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Molecular genetics of avian proteins—III. The egg proteins of an isolated population of jungle fowl, Gallus Gallus L.
Comp. Biochem. Physiol.,
1964, Vol. 12, pp. 389 to 403. Pergamon Press Ltd. Printed in Great Britain
MOLECULAR GENETICS OF AVIAN P R O T E I N S - - I I I . THE EGG PROTEINS OF AN ISOLATED POPULATION OF JUNGLE FOWL, G A L L U S G A L L U S L. C. M. ANN BAKER* Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois (Received
27 February 1964)
Abstract--1. Egg whites and yolks of jungle fowl and of various breeds of domestic fowl have been compared by starch gel electrophoresis. The yolk proteins of jungle fowl and domestic fowl are identical. Some egg-white proteins are identical; other egg-white fractions contain individual, genetically controlled variation common to both jungle fowl and domestic fowl. In addition, nearly three-fifths of the jungle fowl examined had an egg-white protein not yet found in other populations. 2. The "jungle-fowl protein" appears to be part of the globulin fraction G3 and to be controlled by a gene (GaJ) codominant with the Ga A allele at the Ga locus. 3. While the full evolutionary significance of these findings cannot be assessed until there is more information from other species and populations within species, the similarity of the haemoglobins and of the proteins of egg whites, egg yolks and sera of jungle fowl and domestic fowl is so great that it would appear unnecessary to regard the latter as a separate species, G. domesticus. INTRODUCTION THE relationship between domestic fowl and the four species of jungle fowl has been the subject of much speculation. Attempts have been made to clarify the situation by comparing morphological characteristics of jungle fowl with those of domestic fowl and by studying the inheritance of traits in crosses within the genus G a l l u s (Darwin, 1868; Lotsy & Kuiper, 1924; Ghigi, 1927). In addition, some workers have used chemical methods: Spohn & Riddle (1916) compared analyses of egg yolks; McCabe & Deutsch (1952), Sibley (1960) and Lush (1961) used electrophoretic techniques to examine egg whites. None of these workers found any differences between the egg proteins of domestic and jungle fowl, although Lush (1961) reported individual intrageneric variation common to both. Baker & Manwell (1962), however, mentioned that they had found some jungle fowl had a previously unknown variant of egg white, apparently controlled by an allele at the Gz locus. * Present address : Ministry of Agriculture, Fisheries and Food, Government Buildings, Winchester. 389
390
C.M. ANN BAKER
The present paper describes the jungle-fowl G 3 variant in greater detail and reports other pertinent egg protein work. The latter comprises two main studies: first, the initial results of a search to find the jungle-fowl G 3 variant, other protein variants, or both, in the egg white of various breeds of Gallus gallus and, secondly, the preliminary results of a comparison of yolk proteins from domestic and jungle fowl. MATERIALS AND METHODS
Nomenclature The nomenclature of domestic fowl, their morphological characteristics and their egg proteins follow the authorities cited by Baker & Manwell (1962).
Sources of eggs Jungle-fowl (G. gallus) eggs were the gift of Dr. J. H. Bruckner of Cornell University. The Cornell flock of jungle fowl was started in 1940 with an importation from a Hawaiian game farm which had obtained its foundation stock from IndoChina. As far as can be ascertained, the Cornell flock has been closed since its inception. Fayoumi eggs were a present from Dr. A. W. Nordskog of Iowa State University. Three lines of birds selected for different traits were represented: J, selected for high egg production; K, selected for large body size; and L, selected for large egg size. The population from which the lines were derived consisted of about eighty birds imported from Egypt; in the early generations of selection a few birds had been introduced from the Purdue control line. Eggs from line P, the Purdue random-bred control Fayoumis, were examined also. Phoenix, Polish, Sebright and Silkie eggs were given by Mr. A. J. Maggio, a bird fancier of Savoy, Illinois. Mr. Maggio's birds are selected for breed points. Cockerels are exchanged occasionally for males from other breeders' flocks. Bantam eggs were given by two breeders, namely, Mrs. Mary A. Petersen, of Vincennes, Indiana, and Dr. H. Shoemaker, of the Department of Zoology, University of Illinois. Both flocks are bred by random mating. Mrs. Petersen has had her flock for over 15 years; the birds show considerable variation in colour and type. Dr. Shoemaker's bantams are somewhat similar to jungle fowl in colour but lack other jungle-fowl characteristics, such as the eclipse moult. This flock also contains much morphological variation. Dr. H. M. Scott, of the Poultry Department, University of Illinois, gave the author nine New Hampshire hens, each of which represented one of the nine ovoglobulin genotypes reported previously (Baker & Manwell, 1962). These birds provided eggs used as controls. Samples of egg white from individual hens of Thornber strain six (Ogden et al., 1962) were supplied by Mr. A. L. Ogden and Mr. E. M. McDermid.
Treaiment of eggs Egg whites were treated in the way described by Baker & Manwell (1962). Yolk was obtained by three methods: (1) In the earliest experiments a disposable
391
M O L E C U L A R G E N E T I C S O F AVIAN P R O T E I N S - - I I I
pipette was inserted through the vitelline membrane. Contamination with egg white was avoided in most cases by squeezing the pipette bulb as the pipette point touched and punctured the vitelline membrane. However, as will be discussed in more detail later, some contaminated samples of yolk resulted. (2) The vitelline membrane was cut with either a scalpel or a pair of scissors; immediately after f volume of egg yolk mixed with 2 volumes of wcttet~ Centrifuge 30-40 rains, at 2S, O00g.
J
I
I Precipitate PI Dissolve in 9 vols 10"/, NaCI. Dialy se egainst l disfilled wctter.
Filtrate F1
To ! rot. a d d 2 vols. ethet~
I Top layet~ Ether d lipid.
I
I
Middle laver Contains Iipovitellenin. (Fevold d Lausten, 1946).
Filtrate F2. Contains phosvitin. (Mecham d OIIcott, 1949).
I Precipitate P2" Contains lioovitellita (Alderton 6~ Fevo~ 1945)
Bottom layer. Contains livet in. (Kay ¢I Marshal¢ 1928; Shepard c~ Nottle, 1949).
FIG. 1. "Flow sheet" for the separation of the principal fractions of egg yolk. making the incision a sample was taken from the yolk with either a disposable pipette or a medicine dropper. No contamination with white could be detected in any sample of yolk obtained in this way. (3) When a large quantity of yolk was required from a single egg, a more elaborate procedure was used. The entire yolk was separated from the thick and thin whites and then washed in 0.9% sodium chloride to remove all traces of ehalaziferous white. The white-free yolk was dried by rolling on paper towels and wrapped in a paper tissue so that only a small part of the under surface was exposed. A clean incision was made on the exposed portion with a sharp scalpel. The yolk drained into a small beaker while the vitelline membrane stuck to the tissue.
Biochemical techniques Whites and yolks were resolved into their component proteins by Smithies's (1959) technique of vertical starch gel electrophoresis, modified in the ways described elsewhere (Manwell, 1963 ; Manwell & Baker, 1963). The main buffers used were acetate (4.92 g sodium acetate and 3.6 ml glacial acetic acid per 1., gel pH 4.7), barbiturate (1-88 g diethylbarbituric acid and 7"7 g sodium diethyl
392
C.M. ANN BAKER
barbiturate per 1., gel pH 8), Smithies's (1959) borate (gel pH 8-4) and tris-EDTAborate (5.06 g tris-(hydroxymethyl) aminomethane, 0.65 g ethylenediamino-tetraacetic acid and 2.39 g boric acid per 1. ; gel pH 8-6). The partial or complete purification of yolk proteins was carried out in such a way that the major components could be obtained by a comprehensive series of fractionations applied to a single initial sample. The individual steps were modified from well-recognized methods for the isolation of lipovitellin (Alderton & Fevold, 1945), lipovitellenin (Fevold & Lausten, 1946), phosvitin (Mecham & Olcott, 1949) and livetin (Kay & Marshall, 1928; Shepard & Hottle, 1949). The composite fractionation scheme is shown in the form of a flow sheet in Fig. 1. Commercially purified phosvitin was obtained from Nutritional Biochemicals Corporation. After the electrophoresis of whole yolk and some of the purified fractions (especially lipovitellin and lipovitellenin) a considerable amount of material remains in the slot. The residue is smeared over the gel during cutting and even when subsequently blotted off, results in unsightly, non-specific staining. This can be avoided if the residue is removed before slicing, an action easily accomplished by forcing the residual yolk out of the slots with jets of distilled water from a squeeze bottle. Surplus moisture is removed by blotting the gel with a paper towel.
Histochemical techniques These were identical with the methods described by Baker & Manwell (1962). RESULTS
A. Egg-white proteins 1. The identification of the "Jungle-fowl" Ga protein. Although the egg-white proteins have been considered in detail in the first paper of this series (Baker & Manwell, 1962), a simplified summary of the information concerning the genetic control of the ovoglobulins is repeated in this paragraph for the benefit of readers not familiar with the previous work. Genetic variation in the ovoglobulin fractions G~ and G 3 is determined at two loci. The G 2 fraction is controlled by the allelic genes G~c and G~D; birds can be homozygous for either gene, in which case only the globulin controlled by that gene is made, or heterozygous, when both G 2 proteins are produced. The G 3 fraction is similarly controlled by the alleles GaA and G3B. The relative positions of these proteins after starch gel electrophoresis in various buffers are shown in Figs. 2-5. It has been noted already (Baker & Manwell, 1962) that the eggs from some hens in the Cornell flock of jungle fowl contained a protein electrophoretically similar in form to and with a slightly more acidic isoelectric point than G~A. The new Ga variant has been called J (for jungle fowl) or G3J, until such time as it is more fully characterized. The position of G3J in a borate gel is shown in Fig. 2. The control, a G3A G3u, G2c Ga D heterozygote, was a New Hampshire; the other samples were from jungle fowl. G3J can occur either alone or together with G3a. Presumably
MOLECULAR GENETICS OF AVIAN P R O T E I N S - - I I I
393
GaJ and Ga B can occur together also but this phenotype has not been found in the birds available, probably because the frequency of the GaB allele is low. However, it is possible to mimic the GzJ G3B phenotype, just as it is possible to mimic any other heterozygous egg-white phenotype by mixing egg whites from the appropriate homozygotes. Anode
[
,
-~-C
~
Sctmple s l o t s . - - ~
---Albumin&
~'61obuh'ns.
m
~--Lysozyme.
Cat~ode
FIG. 2b. Diagram of part of Fig. 2a to show the positions of the major fractions and the ovoglobulin variants. As G3J has properties sufficiently similar to G3A to make it difficult to distinguish between the two proteins in borate gels sometimes, other buffer systems were used. Of these, the tris-EDTA-borate and the acetate gave particularly good results. The positions of the major components of egg white in the different systems were ascertained by the electrophoresis of purified egg-white proteins. The position of each globulin band was identified in the different systems by comparison of egg whites from birds with known genotypes.
394
C.M. ANN BAKER
In tris-EDTA-borate (Fig. 3) the migration of the proteins is very similar to that in borate. The main differences are that, in the tris-EDTA-borate, the conalbumin and post-conalbumin are separated; the globulin bands are more diffuse but, when viewed by transmitted light, easier to distinguish from each other than Anode
I •3
~
•
Globu{ins.
G2~
m 5arnple s l o t s ~
~=~=~-'oslconaiDurn/n,
~L._Lysozyme" ~2
Cathode
e
FIG. 3b. Diagram of part of Fig. 3a to show the positions of the major fractions and the ovoglobulin variants.
in borate, the albumins are more compact; the three albumins, A~, A s and Aa, are clearly separate when viewed by transmitted light; variation in A 3 is visible; the post-albumins are more compact; and, as suspected from some borate gels, there appears to be variation in the post-albumin. In acetate (Fig. 4) the positions of the proteins are altered. The prealbumins, which have isoelectric points (pI) of less than 4-1 (Rhodes et al., 1959), migrate to the cathode; albumin, with a pI in the region of 4.7 (Fevold, 1951), remains in the vicinity of the slots; conalbumin, pI about 6.8 (Warner & Weber, 1951), migrates rapidly to the anode. As might be expected, the globulins migrate between the conalbumins and the albumin. However, the G 2 proteins do not migrate in acetate as rapidly as their positions in borate or their pI of 6-0, calculated from mobility
(a) FIG. 2a. Phc
-aph of a borate
(b)
(4
Cd)
gel showing variation egg whites.
in the glol
of hens’
W
(4
(4
3a. Photograph of variation in the ovoglobulins of the same eggs as depicted in Fig. 2a after electrophoresis in a tris-EDTA-borate gel.
(4
FIG. 4a. Photograph in Fig.
(b)
(4
(4
of variation in the ovoglobulins of the same eggs as depicted 2a after electrophoresis in an acetate gel, pH 4.7.
FIG. 6a.
Photograph
of egg-yolk
proteins
after
electrophoresis
in a borate
gel.
395
MOLECULAR GENETICS OF AVIAN PROTEINS-III
values by Longsworth et al. (1940), would suggest. In acetate, the G, fraction is slower than the G, fraction; within the G, fraction the relative positions of GsC and GzD are the same as in borate gels. The G, fraction, with a p1 of 5.6 (Longsin acetate buffer. The worth et al., 1940), migrates just behind the conalbumin position of the bands in the G, region is somewhat different in acetate; as suggested by their migration in borate buffer, G, J has the most acid and GsB the least acid p1, GSA being somewhere in between.
Albumins. Samtie
slots_
u--Prealbum;n. Cathode
FIG.
4b.
Diagram
of part
of Fig.
4a to show
and the ovoglobulin
the positions
of the major
fractions
variants.
One of the most significant aspects of the observations of egg white in other buffers was that G, retained its identity under all conditions and can thus be considered a separate entity and not merely misinterpretation of the variation caused in this region by the proximity of GSA, GsB and the mucoid. 2. The occurrence of G,“. Unfortunately the Cornell jungle fowl were not pedigree so it was not possible to use breeding records to support an hypothesis for the inheritance of the jungle-fowl protein. However, none of the observed results contradicted the hypothesis that GsJ is a third allele at the G3 locus: the
C. M. ANN BAKER
396
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_ -
_ ._ -
2
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‘3
2
a
3
\o
MOLECULAR
GENETICS
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PROTEINS-III
397
electrophoretic behaviour of the protein G,’ suggests strongly that it is indeed a a part of G,; the occurrence of GaJ (alone or with GSA) indicates a possible genetic polymorphism ; and the observed genotype frequencies do not differ significantly from those expected. Table 1 summarizes the data for globulin types of isolated populations of chickens examined in an attempt to find G,J or other new variants of G, and G,. So far, there has been no success in this direction. The data support previous but findings that GaA and GzD are the most common alleles in many populations, more information is needed before any conclusions can be reached concerning gene frequencies and their biological significance.
t
I
----a--
5 W
m-A--w
J--
--c--v
m----D--B
,-8~f EsI
+
0
5 (ii)
o-c-0
o__o-D-(--J-_0
+
o-o-s0
5 (iii)
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El Lo-c046 o-;x
-. -0
0
I
FIG. 5. Diagram of the migration of ovoglobulins in: (i) borate, pH 8.4; (ii) trisEDTA-borate, pH 8.6; and (iii) acetate, pH 4.7. The Sebright bantam and jungle fowl depicted each had a GzD protein which appeared normal in borate and trisEDTA-borate gels but differed from the usual GzD in acetate gels.
3. Some other variations observed in egg white. (a) Variation of the protein GzD. Electrophoresis of egg whites in acetate buffer (pH 4.7) revealed that there was individual variation of the protein identified as GzD in borate and tris-EDTAFig. 5 shows the two examples found so far, a Sebright, borate buffer systems. GsB GaB, GaD GzD and a jungle fowl, G,* Ga*, GzD GzD. These are compared with controls made from a New Hampshire egg (GaA GaB, GzC G,‘) and a bantam
C. M. ANN BAKER
398
G,* GaB, G,e GaD and a mixegg (GA GA, GD GzD ) mixed to give the phenotype ture of the same New Hampshire egg and a jungle-fowl egg (GaJ Gs*, GaD GsD) to give a pattern G,J, G,” GaB, GsC GaD. The Sebright, jungle-fowl and control phenotypes are shown in borate (Fig. 5i), tvis-EDTA-borate (Fig. Sii) and acetate (Fig. 5iii) buffer systems. In the original eight slot gels these samples were all run from inner slots so the results cannot be ascribed to the “edge effects” sometimes occurring in the two outer slots of starch gels. In the acetate gel, the Sebright G, D band migrated in a position approximately intermediate between and overlapping with those of G,J and CBA, the jungle-fowl GsD band migrated between the positions of G,c and GZD, overlapping the area of the former. (b) Additional variation in the globulin region. Fig. 2 shows that, in addition to the G, and G, variants, the globulin region has a series of very faint bands. These will be referred to subsequently as sub-A, sub-B (already noted by Baker & Manwell, 1962) and sub-C because they migrate in positions slightly slower than Ga*, GaB and GaC respectively. As G,* and GaB tend to obscure the sub-A region, it is virtually impossible to see sub-A except in G,J homozygotes. A similar situation exists in respect of sub-B, which is masked by GaB and GaC. The separation of GaC and GzD in borate gels is wider than the separation of G,* from G,a or of G,s from G,” so sub-C can be identified comparatively easily in the presence of other protein bands. Sub-A, sub-B and sub-C have been found in many of the populations studied, particularly in the “fancy” breeds. However, as well as the difficulty in finding sub-A and sub-B in the presence of some of the G, and G, variants, there is an additional difficulty in that sub-A, sub-B and sub-C are only revealed when the conditions of electrophoresis, staining and decolorizing are favourable. Hence, the nature and the genetic control or physiological causes of these proteins have not yet been investigated. of G. gallus examined by (c) Conalbumin polymorphism. All the populations the author were found to have the same type of conalbumin, apparently the “conalbumin b” of Ogden et al. (1962). Recently it has been possible to examine egg whites from some of the birds used by Ogden et al., and to confirm that all the chickens screened by the author were of the type bb. of the ovalbumin A, was observed in (d) Albumin polymorphism. Variation However, only the Fayoumis and jungle fowl contributed most populations. This was not applied because the eggs from enough eggs for statistical analysis. these birds had to travel for 2-3 days in hot weather, which resulted in some deterioration. Thus, the possibility that the variation was caused by the environment rather than the genotype of the hen could not be excluded. B. Egg-yolk proteins of a typical borate 1. IdentiJication of yolk proteins. Fig. 6a is a photograph gel of whole egg yolks stained for total protein. The accompanying diagram
MOLECULAR
GENETICS
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399
PROTEINS-III
(Fig. 6b) also shows the proteins which are only visible under special conditions. Further information about all these fractions is given below. Livetin. Electrophoresis of purified livetin revealed most of the proteins found after the electrophoresis of whole yolk. The main difference between whole yolk and livetin was that the latter lacked the trace proteins migrating most rapidly towards the anode.
Phosvtiin region.:
1
I
-
Amylase. Sample slot Catho@ proteins of lipoviteNenin
fXfhode.
FIG. 6b.
Diagram
of egg-yolk
proteins
after electrophoresis
in a borate
gel.
Williams (1962a) has discussed the identity of the proteins in the livetin fraction in some detail. These proteins are divided into three main categories on the basis of electrophoretic mobility, namely 01-, /zL 2nd y-livetin. Williams (1962a) identified the a-livetin as serum albumin, /3-livetin as an cu,-globulin and y-livetin as y-globulin. He also found livetin contained transferrin (1962a, b). In the present study, the protein labelled “serum albumin” had an electrophoretic mobility corresponding to that of serum albumin and the protein labelled “transferrin” had a mobility .corresponding to that of serum transferrin. The livetin “transferrin” also stained green with Nitroso-R salt; however, the large quantity of iron in egg yolk (Romanoff & Romanoff, 1949) causes other proteins to react
400
C. M. ANN BAKER
with the iron stain and makes the identification of transferrin by this method a matter of subjective assessment of the intensity of staining of proteins migrating in a similar manner to serum transferrin. It had been hoped that the “serum albumin” and “transferrin” could be identified immunologically, but unfortunately the conditions in the animal room precluded work as exact as the production of specific antibodies. Lipovitellenin. Most of this fraction did not move out of the sample slot. In some of the first runs of the lipovitellenin fraction a pattern of protein bands corresponding to that of livetin was obtained. This was probably due to contamination as repetition of the steps separating lipovitellenin from livetin removed the livetin-like proteins from the former fraction. Lipovitellenin also had two faint cathodal proteins. No protein bands could be found after the electrophoresis of vitellenin prepared from lipovitellenin by the methods of Fevold & Lausten (1946). Lipovitellin. This fraction did not migrate from the sample slot. Vitellin, prepared from lipovitellin by the methods of Alderton & Fevold (1945), had one faint protein band after electrophoresis. Phosvitin. The phosvitin fraction obviously left the slots but could not be found after staining with nigrosin or amido black 10B. However, when purified phosvitin was run in a position between whole yolks or livetin, the stained gel showed that the samples on either side of the phosvitin were distorted in the “albumin” region by an unstained bulge apparently produced from the phosvitin sample. The same result was obtained when commercially purified phosvitin was used. of whole yolks three Fast anodal trace proteins. After the electrophoresis trace proteins, migrating rapidly to the anode could be identified (Fig. 6b). These were not recovered in any of the fractions described above. egg yolk contains several esterase bands. Most of the Esterase. Chicken esterase activity is in the livetin fraction, where it occurs in the “serum albumin” and “transferrin” regions (Fig. 6b). In addition, the vitellin fraction shows a diffuse esterase activity, extending from the insertion slot to the “albumin” region. This vitellin esterase is not seen in whole yolk. Amylase. Chicken yolk amylase, from its action probably an ol-amylase, is identified by the damage it causes to the slots of starch gels. Of the purified fractions, lipovitellenin, lipovitellin and vitellin had amylase. 2. Variation in egg yolk. At first, when using method (1) for obtaining egg yolk, qualitative and quantit%ive variation was observed in the region of the fast anodal trace proteins. It was found that this variation was caused by contamination with egg white and could be reproduced by the addition of varying After the adoption of method (2) for sampling amounts of white to yolk samples. yolks, no “variation” was observed. A total of 172 yolks from jungle fowl and from various breeds of domestic fowl were identical. However, the examination of yolks from the eggs of other galliform birds revealed marked interspecific differences.
MOLECULAR
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DISCUSSION
Before the evolutionary significance of GsJ or any other egg protein can be fully comprehended, more work is necessary to determine the structure and function of individual proteins and their distribution in domestic and wild stocks of Gallusgallus and other species within the genus. In the present state of knowledge, G,J could be either an original jungle-fowl protein, subsequently lost in many domestic populations, or have arisen by mutation in the Cornell jungle Similarly, the low occurrence of GsB in the fowl or their Hawaiian progenitors. Cornell jungle fowl could be explained as a characteristic in the process of being eliminated or as a recent mutation; the absence of GsC in the flock could be interpreted as loss of the protein from this population or as indicative that GsC is not a jungle-fowl protein but arose in domestic breeds by mutation. Whatever the origin of the egg-white variants, birds hatched in Illinois from Cornell jungle-fowl eggs were observed to have several characteristics popularly associated with wild jungle fowl. Although kept under the same conditions as domestic breeds, the Cornell jungle fowl were more nervous and suspicious than their domestic contemporaries; the tail carriage of the jungle fowl males was usually low, in contrast to the erect tail carriage of most domestic roosters; and at least one jungle fowl male had an eclipse moult. With the exception of G sJ, the egg proteins of the Cornell jungle fowl were the same as the egg proteins found in the available populations of domestic fowl. This similarity of jungle fowl and domestic fowl proteins was found in the blood also. Dr. F. X. Ogasawara, of the University of California at Davis, supplied several blood samples from jungle fowl for other studies (Manwell et al., 1963). These samples were compared with samples from domestic fowl in several ways: the haemoglobin of Dr. Ogasawara’s jungle fowl is electrophoretically identical with the haemoglobin from domestic breeds; the oxygen equilibria of individual haemoglobin samples from adult domestic and jungle fowl are the same in all the cases studied; it has not yet been possible to distinguish jungle fowl from domestic fowl by differences in serum proteins, although other closely related galliform species can be separated from each other by this means. It would appear that whatever the evolutionary significance of G,’ the domestic fowl is so similar to the jungle fowl that the former should be referred to as G. gahs also and not arbitrarily assigned to a separate species, G. domesticus as is done by some authorities. Some comment on the electrophoretic behaviour of the globulin fractions is warranted. In the present work the observed behaviour of the G, and G, globulin fractions in the acetate buffer (pH 4.7) does not agree with the findings of Longsworth et al. (1940) who represented G, as migrating more rapidly than G, at low pH’s. There are two possible reasons for this discrepancy between the two studies. First, the “molecular sieve” action of the starch gel retards the migration of larger molecules, an effect not present in the moving boundary electrophoresis used by Longsworth et al. (1940). Thus it could be that the G, globulins are intrinsically larger than G, globulins; the same reasoning can be applied to the electrophoretic
402
C. M. ANN BAKER
behaviour of the proteins within the G, fraction to account for the slower migration of GzD than G,” at both alkaline and acid pH’s in starch gels. Secondly, as the G, and G, fractions are quantitatively and qualitatively very similar in electrophoresis, and as the main criterion of Longsworth et al. (1940) for distinguishing between the two fractions was electrophoretic mobility, it is possible that the “G2” observed in acid buffers by these workers was actually G, and vice versa. If this were so, the behaviour of G, and G, in the low pH acetate buffers of Longsworth et al. (1940) and of the author could be due to some change in the structure, size or charges (or a combination of these factors) of the molecules of G,, G, or both. However, the behavior of the individual proteins within the G, and G, fractions suggests that the first reason-the action of the molecular sieveis the more likely. The differences observed between some GzD proteins in the acetate buffer are of interest also. Both the Sebright egg and the jungle-fowl egg were under 2 days old (the latter was laid by a pullet hatched from a Cornell jungle-fowl egg in Illinois) so the differences cannot be ascribed to deterioration. It is possible that a GzD polypeptide chain could have been altered in another way, as by the substitution of one amino acid for another, the addition of extra amino acids or by the These alterations could have lack of, or the duplication of, an existing segment. occurred in such a manner as to produce changes in the charge or size of the GzD molecules, only apparent in the low pH acetate gel. Unfortunately, the lack of facilities prevented identification of the birds by trap-nesting so, in this case, it was not possible to use them for further studies. The proteins sub-A, sub-B and sub-C do not seem to be artefacts produced by deterioration as they have been found in fresh eggs. Sub-A, sub-B and sub-C could be polymers of other globulin proteins or, if each of the latter consists of more than one chain, products of chain recombination. Acknozcledgements-This work has been supported by the United States Public Health Service (GM 7939) and from grants made to Dr. Clyde Manwell by the United States National Science Foundation (G13467, G18082 and GB612). The author wishes to record her gratitude to these agencies and to thank the United States Educational Commission in the United Kingdom for a Fulbright Travel Grant. The author thanks Dr. J. H. Bruckner for jungle-fowl eggs; Dr. A. W. Nordskog for Fayoumi eggs; Mr. A. J. Maggio for Phoenix, Polish, Sebright and Silkie eggs; Mrs. Mar> A. Petersen and Dr. H. Shoemaker for bantam eggs; Dr. H. M. Scott for nine New Hampshire hens; Messrs. A. L. Ogden and E. M. McDermid for egg-white samples from Yorkshire, for the Thornber strain six; and Messrs. Thornber Bros., of Mytholmroyd, provision of clerical assistance for the preparation of this manuscript. The author is especially indebted to Dr. C. Manwell for laboratory generously giving his time for advice, criticism and discussion.
facilities
and for
REFERENCES ALDERTON G. & FEVOLD H. L. (1945) Preparation of the egg lipoprotein, Arch. Biochem. 8, 415419. BAKER C. M. A. & MANWELL C. (1962) Molecular genetics of avian proteins. white proteins of the domestic fowl. Brit. Pod. Sci. 3, 161-174.
lipovitellin. 1. The
egg-
MOLECULAR GENETICSOF AVIANPROTEINS-III
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DARWIN C. (1868) The Variation of Animals and Plants under Domestication. John Murray, London. FEVOLD H. L. (1951) Egg proteins. Advanc. Protein Chem. 6, 187-252. FEVOLD H. L. & LAUSTENA. (1946) Isolation of a new lipoprotein, lipovitellenin, from egg yolk. Arch. Biochem. 11, 1-7. GHIGI A. (1927) On the origin of the domestic breeds of poultry and their classification. Proc. 3rd World’s Poultry Sci. Cong. pp. 52-54. KAY H. D. & MARSHALLP. G. (1928) The second protein (livetin) of egg yolk. Bi0chem.J. 22, 1264-1269. LONGSWORTHL. G., CANNANR. K. & MACINNES D. A. (1940) An electrophoretic study of the proteins of egg white. J. Amer. Chem. Sot. 62, 2580-2590. LOTSY J. P. & KUIPER K. (1924) A preliminary statement of the results of Mr. Houwink’s experiments concerning the origin of some domestic animals. V. Genetica 6, 221-282. LUSH I. E. (1961) Genetic polymorphisms in the egg albumin proteins of the domestic fowl. Nature, Lond. 189, 981-984. MCCABE R. A. & DEUTSCHH. F. (1952) The relationships of certain birds as indicated by their egg-white proteins. Auk 69, l-18. MANWELLC. (1963) The blood proteins of cyclostomes. A study in phylogenetic and ontogenetic biochemistry. In The Biology of Myxine (Edited by A. BRODAL& R. FANGE). Universitetsforlaget, Oslo. MANWELLC. & BAKER C. M. A. (1963) Starch gel electrophoresis of sera from some marine arthropods: studies on the heterogeneity of hemocyanin and on a “ceruloplasmin-like protein”. Comp. Biochem. Physiol. 8, 193-208. MANWELL C., BAKER C. M. A. & CHILDEM W. (1963) Molecular genetics of hemoglobin in hybrid fishes and birds-I. The molecular basis for “hybrid vigor”. Comp. Biochem. Physiol. 10, 103-120. MECHAMD. K. & OLCOTT H. S. (1949) Phosvitin, the principal phosphoprotein of egg yolk. J. Amer. Chem. Sot. 71, 3670-3679. OGDENA. L., MORTONJ. R., GILMOUR D. G. & MCDERMID E. M. (1962) Inherited variants in the transferrins and conalbumins of the chicken. Nature, Lond. 195, 1026-1028. RHODES M. V., BENNETT N. & FEENEY R. E. (1959) The flavoprotein-apoprotein system of egg white. J. Biol. Chem. 234, 2054-2060. ROMANOFFA. L. & ROMANOFFA. J. (1949) The Aviun Egg. John Wiley, New York. SHEPARDC. C. & HOTTLE G. A. (1949) Studies of the composition of the livetin fraction of the yolk of hen’s eggs with the use of electrophoretic analysis. J. Biol. Chem. 179, 349-357. SIBLEY C. G. (1960) The electrophoretic patterns of avian egg-white proteins as taxonomic characters. Ibis 102, 215-284. SMITHIES 0. (1959) An improved procedure for starch gel electrophoresis: further variations in the serum proteins of normal individuals. Biochem. r. 61, 629-641. SPOHN A. A. & RIDDLE 0. (1916) Studies on the physiology of reproduction in birds. II. On the chemical composition of white and yellow egg yolk of the fowl and pigeon. Amer.J Physiol. 41, 397-408. WARNERR. C. & WEBER L. (1951) The preparation of crystalline conalbumin. J. Biol. Chem. 191, 173-180. WILLIAMS J. (1962a) Serum proteins and the livetins of hen’s_egg yolk. Biochem. J. 83, 346-355. WILLIAMS J. (1962b) A comparison of conalbumin and transferrin in the domestic fowl. Biochem. J. 83, 355-364.
27
1964, Vol. 12, pp. 389 to 403. Pergamon Press Ltd. Printed in Great Britain
MOLECULAR GENETICS OF AVIAN P R O T E I N S - - I I I . THE EGG PROTEINS OF AN ISOLATED POPULATION OF JUNGLE FOWL, G A L L U S G A L L U S L. C. M. ANN BAKER* Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois (Received
27 February 1964)
Abstract--1. Egg whites and yolks of jungle fowl and of various breeds of domestic fowl have been compared by starch gel electrophoresis. The yolk proteins of jungle fowl and domestic fowl are identical. Some egg-white proteins are identical; other egg-white fractions contain individual, genetically controlled variation common to both jungle fowl and domestic fowl. In addition, nearly three-fifths of the jungle fowl examined had an egg-white protein not yet found in other populations. 2. The "jungle-fowl protein" appears to be part of the globulin fraction G3 and to be controlled by a gene (GaJ) codominant with the Ga A allele at the Ga locus. 3. While the full evolutionary significance of these findings cannot be assessed until there is more information from other species and populations within species, the similarity of the haemoglobins and of the proteins of egg whites, egg yolks and sera of jungle fowl and domestic fowl is so great that it would appear unnecessary to regard the latter as a separate species, G. domesticus. INTRODUCTION THE relationship between domestic fowl and the four species of jungle fowl has been the subject of much speculation. Attempts have been made to clarify the situation by comparing morphological characteristics of jungle fowl with those of domestic fowl and by studying the inheritance of traits in crosses within the genus G a l l u s (Darwin, 1868; Lotsy & Kuiper, 1924; Ghigi, 1927). In addition, some workers have used chemical methods: Spohn & Riddle (1916) compared analyses of egg yolks; McCabe & Deutsch (1952), Sibley (1960) and Lush (1961) used electrophoretic techniques to examine egg whites. None of these workers found any differences between the egg proteins of domestic and jungle fowl, although Lush (1961) reported individual intrageneric variation common to both. Baker & Manwell (1962), however, mentioned that they had found some jungle fowl had a previously unknown variant of egg white, apparently controlled by an allele at the Gz locus. * Present address : Ministry of Agriculture, Fisheries and Food, Government Buildings, Winchester. 389
390
C.M. ANN BAKER
The present paper describes the jungle-fowl G 3 variant in greater detail and reports other pertinent egg protein work. The latter comprises two main studies: first, the initial results of a search to find the jungle-fowl G 3 variant, other protein variants, or both, in the egg white of various breeds of Gallus gallus and, secondly, the preliminary results of a comparison of yolk proteins from domestic and jungle fowl. MATERIALS AND METHODS
Nomenclature The nomenclature of domestic fowl, their morphological characteristics and their egg proteins follow the authorities cited by Baker & Manwell (1962).
Sources of eggs Jungle-fowl (G. gallus) eggs were the gift of Dr. J. H. Bruckner of Cornell University. The Cornell flock of jungle fowl was started in 1940 with an importation from a Hawaiian game farm which had obtained its foundation stock from IndoChina. As far as can be ascertained, the Cornell flock has been closed since its inception. Fayoumi eggs were a present from Dr. A. W. Nordskog of Iowa State University. Three lines of birds selected for different traits were represented: J, selected for high egg production; K, selected for large body size; and L, selected for large egg size. The population from which the lines were derived consisted of about eighty birds imported from Egypt; in the early generations of selection a few birds had been introduced from the Purdue control line. Eggs from line P, the Purdue random-bred control Fayoumis, were examined also. Phoenix, Polish, Sebright and Silkie eggs were given by Mr. A. J. Maggio, a bird fancier of Savoy, Illinois. Mr. Maggio's birds are selected for breed points. Cockerels are exchanged occasionally for males from other breeders' flocks. Bantam eggs were given by two breeders, namely, Mrs. Mary A. Petersen, of Vincennes, Indiana, and Dr. H. Shoemaker, of the Department of Zoology, University of Illinois. Both flocks are bred by random mating. Mrs. Petersen has had her flock for over 15 years; the birds show considerable variation in colour and type. Dr. Shoemaker's bantams are somewhat similar to jungle fowl in colour but lack other jungle-fowl characteristics, such as the eclipse moult. This flock also contains much morphological variation. Dr. H. M. Scott, of the Poultry Department, University of Illinois, gave the author nine New Hampshire hens, each of which represented one of the nine ovoglobulin genotypes reported previously (Baker & Manwell, 1962). These birds provided eggs used as controls. Samples of egg white from individual hens of Thornber strain six (Ogden et al., 1962) were supplied by Mr. A. L. Ogden and Mr. E. M. McDermid.
Treaiment of eggs Egg whites were treated in the way described by Baker & Manwell (1962). Yolk was obtained by three methods: (1) In the earliest experiments a disposable
391
M O L E C U L A R G E N E T I C S O F AVIAN P R O T E I N S - - I I I
pipette was inserted through the vitelline membrane. Contamination with egg white was avoided in most cases by squeezing the pipette bulb as the pipette point touched and punctured the vitelline membrane. However, as will be discussed in more detail later, some contaminated samples of yolk resulted. (2) The vitelline membrane was cut with either a scalpel or a pair of scissors; immediately after f volume of egg yolk mixed with 2 volumes of wcttet~ Centrifuge 30-40 rains, at 2S, O00g.
J
I
I Precipitate PI Dissolve in 9 vols 10"/, NaCI. Dialy se egainst l disfilled wctter.
Filtrate F1
To ! rot. a d d 2 vols. ethet~
I Top layet~ Ether d lipid.
I
I
Middle laver Contains Iipovitellenin. (Fevold d Lausten, 1946).
Filtrate F2. Contains phosvitin. (Mecham d OIIcott, 1949).
I Precipitate P2" Contains lioovitellita (Alderton 6~ Fevo~ 1945)
Bottom layer. Contains livet in. (Kay ¢I Marshal¢ 1928; Shepard c~ Nottle, 1949).
FIG. 1. "Flow sheet" for the separation of the principal fractions of egg yolk. making the incision a sample was taken from the yolk with either a disposable pipette or a medicine dropper. No contamination with white could be detected in any sample of yolk obtained in this way. (3) When a large quantity of yolk was required from a single egg, a more elaborate procedure was used. The entire yolk was separated from the thick and thin whites and then washed in 0.9% sodium chloride to remove all traces of ehalaziferous white. The white-free yolk was dried by rolling on paper towels and wrapped in a paper tissue so that only a small part of the under surface was exposed. A clean incision was made on the exposed portion with a sharp scalpel. The yolk drained into a small beaker while the vitelline membrane stuck to the tissue.
Biochemical techniques Whites and yolks were resolved into their component proteins by Smithies's (1959) technique of vertical starch gel electrophoresis, modified in the ways described elsewhere (Manwell, 1963 ; Manwell & Baker, 1963). The main buffers used were acetate (4.92 g sodium acetate and 3.6 ml glacial acetic acid per 1., gel pH 4.7), barbiturate (1-88 g diethylbarbituric acid and 7"7 g sodium diethyl
392
C.M. ANN BAKER
barbiturate per 1., gel pH 8), Smithies's (1959) borate (gel pH 8-4) and tris-EDTAborate (5.06 g tris-(hydroxymethyl) aminomethane, 0.65 g ethylenediamino-tetraacetic acid and 2.39 g boric acid per 1. ; gel pH 8-6). The partial or complete purification of yolk proteins was carried out in such a way that the major components could be obtained by a comprehensive series of fractionations applied to a single initial sample. The individual steps were modified from well-recognized methods for the isolation of lipovitellin (Alderton & Fevold, 1945), lipovitellenin (Fevold & Lausten, 1946), phosvitin (Mecham & Olcott, 1949) and livetin (Kay & Marshall, 1928; Shepard & Hottle, 1949). The composite fractionation scheme is shown in the form of a flow sheet in Fig. 1. Commercially purified phosvitin was obtained from Nutritional Biochemicals Corporation. After the electrophoresis of whole yolk and some of the purified fractions (especially lipovitellin and lipovitellenin) a considerable amount of material remains in the slot. The residue is smeared over the gel during cutting and even when subsequently blotted off, results in unsightly, non-specific staining. This can be avoided if the residue is removed before slicing, an action easily accomplished by forcing the residual yolk out of the slots with jets of distilled water from a squeeze bottle. Surplus moisture is removed by blotting the gel with a paper towel.
Histochemical techniques These were identical with the methods described by Baker & Manwell (1962). RESULTS
A. Egg-white proteins 1. The identification of the "Jungle-fowl" Ga protein. Although the egg-white proteins have been considered in detail in the first paper of this series (Baker & Manwell, 1962), a simplified summary of the information concerning the genetic control of the ovoglobulins is repeated in this paragraph for the benefit of readers not familiar with the previous work. Genetic variation in the ovoglobulin fractions G~ and G 3 is determined at two loci. The G 2 fraction is controlled by the allelic genes G~c and G~D; birds can be homozygous for either gene, in which case only the globulin controlled by that gene is made, or heterozygous, when both G 2 proteins are produced. The G 3 fraction is similarly controlled by the alleles GaA and G3B. The relative positions of these proteins after starch gel electrophoresis in various buffers are shown in Figs. 2-5. It has been noted already (Baker & Manwell, 1962) that the eggs from some hens in the Cornell flock of jungle fowl contained a protein electrophoretically similar in form to and with a slightly more acidic isoelectric point than G~A. The new Ga variant has been called J (for jungle fowl) or G3J, until such time as it is more fully characterized. The position of G3J in a borate gel is shown in Fig. 2. The control, a G3A G3u, G2c Ga D heterozygote, was a New Hampshire; the other samples were from jungle fowl. G3J can occur either alone or together with G3a. Presumably
MOLECULAR GENETICS OF AVIAN P R O T E I N S - - I I I
393
GaJ and Ga B can occur together also but this phenotype has not been found in the birds available, probably because the frequency of the GaB allele is low. However, it is possible to mimic the GzJ G3B phenotype, just as it is possible to mimic any other heterozygous egg-white phenotype by mixing egg whites from the appropriate homozygotes. Anode
[
,
-~-C
~
Sctmple s l o t s . - - ~
---Albumin&
~'61obuh'ns.
m
~--Lysozyme.
Cat~ode
FIG. 2b. Diagram of part of Fig. 2a to show the positions of the major fractions and the ovoglobulin variants. As G3J has properties sufficiently similar to G3A to make it difficult to distinguish between the two proteins in borate gels sometimes, other buffer systems were used. Of these, the tris-EDTA-borate and the acetate gave particularly good results. The positions of the major components of egg white in the different systems were ascertained by the electrophoresis of purified egg-white proteins. The position of each globulin band was identified in the different systems by comparison of egg whites from birds with known genotypes.
394
C.M. ANN BAKER
In tris-EDTA-borate (Fig. 3) the migration of the proteins is very similar to that in borate. The main differences are that, in the tris-EDTA-borate, the conalbumin and post-conalbumin are separated; the globulin bands are more diffuse but, when viewed by transmitted light, easier to distinguish from each other than Anode
I •3
~
•
Globu{ins.
G2~
m 5arnple s l o t s ~
~=~=~-'oslconaiDurn/n,
~L._Lysozyme" ~2
Cathode
e
FIG. 3b. Diagram of part of Fig. 3a to show the positions of the major fractions and the ovoglobulin variants.
in borate, the albumins are more compact; the three albumins, A~, A s and Aa, are clearly separate when viewed by transmitted light; variation in A 3 is visible; the post-albumins are more compact; and, as suspected from some borate gels, there appears to be variation in the post-albumin. In acetate (Fig. 4) the positions of the proteins are altered. The prealbumins, which have isoelectric points (pI) of less than 4-1 (Rhodes et al., 1959), migrate to the cathode; albumin, with a pI in the region of 4.7 (Fevold, 1951), remains in the vicinity of the slots; conalbumin, pI about 6.8 (Warner & Weber, 1951), migrates rapidly to the anode. As might be expected, the globulins migrate between the conalbumins and the albumin. However, the G 2 proteins do not migrate in acetate as rapidly as their positions in borate or their pI of 6-0, calculated from mobility
(a) FIG. 2a. Phc
-aph of a borate
(b)
(4
Cd)
gel showing variation egg whites.
in the glol
of hens’
W
(4
(4
3a. Photograph of variation in the ovoglobulins of the same eggs as depicted in Fig. 2a after electrophoresis in a tris-EDTA-borate gel.
(4
FIG. 4a. Photograph in Fig.
(b)
(4
(4
of variation in the ovoglobulins of the same eggs as depicted 2a after electrophoresis in an acetate gel, pH 4.7.
FIG. 6a.
Photograph
of egg-yolk
proteins
after
electrophoresis
in a borate
gel.
395
MOLECULAR GENETICS OF AVIAN PROTEINS-III
values by Longsworth et al. (1940), would suggest. In acetate, the G, fraction is slower than the G, fraction; within the G, fraction the relative positions of GsC and GzD are the same as in borate gels. The G, fraction, with a p1 of 5.6 (Longsin acetate buffer. The worth et al., 1940), migrates just behind the conalbumin position of the bands in the G, region is somewhat different in acetate; as suggested by their migration in borate buffer, G, J has the most acid and GsB the least acid p1, GSA being somewhere in between.
Albumins. Samtie
slots_
u--Prealbum;n. Cathode
FIG.
4b.
Diagram
of part
of Fig.
4a to show
and the ovoglobulin
the positions
of the major
fractions
variants.
One of the most significant aspects of the observations of egg white in other buffers was that G, retained its identity under all conditions and can thus be considered a separate entity and not merely misinterpretation of the variation caused in this region by the proximity of GSA, GsB and the mucoid. 2. The occurrence of G,“. Unfortunately the Cornell jungle fowl were not pedigree so it was not possible to use breeding records to support an hypothesis for the inheritance of the jungle-fowl protein. However, none of the observed results contradicted the hypothesis that GsJ is a third allele at the G3 locus: the
C. M. ANN BAKER
396
_
-e-
_ -
_ ._ -
2
‘C m
‘3
2
a
3
\o
MOLECULAR
GENETICS
OF AVIAN
PROTEINS-III
397
electrophoretic behaviour of the protein G,’ suggests strongly that it is indeed a a part of G,; the occurrence of GaJ (alone or with GSA) indicates a possible genetic polymorphism ; and the observed genotype frequencies do not differ significantly from those expected. Table 1 summarizes the data for globulin types of isolated populations of chickens examined in an attempt to find G,J or other new variants of G, and G,. So far, there has been no success in this direction. The data support previous but findings that GaA and GzD are the most common alleles in many populations, more information is needed before any conclusions can be reached concerning gene frequencies and their biological significance.
t
I
----a--
5 W
m-A--w
J--
--c--v
m----D--B
,-8~f EsI
+
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o-c-0
o__o-D-(--J-_0
+
o-o-s0
5 (iii)
Sl
El Lo-c046 o-;x
-. -0
0
I
FIG. 5. Diagram of the migration of ovoglobulins in: (i) borate, pH 8.4; (ii) trisEDTA-borate, pH 8.6; and (iii) acetate, pH 4.7. The Sebright bantam and jungle fowl depicted each had a GzD protein which appeared normal in borate and trisEDTA-borate gels but differed from the usual GzD in acetate gels.
3. Some other variations observed in egg white. (a) Variation of the protein GzD. Electrophoresis of egg whites in acetate buffer (pH 4.7) revealed that there was individual variation of the protein identified as GzD in borate and tris-EDTAFig. 5 shows the two examples found so far, a Sebright, borate buffer systems. GsB GaB, GaD GzD and a jungle fowl, G,* Ga*, GzD GzD. These are compared with controls made from a New Hampshire egg (GaA GaB, GzC G,‘) and a bantam
C. M. ANN BAKER
398
G,* GaB, G,e GaD and a mixegg (GA GA, GD GzD ) mixed to give the phenotype ture of the same New Hampshire egg and a jungle-fowl egg (GaJ Gs*, GaD GsD) to give a pattern G,J, G,” GaB, GsC GaD. The Sebright, jungle-fowl and control phenotypes are shown in borate (Fig. 5i), tvis-EDTA-borate (Fig. Sii) and acetate (Fig. 5iii) buffer systems. In the original eight slot gels these samples were all run from inner slots so the results cannot be ascribed to the “edge effects” sometimes occurring in the two outer slots of starch gels. In the acetate gel, the Sebright G, D band migrated in a position approximately intermediate between and overlapping with those of G,J and CBA, the jungle-fowl GsD band migrated between the positions of G,c and GZD, overlapping the area of the former. (b) Additional variation in the globulin region. Fig. 2 shows that, in addition to the G, and G, variants, the globulin region has a series of very faint bands. These will be referred to subsequently as sub-A, sub-B (already noted by Baker & Manwell, 1962) and sub-C because they migrate in positions slightly slower than Ga*, GaB and GaC respectively. As G,* and GaB tend to obscure the sub-A region, it is virtually impossible to see sub-A except in G,J homozygotes. A similar situation exists in respect of sub-B, which is masked by GaB and GaC. The separation of GaC and GzD in borate gels is wider than the separation of G,* from G,a or of G,s from G,” so sub-C can be identified comparatively easily in the presence of other protein bands. Sub-A, sub-B and sub-C have been found in many of the populations studied, particularly in the “fancy” breeds. However, as well as the difficulty in finding sub-A and sub-B in the presence of some of the G, and G, variants, there is an additional difficulty in that sub-A, sub-B and sub-C are only revealed when the conditions of electrophoresis, staining and decolorizing are favourable. Hence, the nature and the genetic control or physiological causes of these proteins have not yet been investigated. of G. gallus examined by (c) Conalbumin polymorphism. All the populations the author were found to have the same type of conalbumin, apparently the “conalbumin b” of Ogden et al. (1962). Recently it has been possible to examine egg whites from some of the birds used by Ogden et al., and to confirm that all the chickens screened by the author were of the type bb. of the ovalbumin A, was observed in (d) Albumin polymorphism. Variation However, only the Fayoumis and jungle fowl contributed most populations. This was not applied because the eggs from enough eggs for statistical analysis. these birds had to travel for 2-3 days in hot weather, which resulted in some deterioration. Thus, the possibility that the variation was caused by the environment rather than the genotype of the hen could not be excluded. B. Egg-yolk proteins of a typical borate 1. IdentiJication of yolk proteins. Fig. 6a is a photograph gel of whole egg yolks stained for total protein. The accompanying diagram
MOLECULAR
GENETICS
OF AVIAN
399
PROTEINS-III
(Fig. 6b) also shows the proteins which are only visible under special conditions. Further information about all these fractions is given below. Livetin. Electrophoresis of purified livetin revealed most of the proteins found after the electrophoresis of whole yolk. The main difference between whole yolk and livetin was that the latter lacked the trace proteins migrating most rapidly towards the anode.
Phosvtiin region.:
1
I
-
Amylase. Sample slot Catho@ proteins of lipoviteNenin
fXfhode.
FIG. 6b.
Diagram
of egg-yolk
proteins
after electrophoresis
in a borate
gel.
Williams (1962a) has discussed the identity of the proteins in the livetin fraction in some detail. These proteins are divided into three main categories on the basis of electrophoretic mobility, namely 01-, /zL 2nd y-livetin. Williams (1962a) identified the a-livetin as serum albumin, /3-livetin as an cu,-globulin and y-livetin as y-globulin. He also found livetin contained transferrin (1962a, b). In the present study, the protein labelled “serum albumin” had an electrophoretic mobility corresponding to that of serum albumin and the protein labelled “transferrin” had a mobility .corresponding to that of serum transferrin. The livetin “transferrin” also stained green with Nitroso-R salt; however, the large quantity of iron in egg yolk (Romanoff & Romanoff, 1949) causes other proteins to react
400
C. M. ANN BAKER
with the iron stain and makes the identification of transferrin by this method a matter of subjective assessment of the intensity of staining of proteins migrating in a similar manner to serum transferrin. It had been hoped that the “serum albumin” and “transferrin” could be identified immunologically, but unfortunately the conditions in the animal room precluded work as exact as the production of specific antibodies. Lipovitellenin. Most of this fraction did not move out of the sample slot. In some of the first runs of the lipovitellenin fraction a pattern of protein bands corresponding to that of livetin was obtained. This was probably due to contamination as repetition of the steps separating lipovitellenin from livetin removed the livetin-like proteins from the former fraction. Lipovitellenin also had two faint cathodal proteins. No protein bands could be found after the electrophoresis of vitellenin prepared from lipovitellenin by the methods of Fevold & Lausten (1946). Lipovitellin. This fraction did not migrate from the sample slot. Vitellin, prepared from lipovitellin by the methods of Alderton & Fevold (1945), had one faint protein band after electrophoresis. Phosvitin. The phosvitin fraction obviously left the slots but could not be found after staining with nigrosin or amido black 10B. However, when purified phosvitin was run in a position between whole yolks or livetin, the stained gel showed that the samples on either side of the phosvitin were distorted in the “albumin” region by an unstained bulge apparently produced from the phosvitin sample. The same result was obtained when commercially purified phosvitin was used. of whole yolks three Fast anodal trace proteins. After the electrophoresis trace proteins, migrating rapidly to the anode could be identified (Fig. 6b). These were not recovered in any of the fractions described above. egg yolk contains several esterase bands. Most of the Esterase. Chicken esterase activity is in the livetin fraction, where it occurs in the “serum albumin” and “transferrin” regions (Fig. 6b). In addition, the vitellin fraction shows a diffuse esterase activity, extending from the insertion slot to the “albumin” region. This vitellin esterase is not seen in whole yolk. Amylase. Chicken yolk amylase, from its action probably an ol-amylase, is identified by the damage it causes to the slots of starch gels. Of the purified fractions, lipovitellenin, lipovitellin and vitellin had amylase. 2. Variation in egg yolk. At first, when using method (1) for obtaining egg yolk, qualitative and quantit%ive variation was observed in the region of the fast anodal trace proteins. It was found that this variation was caused by contamination with egg white and could be reproduced by the addition of varying After the adoption of method (2) for sampling amounts of white to yolk samples. yolks, no “variation” was observed. A total of 172 yolks from jungle fowl and from various breeds of domestic fowl were identical. However, the examination of yolks from the eggs of other galliform birds revealed marked interspecific differences.
MOLECULAR
GENETICS
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PROTEINS-III
401
DISCUSSION
Before the evolutionary significance of GsJ or any other egg protein can be fully comprehended, more work is necessary to determine the structure and function of individual proteins and their distribution in domestic and wild stocks of Gallusgallus and other species within the genus. In the present state of knowledge, G,J could be either an original jungle-fowl protein, subsequently lost in many domestic populations, or have arisen by mutation in the Cornell jungle Similarly, the low occurrence of GsB in the fowl or their Hawaiian progenitors. Cornell jungle fowl could be explained as a characteristic in the process of being eliminated or as a recent mutation; the absence of GsC in the flock could be interpreted as loss of the protein from this population or as indicative that GsC is not a jungle-fowl protein but arose in domestic breeds by mutation. Whatever the origin of the egg-white variants, birds hatched in Illinois from Cornell jungle-fowl eggs were observed to have several characteristics popularly associated with wild jungle fowl. Although kept under the same conditions as domestic breeds, the Cornell jungle fowl were more nervous and suspicious than their domestic contemporaries; the tail carriage of the jungle fowl males was usually low, in contrast to the erect tail carriage of most domestic roosters; and at least one jungle fowl male had an eclipse moult. With the exception of G sJ, the egg proteins of the Cornell jungle fowl were the same as the egg proteins found in the available populations of domestic fowl. This similarity of jungle fowl and domestic fowl proteins was found in the blood also. Dr. F. X. Ogasawara, of the University of California at Davis, supplied several blood samples from jungle fowl for other studies (Manwell et al., 1963). These samples were compared with samples from domestic fowl in several ways: the haemoglobin of Dr. Ogasawara’s jungle fowl is electrophoretically identical with the haemoglobin from domestic breeds; the oxygen equilibria of individual haemoglobin samples from adult domestic and jungle fowl are the same in all the cases studied; it has not yet been possible to distinguish jungle fowl from domestic fowl by differences in serum proteins, although other closely related galliform species can be separated from each other by this means. It would appear that whatever the evolutionary significance of G,’ the domestic fowl is so similar to the jungle fowl that the former should be referred to as G. gahs also and not arbitrarily assigned to a separate species, G. domesticus as is done by some authorities. Some comment on the electrophoretic behaviour of the globulin fractions is warranted. In the present work the observed behaviour of the G, and G, globulin fractions in the acetate buffer (pH 4.7) does not agree with the findings of Longsworth et al. (1940) who represented G, as migrating more rapidly than G, at low pH’s. There are two possible reasons for this discrepancy between the two studies. First, the “molecular sieve” action of the starch gel retards the migration of larger molecules, an effect not present in the moving boundary electrophoresis used by Longsworth et al. (1940). Thus it could be that the G, globulins are intrinsically larger than G, globulins; the same reasoning can be applied to the electrophoretic
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C. M. ANN BAKER
behaviour of the proteins within the G, fraction to account for the slower migration of GzD than G,” at both alkaline and acid pH’s in starch gels. Secondly, as the G, and G, fractions are quantitatively and qualitatively very similar in electrophoresis, and as the main criterion of Longsworth et al. (1940) for distinguishing between the two fractions was electrophoretic mobility, it is possible that the “G2” observed in acid buffers by these workers was actually G, and vice versa. If this were so, the behaviour of G, and G, in the low pH acetate buffers of Longsworth et al. (1940) and of the author could be due to some change in the structure, size or charges (or a combination of these factors) of the molecules of G,, G, or both. However, the behavior of the individual proteins within the G, and G, fractions suggests that the first reason-the action of the molecular sieveis the more likely. The differences observed between some GzD proteins in the acetate buffer are of interest also. Both the Sebright egg and the jungle-fowl egg were under 2 days old (the latter was laid by a pullet hatched from a Cornell jungle-fowl egg in Illinois) so the differences cannot be ascribed to deterioration. It is possible that a GzD polypeptide chain could have been altered in another way, as by the substitution of one amino acid for another, the addition of extra amino acids or by the These alterations could have lack of, or the duplication of, an existing segment. occurred in such a manner as to produce changes in the charge or size of the GzD molecules, only apparent in the low pH acetate gel. Unfortunately, the lack of facilities prevented identification of the birds by trap-nesting so, in this case, it was not possible to use them for further studies. The proteins sub-A, sub-B and sub-C do not seem to be artefacts produced by deterioration as they have been found in fresh eggs. Sub-A, sub-B and sub-C could be polymers of other globulin proteins or, if each of the latter consists of more than one chain, products of chain recombination. Acknozcledgements-This work has been supported by the United States Public Health Service (GM 7939) and from grants made to Dr. Clyde Manwell by the United States National Science Foundation (G13467, G18082 and GB612). The author wishes to record her gratitude to these agencies and to thank the United States Educational Commission in the United Kingdom for a Fulbright Travel Grant. The author thanks Dr. J. H. Bruckner for jungle-fowl eggs; Dr. A. W. Nordskog for Fayoumi eggs; Mr. A. J. Maggio for Phoenix, Polish, Sebright and Silkie eggs; Mrs. Mar> A. Petersen and Dr. H. Shoemaker for bantam eggs; Dr. H. M. Scott for nine New Hampshire hens; Messrs. A. L. Ogden and E. M. McDermid for egg-white samples from Yorkshire, for the Thornber strain six; and Messrs. Thornber Bros., of Mytholmroyd, provision of clerical assistance for the preparation of this manuscript. The author is especially indebted to Dr. C. Manwell for laboratory generously giving his time for advice, criticism and discussion.
facilities
and for
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lipovitellin. 1. The
egg-
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