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An electrophoretic analysis of plasma proteins of selected birds and snakes
Comp. Biochem. Physiol. Vol. 71B, pp. 313 to 316, 1982
0305-0491/82/020313-04503.00/0 Pergamon Press Ltd
Printed in Great Britain
AN ELECTROPHORETIC ANALYSIS OF PLASMA PROTEINS OF SELECTED BIRDS A N D SNAKES A. W. ROURKE Biology Department, University of Idaho, Moscow, ID 83843, U.S.A.
(Received 25 June 1981) Abstract--1. Two dimensional gel electrophoresis of plasma proteins of eight species of vertebrates revealed that this technique is of value in phylogenetic studies. 2. The technique was able to determine specific patterns at the subspecies level in the order Squamata of the class Reptilia and generally supported traditional taxonomic inter-relationships of the family Accipitridae and order Passeriformes in the class Aves.
INTRODUCTION There is an increasing number of reports in the literature attempting to determine the relatedness of populations within a species and between species using a variety of electrophoretic techniques. The vast majority of these reports analyze protein patterns and usually concentrate on constitutive proteins (Highton & Larson, 1979; Aya!a, 1975). It is common for such analyses to agree with classical analysis of the populations in question, but there are exceptions to this generality (Nolan et al., 1975). Some of our recent efforts have used two dimensional gel electrophoresis of plasma proteins and standard morphological parameters to analyze populations of trout where species interbreeding had occurred (Salmo clarki x Salmo gairdneri) (Rourke & Wallace, 1978). The data generated in this study seemed to indicate that this electrophoretic technique which analyzes both constitutive and nonconstitutive protein components of plasma, holds some promise for studies attempting to determine the relatedness of various groups of animals. The present studies represent an extension of this procedure to higher vertebrates. Groups of animals within two classes were chosen which contain taxonomic groups thought to be closely related and distantly related. Selections were made in an effort to determine the worth of two dimensional gel electrophoresis in helping to elucidate relatedness. One set of animals consisted of four species of mature female snakes from three different families. Thamnophis eleoans represented the family Colubridae, Naja naja represented the family Elapidae, and the family Viperidae was represented by two subspecies of Crotalis viridis, C.v. lutosus and C.v. ore#anus. In addition to these members of the Order Squamata, four different species of birds were examined. These included the starling, Sturnus vulgaris, Cooper's hawk, Accipiter cooperi, the Goshawk, Accipter gentilis and the red tail hawk, Buteo jamaicensis. These four birds were selected so as to have two species of one genus, three members in one family, Accipitridae, and one species from the Order Passeriformes, an order with a number of recentlyderived, rapidly evolving species (Darlington, 1966). Selection of both birds and snakes was conducted in a fashion to allow examination of organisms that are
presumably closely related as well as organisms that are not as closely related phylogenetically. MATERIALS AND METHODS Avian blood was collected from the wing vein of wild birds. The blood was collected into heparinized syringes and placed on ice in the field. The samples were brought to the laboratory within 48 hr, centrifuged to remove cells, and the supernatants stored for not more than five weeks at -80°C. When samples were to be electrophoresed, they were thawed, protein determinations were carried out using the BioRad procedure, and the two dimensional gels were run, stained and destained as previously described (Rourke & Wallace, 1978). In these procedures proteins were separated in the first dimension disc gel as a function of charge. All gels were run with the anode in the bottom reservoir and the cathode in the upper reservoir, Following this run, the gels were removed and placed on top of Pharmacia slab gels PAA/4. These gels separated proteins in the 50,000-2,000,000 tool. wt range. The disc gels were always laid on top of the slab gels so that the end of the disc gel near the cathode of the first run was in the upper left hand corner of the slab gel. All snake blood samples were collected by cardiac puncture. The samples were treated and analyzed in a fashion identical to that for bird blood but the snake samples were collected and analyzed before the bird samples. RESULTS
Snake data The procedures employed were able to detect differences in patterns at the supspecies level. While the amount of protein analyzed from C. viridis lutosus was nearly equal to that analyzed for C. viridis ore#anus, (~200?tg) C.v. ore#anus had one pronounced intermediate molecular weight protein not found in the plasma of C.v. lutosus (spot 2, Fig. 1), and one less well defined protein of higher molecular weight (spot 1). In addition, there was one protein with a mol. wt lower than protein 2 that migrated toward the anode in the first dimension and was found only in C.v. lutosus, spot 3. The bulk of the proteins in these two subspecies including the high molecular weight proteins at the top of the gel and the lowest molecular weight proteins at the bottom of the gel, spots 5 and 6, were very similar in their migratory patterns.
313
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A.W. ROURKE
Fig. I. Electrophoretic patterns of plasma proteins in samples prepared from C.v. lutosus (a), C.v. oreganus (b), N. naja (c) and T. eleganus (d). Enough plasma was run on each gel to give about 200 #g of protein and the gels were stained with Coomassie Blue. The general pattern displayed by T. elegans bears definite similarities with the two subspecies of rattlesnakes, but there are also clear differences. T. eleoans does not appear to have any proteins at areas 2 and 3. In addition, T. elegans has a low tool. wt protein that remained near the cathode during electrophoresis in the first dimension, spot 4. T. elegans does not display the same two spot pattern (5, 6) characteristic of the lowest mol. wt proteins found in the subspecies of C.
viridis. The pattern displayed by Naja naja is different from that displayed by the three other snakes. The major overriding difference is that so many proteins of various molecular weights remained at the cathode end of the first dimension gel. It appears that Naja naja has a spot in common with T. elegans (4) and that the profiles of these two snakes for the lowest mol. wt components bear more similarities to each other than to either subspecies of C. viridis. Interestingly, Naja naja also has more proteins of intermediate mol. wt that have migratory patterns like those found in the rattlesnakes, spots 1 3. It is not, however, possible to determine if the mobilities of these areas in Naja correspond exactly to those of the C. viridis subspecies. Thus the pattern of Naja is clearly unique, but it does bear some resemblance to those of T. elegans as well as C.v. oreganus and C.v.
lutosus. Bird data Although the same amount of protein was run on these gels as the snake gels, the patterns of the bird protein are not as complicated as those seen for the snakes and in general the members of the family Accipitridae showed more similarities to each other than to the one representative of the Order Passeriformes,
Sturnis vulgaris. While the starling had the most complex pattern, it clearly showed similarities to those of the three hawks examined (Fig. 2). The most intense lower tool. wt fraction found on all gels has been labelled as 1. This spot has a long tail toward the cathode end of the first gel in A. cooperii and S. vulgaris and a small tail in the gel for B. jamaicensis. Only A. gentilis has a tail that is towards the anode end of the first gel. Thus, two members of Accipitridae have characteristics in common with S. vulgaris. The next major spot has been labelled 2 and it is clearly similar in all hawks. In addition, the location and staining of a less prevalent band, spot 3, is quite similar in the hawk samples and appears identical in the two species of Accipiter. The starling is clearly different relative to areas 2 and 3 in that both areas appear to have two peaks and because spot 3 stains more intensely in S. vulgaris than in any of the hawks. The two Accipiter species show similar patterns in the region of spot 4, while B. jamaicensis and S. vulgaris show definite similarities in the area of spot 4. Finally, S. vulgaris has two bands just above spot 3, a situation also found in A. cooperii. The results generally indicate the greatest degree of similarity among the three members of the family Accipitridae, but S. vulgaris showed definite similarities with B. jamaicensis (spots 4 and 1); Accipter cooperil (spots 1 and above 3) and Accipter gentilis (general pattern), DISCUSSION
Phylogenetic interrelationships are best based on a variety of data. It is all too possible to get false impressions if one relies on one type of data. There are
Electrophoresis of vertebrate plasma proteins
315
Fig. 2. Electrophoretic patterns of plasma proteins in samples prepared from A. cooperii (a), A. gentilis (b), B. jamaicensis (c) and S. vulgaris (d). Conditions as in Fig. 1.
data available from birds that make it clear that there are instances when there is not a strict correlation between degree of genetic differences and degree of difference at the organismal level (Nolan et al., 1975). The results of Nolan et al. indicate that the protein differences between turkey-chicken profiles and those of pheasant-chickens are not those anticipated on the basis of classical bird taxonomy. While it is common to assume that protein evolution and organismal evolution proceed in unison, it may well be that these two types of evolution can proceed independently. It is becoming clear that mutations in key regulatory elements could possibly have had profound influences on organismal evolution and yet not necessarily result in a great influence on the evolution of proteins per se (Kolata, 1980). It was thus of considerable interest to examine some organisms with fairly well established taxonomic interrelationships with a procedure that examines many proteins at once. Although the phylogenetic interrelationships of the snake families is far from clear cut, it is generally believed that the Colubridae, Elapidae and Viperidae are closely related and that the Colubidae gave rise to both the Elapidae and the Viperidae (Goin et al., 1978; Kardong, 1981). The results of the present study are consistent with this as the general pattern of T. elegans, C.v. lutosus and C.v. oreganus are similar and yet T. elegans and N. naja show similarities at spot 4. The overall profile of the cobra is, however, different from the other snakes in that the amount of material of various tool. wt that remained at the cathode end of the first dimension gel is far greater than that observed in the other three snakes. The data are consistent with the notion that organismal evolution is reflected by the changes that have occurred in various proteins. It would be possible, however, to examine a restricted group of proteins and come to a C'.B.P. 71/2B
K
different conclusion. If one had examined only spot 2 of figure one, one would have found a protein or class of proteins showing considerable dissimilarities in two subspecies of rattlesnakes. The overall profiles displayed by the four species of birds assayed are clearly far simpler and more defined than that of the simplest snake profile displayed by T. elegans. There are clear similarities between the starling and the redtail hawk (spots 1 and 4), and the starling and the cooper's hawk (spot 1). Similarly all three hawks show similarities in spots 2 and 3 and these birds are more similar to one another than to the profile of the starling. Thus the data are once again consistent with the concept that organismal evolution is reflected by protein evolution. Perhaps the most interesting area on these gels is spot 1 (Fig. 2). The tail on this spot is towards the cathode of the first dimension gel in A. cooperii, B. jamaicensis, and S. vulgaris while the tail is anodal in A. gentilis. Thus if one were to examine just this area, one might conclude that certain interrelationships exist which are not those normally accepted. The data gathered for both birds and snakes point to the utility of employing two dimensional gels in studying animal interrelationships. It is not clear which spots represent constitutive proteins and which do not. Interpretation is further hampered by the fact that posttranslation modification such as glycosylation, phosphorylation and enzymatic cleavage can make profile interpretations difficult. However, the present study has shown differences at the subspecies level and is generally consistent with established taxonomic patterns. Further interpretation of such profiles is now theoretically possible at the DNA level as the gene encoding any protein detected as a direct spot on a two dimensional gel can, in principle, be isolated. This offers the interesting possibility of examining
316
A.W. ROURKE
what types of changes in D N A are responsible for the unexpected types of profiles exemplified by spot 1 of the bird plasma samples. Acknowledgements--The author thanks R. J. Naskali for assistance in photographing the gels. Darwin Vest and Kevin Moore supplied technical assistance in bleeding the animals used in this study. REFERENCES
AVALAF. J. (1975) Genetic Differentiation During the Speciation Process. Evol. Biol. 8, 1-78. DARLIN6TON P. J. (1966) Zoogeography the Geographical Distribution of Animals, 4th edn, pp. 274-275. Wiley, New York.
GOIN C. J., GOIN O. B. & ZUG G. R. (1978) Introduction to Herpetology, 3rd edn, pp. 309-310. Freeman, San Francisco. HIGHTON R. & LARSON A. (1979) The genetic relationships of the salamanders of the genus Plethodon. Syst. Zool. 28, 579-599. KARDONG K. (1981) personal communication. KOLATA G. B. (1980) Genes in pieces. Science 207, 392 393. NOLAN R. A., BRUSH A. H., ARMHEI~ N. & WILSON A. C. (1975) An inconsistency between protein resemblance and taxonomic resemblance:immunological comparison of diverse proteins from gallinaceous birds. Condor 77, 154-159. ROURKE A. W. & WALLACE R. L. (1978) A morphological and electrophoretic comparison of Henry's Lake Salmo clarki and Salmo clarki x Salmo gairdneri hybrids. Comp. Biochem. Physiol. 60B, 447~,51.
0305-0491/82/020313-04503.00/0 Pergamon Press Ltd
Printed in Great Britain
AN ELECTROPHORETIC ANALYSIS OF PLASMA PROTEINS OF SELECTED BIRDS A N D SNAKES A. W. ROURKE Biology Department, University of Idaho, Moscow, ID 83843, U.S.A.
(Received 25 June 1981) Abstract--1. Two dimensional gel electrophoresis of plasma proteins of eight species of vertebrates revealed that this technique is of value in phylogenetic studies. 2. The technique was able to determine specific patterns at the subspecies level in the order Squamata of the class Reptilia and generally supported traditional taxonomic inter-relationships of the family Accipitridae and order Passeriformes in the class Aves.
INTRODUCTION There is an increasing number of reports in the literature attempting to determine the relatedness of populations within a species and between species using a variety of electrophoretic techniques. The vast majority of these reports analyze protein patterns and usually concentrate on constitutive proteins (Highton & Larson, 1979; Aya!a, 1975). It is common for such analyses to agree with classical analysis of the populations in question, but there are exceptions to this generality (Nolan et al., 1975). Some of our recent efforts have used two dimensional gel electrophoresis of plasma proteins and standard morphological parameters to analyze populations of trout where species interbreeding had occurred (Salmo clarki x Salmo gairdneri) (Rourke & Wallace, 1978). The data generated in this study seemed to indicate that this electrophoretic technique which analyzes both constitutive and nonconstitutive protein components of plasma, holds some promise for studies attempting to determine the relatedness of various groups of animals. The present studies represent an extension of this procedure to higher vertebrates. Groups of animals within two classes were chosen which contain taxonomic groups thought to be closely related and distantly related. Selections were made in an effort to determine the worth of two dimensional gel electrophoresis in helping to elucidate relatedness. One set of animals consisted of four species of mature female snakes from three different families. Thamnophis eleoans represented the family Colubridae, Naja naja represented the family Elapidae, and the family Viperidae was represented by two subspecies of Crotalis viridis, C.v. lutosus and C.v. ore#anus. In addition to these members of the Order Squamata, four different species of birds were examined. These included the starling, Sturnus vulgaris, Cooper's hawk, Accipiter cooperi, the Goshawk, Accipter gentilis and the red tail hawk, Buteo jamaicensis. These four birds were selected so as to have two species of one genus, three members in one family, Accipitridae, and one species from the Order Passeriformes, an order with a number of recentlyderived, rapidly evolving species (Darlington, 1966). Selection of both birds and snakes was conducted in a fashion to allow examination of organisms that are
presumably closely related as well as organisms that are not as closely related phylogenetically. MATERIALS AND METHODS Avian blood was collected from the wing vein of wild birds. The blood was collected into heparinized syringes and placed on ice in the field. The samples were brought to the laboratory within 48 hr, centrifuged to remove cells, and the supernatants stored for not more than five weeks at -80°C. When samples were to be electrophoresed, they were thawed, protein determinations were carried out using the BioRad procedure, and the two dimensional gels were run, stained and destained as previously described (Rourke & Wallace, 1978). In these procedures proteins were separated in the first dimension disc gel as a function of charge. All gels were run with the anode in the bottom reservoir and the cathode in the upper reservoir, Following this run, the gels were removed and placed on top of Pharmacia slab gels PAA/4. These gels separated proteins in the 50,000-2,000,000 tool. wt range. The disc gels were always laid on top of the slab gels so that the end of the disc gel near the cathode of the first run was in the upper left hand corner of the slab gel. All snake blood samples were collected by cardiac puncture. The samples were treated and analyzed in a fashion identical to that for bird blood but the snake samples were collected and analyzed before the bird samples. RESULTS
Snake data The procedures employed were able to detect differences in patterns at the supspecies level. While the amount of protein analyzed from C. viridis lutosus was nearly equal to that analyzed for C. viridis ore#anus, (~200?tg) C.v. ore#anus had one pronounced intermediate molecular weight protein not found in the plasma of C.v. lutosus (spot 2, Fig. 1), and one less well defined protein of higher molecular weight (spot 1). In addition, there was one protein with a mol. wt lower than protein 2 that migrated toward the anode in the first dimension and was found only in C.v. lutosus, spot 3. The bulk of the proteins in these two subspecies including the high molecular weight proteins at the top of the gel and the lowest molecular weight proteins at the bottom of the gel, spots 5 and 6, were very similar in their migratory patterns.
313
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A.W. ROURKE
Fig. I. Electrophoretic patterns of plasma proteins in samples prepared from C.v. lutosus (a), C.v. oreganus (b), N. naja (c) and T. eleganus (d). Enough plasma was run on each gel to give about 200 #g of protein and the gels were stained with Coomassie Blue. The general pattern displayed by T. elegans bears definite similarities with the two subspecies of rattlesnakes, but there are also clear differences. T. eleoans does not appear to have any proteins at areas 2 and 3. In addition, T. elegans has a low tool. wt protein that remained near the cathode during electrophoresis in the first dimension, spot 4. T. elegans does not display the same two spot pattern (5, 6) characteristic of the lowest mol. wt proteins found in the subspecies of C.
viridis. The pattern displayed by Naja naja is different from that displayed by the three other snakes. The major overriding difference is that so many proteins of various molecular weights remained at the cathode end of the first dimension gel. It appears that Naja naja has a spot in common with T. elegans (4) and that the profiles of these two snakes for the lowest mol. wt components bear more similarities to each other than to either subspecies of C. viridis. Interestingly, Naja naja also has more proteins of intermediate mol. wt that have migratory patterns like those found in the rattlesnakes, spots 1 3. It is not, however, possible to determine if the mobilities of these areas in Naja correspond exactly to those of the C. viridis subspecies. Thus the pattern of Naja is clearly unique, but it does bear some resemblance to those of T. elegans as well as C.v. oreganus and C.v.
lutosus. Bird data Although the same amount of protein was run on these gels as the snake gels, the patterns of the bird protein are not as complicated as those seen for the snakes and in general the members of the family Accipitridae showed more similarities to each other than to the one representative of the Order Passeriformes,
Sturnis vulgaris. While the starling had the most complex pattern, it clearly showed similarities to those of the three hawks examined (Fig. 2). The most intense lower tool. wt fraction found on all gels has been labelled as 1. This spot has a long tail toward the cathode end of the first gel in A. cooperii and S. vulgaris and a small tail in the gel for B. jamaicensis. Only A. gentilis has a tail that is towards the anode end of the first gel. Thus, two members of Accipitridae have characteristics in common with S. vulgaris. The next major spot has been labelled 2 and it is clearly similar in all hawks. In addition, the location and staining of a less prevalent band, spot 3, is quite similar in the hawk samples and appears identical in the two species of Accipiter. The starling is clearly different relative to areas 2 and 3 in that both areas appear to have two peaks and because spot 3 stains more intensely in S. vulgaris than in any of the hawks. The two Accipiter species show similar patterns in the region of spot 4, while B. jamaicensis and S. vulgaris show definite similarities in the area of spot 4. Finally, S. vulgaris has two bands just above spot 3, a situation also found in A. cooperii. The results generally indicate the greatest degree of similarity among the three members of the family Accipitridae, but S. vulgaris showed definite similarities with B. jamaicensis (spots 4 and 1); Accipter cooperil (spots 1 and above 3) and Accipter gentilis (general pattern), DISCUSSION
Phylogenetic interrelationships are best based on a variety of data. It is all too possible to get false impressions if one relies on one type of data. There are
Electrophoresis of vertebrate plasma proteins
315
Fig. 2. Electrophoretic patterns of plasma proteins in samples prepared from A. cooperii (a), A. gentilis (b), B. jamaicensis (c) and S. vulgaris (d). Conditions as in Fig. 1.
data available from birds that make it clear that there are instances when there is not a strict correlation between degree of genetic differences and degree of difference at the organismal level (Nolan et al., 1975). The results of Nolan et al. indicate that the protein differences between turkey-chicken profiles and those of pheasant-chickens are not those anticipated on the basis of classical bird taxonomy. While it is common to assume that protein evolution and organismal evolution proceed in unison, it may well be that these two types of evolution can proceed independently. It is becoming clear that mutations in key regulatory elements could possibly have had profound influences on organismal evolution and yet not necessarily result in a great influence on the evolution of proteins per se (Kolata, 1980). It was thus of considerable interest to examine some organisms with fairly well established taxonomic interrelationships with a procedure that examines many proteins at once. Although the phylogenetic interrelationships of the snake families is far from clear cut, it is generally believed that the Colubridae, Elapidae and Viperidae are closely related and that the Colubidae gave rise to both the Elapidae and the Viperidae (Goin et al., 1978; Kardong, 1981). The results of the present study are consistent with this as the general pattern of T. elegans, C.v. lutosus and C.v. oreganus are similar and yet T. elegans and N. naja show similarities at spot 4. The overall profile of the cobra is, however, different from the other snakes in that the amount of material of various tool. wt that remained at the cathode end of the first dimension gel is far greater than that observed in the other three snakes. The data are consistent with the notion that organismal evolution is reflected by the changes that have occurred in various proteins. It would be possible, however, to examine a restricted group of proteins and come to a C'.B.P. 71/2B
K
different conclusion. If one had examined only spot 2 of figure one, one would have found a protein or class of proteins showing considerable dissimilarities in two subspecies of rattlesnakes. The overall profiles displayed by the four species of birds assayed are clearly far simpler and more defined than that of the simplest snake profile displayed by T. elegans. There are clear similarities between the starling and the redtail hawk (spots 1 and 4), and the starling and the cooper's hawk (spot 1). Similarly all three hawks show similarities in spots 2 and 3 and these birds are more similar to one another than to the profile of the starling. Thus the data are once again consistent with the concept that organismal evolution is reflected by protein evolution. Perhaps the most interesting area on these gels is spot 1 (Fig. 2). The tail on this spot is towards the cathode of the first dimension gel in A. cooperii, B. jamaicensis, and S. vulgaris while the tail is anodal in A. gentilis. Thus if one were to examine just this area, one might conclude that certain interrelationships exist which are not those normally accepted. The data gathered for both birds and snakes point to the utility of employing two dimensional gels in studying animal interrelationships. It is not clear which spots represent constitutive proteins and which do not. Interpretation is further hampered by the fact that posttranslation modification such as glycosylation, phosphorylation and enzymatic cleavage can make profile interpretations difficult. However, the present study has shown differences at the subspecies level and is generally consistent with established taxonomic patterns. Further interpretation of such profiles is now theoretically possible at the DNA level as the gene encoding any protein detected as a direct spot on a two dimensional gel can, in principle, be isolated. This offers the interesting possibility of examining
316
A.W. ROURKE
what types of changes in D N A are responsible for the unexpected types of profiles exemplified by spot 1 of the bird plasma samples. Acknowledgements--The author thanks R. J. Naskali for assistance in photographing the gels. Darwin Vest and Kevin Moore supplied technical assistance in bleeding the animals used in this study. REFERENCES
AVALAF. J. (1975) Genetic Differentiation During the Speciation Process. Evol. Biol. 8, 1-78. DARLIN6TON P. J. (1966) Zoogeography the Geographical Distribution of Animals, 4th edn, pp. 274-275. Wiley, New York.
GOIN C. J., GOIN O. B. & ZUG G. R. (1978) Introduction to Herpetology, 3rd edn, pp. 309-310. Freeman, San Francisco. HIGHTON R. & LARSON A. (1979) The genetic relationships of the salamanders of the genus Plethodon. Syst. Zool. 28, 579-599. KARDONG K. (1981) personal communication. KOLATA G. B. (1980) Genes in pieces. Science 207, 392 393. NOLAN R. A., BRUSH A. H., ARMHEI~ N. & WILSON A. C. (1975) An inconsistency between protein resemblance and taxonomic resemblance:immunological comparison of diverse proteins from gallinaceous birds. Condor 77, 154-159. ROURKE A. W. & WALLACE R. L. (1978) A morphological and electrophoretic comparison of Henry's Lake Salmo clarki and Salmo clarki x Salmo gairdneri hybrids. Comp. Biochem. Physiol. 60B, 447~,51.