernating hemoglobins of 13-lined ground squirrels (Citellus tridecemlineatus) by isoelectric focusing (IEF) pH 7.0-9.0 indicated that this molecule is extremely responsive to the various activity states of this hibernator. 2. Major alterations of ground squirrel hemoglobin occur with the varying activity states as evidenced by the distinctive changes in the isoelectric points (pIs) of these protein components. 3. Hemoglobin from winter-hibernating or summer-induced hibernating ground squirrels does not revert to a fetal type of hemoglobin. 4. The presence of an additional hemoglobin peak pI 6.55 in the summer-induced hibernator may serve as a possible assay for hibernation inducing trigger(s) (HIT) molecules under study in our laboratory.