GENETICS OF HORSE ACIDIC PREALBTJMINS

M. BRAEND*

Department of Medicine, The Veterinary College of Norway, Oslo, Norway

Received March 4, 1969

HE term “prealbumin” was originally applied by SMITHIES (1955) to those zones of protein which migrate ahead of albumin in alkaline starch gel. Using

acidic starch gel, BRAEND and EFREMOV (1965) and GAHNE (1966) detected nu- merous protein zones in horse serum that migrated ahead of albumin. Also, FAGERHOL and BRAEND (1965) demonstrated a multiplicity of prealbumin zones in human serum. GAHNE (1966) showed that the most anodal series of equine prealbumin zones belong to one genetic system, designated Pr, controlled by four codominant autosomal alleles. BRAEND (1967 a,b) described what appeared to be four additional systems of acidic prealbumins of horses and tentatively named them Xc, Xd, Xh and Xk, in decreasing order of mobility. The purpose of the present report is to describe a technique which permits good resolution of the prealbumin zones of horse serum, to present evidence for additional alleles at the Pr locus, and to present evidence on the genetic control of phenotypes in the Xk system.

MATERIALS A N D METHODS

Serum samples, stored at -20°C when not in use, were obtained from 44 D0le stallions, 78 Dsle mares and foals, 68 Norwegian Trotter stallions, 43 Norwegian Trotter mares, 50 Warm- blood stallions and 5 Warmblood mares. In addition, 30 horses representing a variety of breeds were sampled monthly over a period of 6 months to establish the repeatability of Pr and Xk phenotypes. The family material comprised of 58 mares and their 65 offspring sired by a total of 5 different stallions.

Gels were prepared from commercial Norwegian potato starch hydrolyzed for 75 min using 600 g of starch and 12 ml of HC1 in 1200 ml of acetone. Hydrolysis was performed at tempera- tures of 37”C, 44°C and 48°C.

The gel buffer (pH 4.8) consisted of 27 to 30 ml of 0.05 M citric acid, 12 to 13.5 ml of 0.2 M

Tris (Sigma 7-9) and RH,0 to 175 al . Twenty to 21 g of the staruh hydrolyzed at 44 or 48°C were mixed with 175 ml of the buffer. This mixture was boiled for 3 min, degassed and poured into a gel frame which measured 22 x 13 x 0.46 cm. If the gels appeared to be too thin, then about 10 to 15% of the starch used in their preparation was obtained from that hydrolyzed at 37°C.

The serum samples were adsorbed onto 1 x 0 5 cm strips of filter paper of about 1 mm in thickness and these were inserted into a cut made 4 cm from the cathodic bridge. The electrolyte vessels each contained 1 liter of electrolyte (pH 6 0) composed of 74 g of H,BO, and 1 g of NaOH in one liter of H,O. The bridges were composed of 12 layers of commercial Norwegian filter paper having a thickness intermediate to Whatman No. 1 and No. 3. These were overlaid on the gel to a distance of 2 cm and the rest of the gel was covered with a thin polyethylene sheet. The initial output of power was 300 v corresponding to 100 v between the bridges. The filter paper

* Present address Department of Animal Productlon, Faculty of Vetennary Science, P. 0 Kabete, Kenya, East Africa

Genetics 65 : 495-503 July 1970

496 M. BRAEND

strips were removed after 15 min. When the borate boundary had reached a position 2 cm behind the line of insertion, the voltage was increased to 400. A further increase, to 500 v, was made when the borate boundary reached a point 2 cm beyond the line of insertion (origin). The run- ning time, about 4 hr, was complete when the borate boundary reached a point 7 cm beyond the origin. The gels were sliced horizontally and the bottom halves were stained with amido black as described by SMITHIES (1955).

RESULTS

The aforementioned technique, as illustrated by Figures 2, 3 and 4, permitted the resolution of a large number of protein zones in the prealbumin region of the gels. Those photos also illustrate what would appear to be an almost overwhelm- ing amount of phenotypic variation. It is in part for the latter reason that atten- tion in this report is focused only on the most anodal series of zones, which belong to the Pr system, and the most cathodal set of bands, which belongs to the Xk system.

The Pr system: GAHNE (1966) observed eight phenotypes in the Pr system and showed that those phenotypes could be accounted for on the basis of four codomi- nant autsomal alleles designated PF, PIJ, PrL, and PP. With the technique used by GAHNE, each of the four alleles appeared to control a three-zone pattern in which the intermediate zone exhibited the most intensive staining. Thus the basic zone patterns, as seen in the homozygous phenotypes F’F, 11, LL and SS, would differ only with respect to the relative electrophoretic position of the three zones. With the present technique, which is capable of resolving many more Pr pheno- types, and therefore called for the postulation of four additional alleles which I have designated PrV, PrT, PrU and Prw in accordance with previous suggestions

ST IL W W UU S S NN LL FF +

- + Start ------- FIGURE 1.-A sohematic presentation of the appearance of prealbumin zones in phenotypes

FF, LL, NN, SS, UU, WW, IL and ST of the Pr system of horses.

HORSE PREALBUMINS 49 7

for nomenclature (BRAEND 1965). the basic zone patterns controlled by the indi- vidual alleles varied considerably not only with respect to number of zones but also with respect to staining intensity of the zones.

Optimum resolution of all the zones in the Pr region of the gels was difficult to attain. While the more anodal Pr zones were usually the most sharply defined, those immediately behind the borate boundary did not always separate clearly. Nevertheless, with the inclusion of appropriate known control samples in each gel it became a re!atively easy matter to diagnose the 27 Pr phenotypes encoun- tered in this study. All except phenotype NT are represented in Figures 2 ,3 and 4.

As an aid in phenotypic classification, the basic zone patterns controlled by alleles Pr", PrL, Pry Pr", Pr" and Prlr. as seen in their respective homozygous phenotypes, are schematically shown in Figure 1. In the absence of samples from horses homozygous for alleles Pr' and PrT, two heterozygous patterns, IL and ST, are shown in Figure 1. Thus it would appear that each of the alleles PrF, Prr, Pr', and Pr"' controls three protein zones whereas Pr'. Pry, Pr' and Pry would appear to control one major zone with indications of an additional weakly stain- ing. faster zone for alleles Pry and PrS as seen most clearly in their respective homozygous phenotypes.

With these basic patterns (Figure 1) in mind some additional remarks relat- ing to the descriptive aspects of Pr zones are now in order. The fastest of the three zones controlled by allele Pr" appeared rather broad (Figure 2 ) and might, with appropriate technique, be shown to be a doublet. Although, as shown sche- matically in Figure 1 and as further illustrated in various phenotypes in Figures

4 Alb *

Pr types

Xk types

+

T

S t a r t

LS LN LL IU IS IN IL FW FS FN FL FF

KS FS KK KK KK KK KK KK FK FK KS KK FIGURE 2.-A photograph of a gel showing the apprearance of various phenotypes in each

of the genetic systems Pr and Xk of horse prealbumins.

498 M. BRAEND

+

fi t ypes WW TW W SW ST SS Nw XU N S NN LW LT FIGURE 3.-A photograph of a gel showing 12 additional phenotypes in the Pr system of

equine prealbumins. All 12 samples were of like phenotype (Xk KK) in the Xli system.

2, 3 and 4, the intermediate zone controlled by allele Pr', would appear to be the most densely staining of the three, there were certain phenotypes, for example, LU and LW of Figme 4, in which all three L zones appeared to stain with about

. .

+

T

t Star t

Pr types SU LS LU UU LL LW LW LU FIGURE 4.-A photograph of a gel showing further differences in Pr phenotypes of horse

serum. All samples were of like phenotype (Xk KK) in the Xk system.

HORSE PREALBUMINS 499

equal intensity. Variation between gels with respect to the degree of separation of Pr zones may be illustrated by comparing phenotype LS in Figure 2 with LS in Figure 4. In the latter gel there was a clear separation between the slowest L zone and the major S zone, but this was not the case in the gel of Figure 3. Atten- tion is called to the fact that the intermediate L zone in sample LS of Figure 4 would appear to be Flightly slower than the intermediate L zone in the adjacent LU phenotype. Attention is also called to the observation that the three zones controlled by allele PrW appeared weaker in phenotype SW than in phenotype TW, as may be seen in Figure 3. All such idiosyncrasies had to be taken into consideration when arriving at the proper diagnosis of Pr phenotypes.

Among the 65 foals and their parents (58 mares and 5 stallions) there were 29 mating classes, as shown in Table 1. Obviously, with that number of mating classes and 13 represented by only one offpring each, the family data were inade- quate for the purpose of comparing observed segregation ratios with expected. Nevertheless, the observed phenotypes in the offspring were only those which

TABLE 1

Inheritance of Pr phenotypes of horses in 29 different mating classes ~

Matlng classes Number of offspring/phenotypes

1. L L X L L 2. N N x N N 3. F F x L L 4. L L x N N 5. L S X L L 6. L W x L L 7. N F x N N 8. N L x N N 9. S L X ss

IO. UL x uu 11. LW X L W 12. NN x LS 13. NN x LW 14. ss X L W 15. L F X LS 16. LN X-LS 17. LI x LW 18. LN x LW 19. LS x LW 20. L T x LW 21. SN x SL 22. UN x UL 23. WN x WL 24. IN x LS 25. IN x LU 26. IN x LW 27. IS x LW 28. NS x LW 29. ST x LW

2/LL 3/NN

2/FL 2/LN 1/Ls 1/LW

2/NN, 1/NF 1/NL

l/SS, 1/SL 2/UL

1/LL 1 /NL I/NL, 2/NW I/SL, 2 / s w

I/NS, 2/LN 1 /LI 1 /LW

2/LL, 2/LS l/LL, 2/Tw, 1/LW 2/SS, 1/NS 1 /uu 1/WW

I/LL

1/IS l/NU, 1/NL 1/NW 1/IL I/SW, 4/SL, 3/NW, 3/NL 1/Tw, 2/SL

500 M. BRAEND

TABLE 2

Gene frequenices in the Pr system for two breeds of horses

Alleles

Breeds PrF Pr‘ PrL PrN Prs PrT PrW

Dele 0.102 0.OM 0.070 0.635 0.156 0.008 0.025 Nqrwegian Trotter 0.032 0.046 0.207 0.203 0.374 0.063 0.077

would be expected assuming that the Pr phenotypes are controlled by codominant alleles.

Among 122 horses of the D0le breed, there were 2/(of type) FL, 18/FN, 5/FS, l/IN, 2/LL, lO/LN, 1/LS, 49/NN, 22/NS, 2/NT, 4/NW, 4/SS and 2/SW, whereas among 111 horses of the Norwegian Trotter breed there were 4/FL, l/FN, I/FS, l/FW, 5/IL, 3/IN, 2/IS, 4/LL, BAN, lO/LS, 6/LT, 5/LW, 5/”, 18/NS, 4/NT, I/NW, 20/SS, 4/ST, SJSW and 1/WW. Allele Pru was not repre- sented in any of these 233 horses. From these data, estimates of the frequencies of alleles PrF, Prl, PrL, PrN, PrS, PrT and PrW were obtained for the two breeds and are shown in Table 2. Although the Norwegian Trotter was originally de- veloped as as subline of the Dale, a breed of draft horses, it is evident that the two differ significantly from one another with respect to the frequencies of Pr alleles. None of the 55 Warmbloods, a breed akin to the Thoroughbred, possessed alleles PrF and PrW whereas the frequency of PrU in that breed was estimated to be about 0.5.

When the allele frequencies shown in Table 2 are used to compute expected numbers in each of the 28 phenotypes, i t can be shown, for each of the two breeds, that the observed numbers compare very well with the expected numbers, thereby supporting the theory of inheritance based on a series of codominant autosomal alleles.

The Xk system: A total of five phenotypes designated FF, FK, FS, KK and KS has so far been identified in this system. Two of these phenotypes, namely, FF and KK, are each characterized by a single zone of protein whereas each of the phenotypes FK, FS and KS is characterized by a pair of zones. Four of these phenotypes, namely, FK, FS, KK and KS, are shown in Figure 2. It is proposed

TABLE 3

Znheritance of X k phenotypes of horses in seven different mating classes ~~ ~~~ ~ ~

Mabng classes Number of offspnng/phenotypes

1. K K X K K 2. K K x F K 1 /KK 3. K K X K S 7/KK, 4/KS 4. K K x F S 2/FK, 1/KS 5. FK x FS 3/FK 6. FS x F S 1 /FS 7. KS x KS 1/KK, I/KS

HORSE PREALBUMINS

TABLE 4

Observed and expected numbers of X k phenotypes in two breeds*

501

Phenotypes

FF FK FS KK KS SS Total

Observed 3 30 4 77 8 0 122 Expected 3.28 31.47 1.97 75.54 9.45 0.30 D01e

Obsenred 0 3 0 93 15 0 111 Expected 0.02 2.75 0.20 93.73 13.79 0.51 Norwegian Trotter

* The gene frequencies p , q and r of alleles XkF, XkK and XkS were 0.16, 0.79 and 0.05, respec- tively, for the D0le breed and 0.01, 0.92 and 0.07, respectively, for the Norwegian Trotter breed.

that the five Xk phenotypes can be accounted for on the basis of three codominant autosomal alleles designated Xk”, X k K and XkS, where X stands for a genetic system of proteins of unknown nature and k stands for the relative electro- phoretic position of the zones controlled by these alleles in contrast with the tentatively identified systems designated Xc, Xd and Xh (BRAEND 1967 a, b). The superscript symbols F, K and S indicate relative electrophoretic positions, in decreasing order, of the individual zones controlled by the three alleles.

The family data on the Xk system are shown in Table 3 and the data on gene frequencies in the D0le breed and Norwegian Trotter are summarized in Table 4. These data are in good agreement with the theory of Xk phenotypes based on three codominant autosomal alleles. The high frequency of allele Xk” in both D ~ l e s and Trotters and the fact that all 55 Warmbloods were homozy- gous for that allele will account for the repetitive occurrence of phenotype KK in Figures 2 , 3 and 4.

DISCUSSION

At this point the reader will no doubt have raised questions concerning the nature of this great multiplicity of protein zones in the prealbumin region of the gels and he may also be inquisitive about genetic information, i f any, on the tentatively identified systems designated Xc, Xd and Xh. There is as yet no information on the nature of these zones. However, based on the studies of the Pi system of man (FAGERHOL 1967; FAGERHOL and LAURELL 1967), it seems probable to the author that some of the zones may represent alphal-antitrypsin. These proteins occur in multiple zones anodal to albumin in acid (pH 5) gels. Some may be other aZph,-globulins, and in that connection it may be recalled that POULIK and SMITHIES (1958) were able to separate human aZpha,-globulins from albumin using two-dimensional electrophoresis.

There is at this writing no information on the inheritance of zones in the tentatively identified systems Xc, Xd and Xh. In the author’s opinion genetic analysis of the large number of zones occupying the region of the gels between the slowest Pr zone, W, and the fastest Xk zone, F, will be forthcoming but such

502 M. BRAEND

analysis will undoubtedly require further modifications in buffers and general technique in order to focus better on the zones in that region. Clearly, the Xk system, as shown in this report, is separate from the Pr system and each is separate from the albumin and transferrin systems of horses which were pre- viously reported by STORMONT and SUZUKI (1963) and BRAEND and STORMONT (1964). For example, all of the Warmblood horses of the present study were of one phenotype in the Xk system but they vaned considerably with respect to their albumin and transferrin phenotypes.

I want to thank Mrs. SIGRUN UNDELAND AAHTHUN for her skilful technical assistance. This study was supported by grants from the Agricultural Research Council of Norway and Det Norske Travselskap (The Norwegian Trotter Association).

SUMMARY

A method of electrophoresis in starch gel which seemingly maximizes the number of prealbumin zones in horse serum is described. The most anodal zones belong to a previously described genetic system designated Pr. With the present technique it has been possible to extend the number of Pr phenotypes from eight to 27, thereby requiring the postulation of four additional alleles at the Pr locus. The previously indicated alleles were designated Pr", PIJ, PrL and PrS, and the new alleles are designated PIN, PIT, PrU and Prw. All act as autosomal codominants. Alleles Prl' and Pr" appear to control but a single zone of protein. Alleles PrN and PrS appear to control two zones, a weakly staining zone followed in each instance by a strongly staining zone. Alleles Pr", PrI, PrL and Prw appear to control three-zone patterns which differ from one another not only with respect to relative electrophoretic position of the zones but also with respect to staining intensity of the zones. Evidence is also presented for a second genetic system of equine prealbumins designated the Xk system and involving codomi- nant autosomal alleles, Xk", X k K and Xk". Each allele appears to control but a single zone of protein and all phenotypes except XkSS were observed. Both family data and gene frequency data agree with the genetic theory involving these two systems. There were significant differences between the three breeds of horses ( D ~ l e , Norwegian Trotter and Warmbloods) with respect to the fre- quencies of Pr alleles and X k alleles.

LITERATURE CITED

BRAEND, M., 1965 Nomenclature of polymorphic protein systems. Nature 206: 1067. -, 1967a Variation of horse prealbumins in acidic starch gels. Acta Vet. Scand. 8: 193-1%. -, 1967b Polimorfismo bioquimico sanguine0 del caballo. Arch. Zootecnia 16 : 369-386.

Haemoglobins, haptoglobins and albumins of horses. Proc. 9th Europ. Anim. Blood Group Conf. 253-2.59. Publishing House of the Czechoslovak Acad- emy of Sciences, Prague.

Studies on hemoglobin and transferrin types of horses. Nord. Vet-Med. 16: 31-37.

Serum Pi types in Norwegians. Acta Pathol. Microbiol. Scand. 70: 421428.

BRAEND, M. and G. EFREMOV, 1965

BRAEND, M. and C. STORMONT, 1964

FAGERHOL, M. K., 1967

HORSE PREALBUMINS 503

Serum prealbumin: Polymorphism in man. Science 149: 986987.

The polymorphism of “prealbumins” and &,-anti- trypsin in human sera. Clin. Chim. Acta 16: 199-203.

Studies on the inheritance of electrophoretic forms of transferrins, albumins, prealbumins and plasma esterases of horses. Genetics 53: 681-694.

Comparison and combination of the starch-gel and filter-paper electrophoretic methods applied to human sera: Two dimensional electrophoresis. Biochem. J. 68: 636643.

Zone electrophoresis in starch gels: Group variations in the serum proteins of normal human adults. Biochem. J. 61 : 629-Wl.

Genetic control of albumin phenotypes of horses. Proc. Soc. Exptl. Biol. Med. 114: 673-675.

FAGERHOL, M. K. and M. BRAEND, 1965

FAGERHOL, M. K. and C. B. LAURFLL, 1967

GAHNE, B., 1966

POULIK, M. D. and 0. SMITHIES, 1955

SMITHIES, O., 1955

STORMONT, C. and Y. SUZUKI, 1963