A survey of tissue-specific isozyme expressions during ... - Springer Link

Biochemical Genetics, Vol. 25, ?Cos.3/4, 1987

A Survey of Tissue-Specific Isozyme Expressions During Chicken Ontogeny S. C. Lougheed 1'2 and B. W. C. Rosser 1'3

Received 11 Dec. 1985--Final5 Dec. 1986

The ontogenetic trends in the expression of 25 isozymes in liver, g: td, heart, and pectoralis muscle of White Leghorn chickens were examined using starch gel electrophoresis. Little change in expression during development was evident in liver S-AAT-A, GPI-A, S-ICDH-A, S-MDH-A and M-MDH-A, in gizzard S-ACON-A, ADH-A, GPI-A, HK-1, HK-3, ME-A PEP-l, and PGM-A, in heart ADH-A, HK-1, HK-3, ME-A, PEP-2, PGM-A, and LDH-A, in pectoralis M-ACON-A, S-ACON-A, ADH-A, HK-1, HK-3, ME-A, PEP-2, and PGM-A, and in liver, gizzard, and heart M-ACON-A, ALD-A, CK-A, G3PDH-A, HK-I, and PGDH-A. Increasing levels of activity were demonstrated in liver ADH-A, ME-A, and PEP-2, in heart M-MDH-A, S-ICDH-A, M-ICDH, and M-AA T-A, and in pectoralis LDH-A, LDH-B, G3PDH-3, ALD-A, CK-A, HK-2, and PGM-B. There was a decrease in the activity of HK-1 in liver and in PEP-1 and PGDH-A in pectoralis muscle throughout development. While CK-C is active in the embryonic pectoralis, CK-A is restricted to later developmental stages. Isozyme expressions in regions of the pectoralis containing fast and slow muscle fibers in 7-month-posthatch individuals were noted and found to be identical. The results underscore the need to use similar developmental stages and tissue samples in comparative electrophoretic studies of birds. KEY WORDS: ontogeny; chicken; isozymes.

This study was supported by Natural Sciences and Engineering Research Council of Canada Grant A9866 to J.P.B. 1 Department of Zoology, University of Guelph, Guelph, Ontario N 1G 2W 1, Canada. 2 Present address: Department of Zoology, University of Western Ontario, London, Ontario N6A 5B7, Canada. 3 Present address: Department of Anatomy, University of Maryland School of Medicine, Baltimore, Maryland 21201. 275 0006-2928/87/0200-0275505.00/0

© 1987 Plenum Publishing Corporation

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INTRODUCTION Many isozymes demonstrate unique spatial and temporal ontogenetic trends of expression within a species (Buth, 1984). Such trends have been documented in a variety of isozymes for numerous taxa (Masters and Holmes, 1972; Ureta, 1982; Goldspink and Lewis, 1985). Few studies, however, have dealt with developmental profiles of avian isozyme expression during development, and these studies have been restricted in the numbers of enzymes and tissues examined (see Matson, 1984). Leung and Haley (1974) have studied the trends in PGDH 4 (EC 1.1.1.43) and G6PDH (EC 1.1.1.49) expression during the embryogenesis of chicken-quail hybrids, Meyerhof and Haley (1975) have studied the ontogenetic patterns of LDH (EC 1.1.1.27) in chicken-quail hybrids, and Wittenberger and Coprean (1981) have studied the postclosional changes in glycogen phosphorylase activity in chick pectoralis muscle. The objective of the present study is to delineate more fully the occurrence and tissue specificities of a wide variety of isozymes during the development of the chicken (Gallus gallus). Additionally, this study provides an indication of those avian tissues which may provide maximum activity of certain enzymes. MATERIALS AND METHODS Two White Leghorns from each of four developmental stages were obtained from a local supplier: 10-day embryo, 1 day posthatch, 7 days posthatch, and 7 months posthatch. These were sacrificed by an intraperitoneal injection of a euthanasic solution (T-61; Hoechst Canada Inc., Montreal). Samples of liver, m. pectoralis, gizzard, and heart were immediately removed from each bird. Additionally, in the 7-month-posthatch individuals, samples containing fasttwitch (100%) and slow-twitch (30% slow, 70% fast) muscle fibers, respectively, were removed from the superficial and deep regions of the pectoralis muscle (see Matsuda et al., 1983). The volumes of all samples obtained were approximately equal. An equal volume of grinding solution [1.21 g Tris, 0.37 g EDTA. H20, 40 mg NADP in 1.0 liter deionized H20 with the p H adjusted with approximately 18 drops stock HC1 (Selander et al., 1971)] was added to each sample. Samples were maintained at - 8 0 ° C until they were used. Prior to electrophoresis, the still-frozen samples were homogenized using a glass rod and then clarified by centrifugation in a Fisher Micro-centrifuge, Model 235A, for 2 min. Wicks used to load a portion of each supernatant into the 12% horizontal starch gels were cut from Whatman No. 3 filter paper. This

4See Table I for enzymeabbreviations.

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277

study involved the use of the three electrophoretic buffers listed in Table 2. A total of 20 enzyme stains was employed, following Shaw and Prasad (1970) or Selander et al. (1971) (Table I). Electrophoretic methods were those of Bogart (!982). RESULTS AND DISCUSSION

The resolution and/or staining intensities of four of the enzymes assayed were too poor to establish properly any tissue-specific trends: MPI, ACP, AP, and G6PDH. The banding patterns of various esterases were complex and thus tissue-specific trends for individual EST isozymes were not scored. However, a general trend toward maximum EST activity (most bands and greatest activity compared to other tissues) was noted in liver. For the remaining 15 enzyme systems, a total of 25 presumptive isozymes was scored. Representative results are shown in Figs. 1 through 3. Increasing or decreasing trends in Table I. Summary of Enzymes Studied and Electrophoretic Conditions Used for Each

Enzyme (abbreviation)

EC No. ~

Acid phosphatase (ACP) Aconitase (ACON) Alcohol dehydrogenase (ADH) Aldolase (ALD) Aspartate aminotransferase (AAT) Creatine kinase (CK) Cytosol aminopeptidase (AP) Esterase (EST) c Glucose-6-phosphate dehydrogenase (G6PDH) Glucose-6-phosphate isomerase (GPI) Glycerol-3-phosphate dehydrogenase (G3PDH) Hexokinase (HK) Isoeitrate dehydrogenase (ICDH) Lactate dehydrogenase (LDH) Malate dehydrogenase (MDH) Mannose-6-phosphate isomerase (MPI) NADP-dependent malate dehydrogenase (ME) Peptidase (PEP) a Phosphoglucomutase (PGM) Phosphogluconate dehydrogenase (PGDH)

3.1.3.2 4.2.1.3 1.1.1.1 4.1.2.13 2.6.1.1 2.7.3.2 3.4.11.1 3.1.1.1 1.1.1.49 5.3.1.9 1.1.1.8 2.7.1.1 1.1.1.42 1.1.1.27 1.1.1.37 5.3.1.8 1.1.1.40 3.4A 1 or .13 5.4.2.2 1.1.1.44

Number of Electrophoretic loci resolved bufferb ? 2 1 1 2 2 ? ? ? 1 1 3 2 2 2 ? 1 2 2 1

A A B B C A B A B B A C B C A A A C A C

~Source: Nomenclature Committee of the International Union of Biochemistry (1984). hA, morpholine citrate gel and electrode buffers adjusted to pH 6.5 (Clayton and Tretiak, 1972); B, continuous Tris-citrate I buffer (Selander et al., 197t); C, discontinuous Tris-citrate (Poulik) buffer (Selander et al., 1971). cAlpha-naphthyl acetate used as a substrate for esterase. aPeptides used as substrates for peptidase: gIycyl-L-leucine, L-valyl-L-tyrosine, L-tryptophylL-alanine, L-leueyl-L-tyrosine, L-leucyl-/%alanine, and L-leucyl-L-alanine.

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::ji!!~!:!ii!; HK 1 ~_~HK

2

/ H K

3

ORIGIN

MUSCLE HEART LIVER

GIZZARD

Fig. 1. Discontinuous Tris-citrate starch gel (pH 6.7) stained for hexokinase. Lanes l, 6, 10, and 14 represent tissue homogenates from a 10-day embryo. Lanes 2, 7, 11, and 15 represent tissue homogenates from a 1-day-posthatch individual. Lanes 3, 8, 12, and 16 represent tissue homogenates from a 7-day-posthatch individual. Lanes 4, 5, 9, 13, and 17 represent tissue homogenates from a 7-month-posthatch individual. Lanes 4 and 5 represent slow- and fast-twitch muscle samples, respectively.

the activities of the resolved electrophoretic products are indicated in Table II. Temporal and spatial trends in activities are discussed in turn for each enzyme.

Aconitase (EC 4.2.1.3). Two forms of aconitase commonly occur in vertebrates, a mitochondrial and a soluble form, encoded by separate autosoreal loci (Harris and Hopkinson, 1976). Table |I indicates that M-ACON-A is active in liver in all ontogenetic stages but not in the other three tissues examined. This is in contrast to S-ACON-A, which appears to be active in all four tissues throughout development. S-ACON-A is most active in liver and heart, which reduced levels in gizzard and m. pectoralis. Alcohol Dehydrogenase (EC 1.1.1.1). Fisher et al. (1980) have observed that the majority of chordate species possesses a single ADH isozyme. In G. gallus ADH-A activity is restricted to liver (Table II). No activity was detected in 10-day embryo liver, gradually increasing to maximal activity in the 7-day-posthatch and 7-month-posthatch individuals. This increasing trend of ADH activity in liver concurs with observations of ontogenetic trends in overall ADH activity levels in humans (Masters and Holmes, 1972; Harris and Hopkinson, 1976).

lsozyme ExpressionsDuring ChickenOntogeny

279

____-- S M D H - A

OR,G

N ~ M -

MUSCLE

HEARr

LIVER

MDI-t -A

GIZZARD

Fig. 2. Morpholine citrate starch gel ( p H 6.5) stained for malate dehydrogenase. The order of the lanes is the same as that in Fig. 1.

Aldolase (EC 4.1.2.13). Masters and Holmes (1972) suggested that there are at least two avian aldolase isozymes. ALD-C, the predominant isozyme in embryonic tissues, is replaced in later ontogenetic stages by ALD-A (Masters and Holmes, 1972). In the present study, only one zone of aldolase activity was detected (Table II). ALD activity occurred in m. pectoralis tissue of 7-month-posthatch individuals and of adults. It is probable, therefore, that ALD-A was detected.

- ~

~ ~" ~

~ ~

~

PEP 1

--PEP

'

2

ORIGIN

1

2 3 4 5 6 7 8 91011121314151617

MUSCLE

HEART

LIVER

GIZZARD

Fig. 3. Discontinuous Tris-citrate starch gel ( p H 6.7) stained for peptidase. The order of the lanes is the same as that in Fig. 1.

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Lougheed and Rosser

Table II. Relative Staining Intensitiesa of Various Isozymes in Four Different Tissues from Four Ontogenetic Stagesb of White Leghorns (Gallus gallus) I

I

Liver

Gizzard

Heart

M. pectoralis

Locus

A

B

C

D

A

B

C

D

A

B

C

D

A

B

C

D-S

D-F

M-ACON-A S-ACON-A ADH-A ALD-A S-AAT-A M-AAT-A CK-A CK-C GPI-A G3PDH-A HK-1 HK-2 HK-3 S-ICDH-A M-ICDH-A LDH-A LDH-B S-MDH-A M-MDH-A ME-A PEP-1 PEP-2 PGM-A PGM-B PGDH-A

3 2 0 0 3 1 0 0 2 0 3 0 0 3 0 4 0 3 3 0 3 0 2 0 3

3 3 2 0 3 1 0 0 2 0 3 0 1 3 3 4 0 3 3 0 4 1 3 2 3

3 3 3 0 3 2 0 1 2 0 2 0 1 3 3 3 ! 3 3 3 4 2 3 2 3

3 2 3 0 3 2 0 1 2 0 2 0 2 3 3 1 2 3 3 3 4 3 3 2 3

0 1 0 0 2 0 0 2 1 0 2 0 0 0 0 1 0 2 2 0 3 0 0 0 3

0 1 0 0 3 1 0 4 1 0 2 0 0 2 2 2 2 3 3 0 3 1 0 2 3

0 1 0 0 3 1 0 4 1 0 2 0 0 2 2 1 0 3 3 0 3 1 0 2 3

0 1 0 0 3 1 0 4 1 0 2 0 0 2 2 1 0 3 3 0 3 1 0 2 3

0 2 0 0 3 0 0 3 2 0 2 0 0 0 0 3 0 2 2 0 2 0 0 1 3

0 3 0 0 3 1 0 3 2 0 2 0 0 1 2 3 0 3 3 0 2 0 0 2 3

0 3 0 0 4 2 0 4 3 0 2 0 0 1 2 3 0 3 4 0 3 0 0 2 3

0 3 0 0 4 2 0 4 3 0 2 0 0 2 3 3 0 3 4 0 2 0 0 2 3

0 1 0 0 2 0 0 2 2 0 0 0 0 0 0 0 0 1 1 0 3 0 0 0 3

0 1 0 0 2 0 2 0 2 1 0 0 0 2 0 0 1 2 2 0 2 0 0 1 2

0 1 0 3 2 0 3 0 3 3 0 2 0 2 2 1 3 2 2 0 2 0 0 3 1

0 1 0 3 3 1 3 0 3 4 0 2 0 2 2 2 3 2 2 0 1 0 0 4 1

0 1 0 3 3 1 3 0 3 4 0 2 0 2 2 2 3 2 2 0 1 0 0 4 1

alntensity was scored subjectively based on a visual assessment of each gel. Staining intensities ranged from no activity (0) to maximum (4). bThe ontogenetie stages are 10-day embryo (A), 1 day posthatch (B), 7 days posthatch (C), and 7 months posthatch (D). D-S and D-F refer to slow- and fast-contracting fibers, respectively, from the 7-month-posthatch individuals.

Aspartate Aminotransferase (EC 2.6.1.1).

T w o a u t o s o m a l loci e n c o d e

t h e m i t o c h o n d r i a l a n d s o l u b l e f o r m s o f t h i s e n z y m e in h u m a n s ( H a r r i s a n d H o p k i n s o n , 1976). S i m i l a r l y , t w o A A T i s o z y m e s h a v e b e e n f o u n d in a v a r i e t y of other vertebrates (Avise

et al.,

1980a; B o g a r t

C r a b t r e e , 1985). S - A A T - A is e v i d e n t in all f o u r

et al., 1985; M u r p h y a n d G. gallus t i s s u e s e x a m i n e d

a n d in all o n t o g e n e t i c s t a g e s ( T a b l e I I ) . In g i z z a r d , h e a r t , a n d m . p e c t o r a l i s , t h e r e is a s l i g h t i n c r e a s e in a c t i v i t y in t h e l a t e r s t a g e s . T h e m i t o c h o n d r i a l f o r m o f t h e e n z y m e is less i n t e n s e l y s t a i n e d in all t i s s u e s . A g r a d u a l i n c r e a s e in M - A A T - A a c t i v i t y is n o t e d in all t i s s u e s t h r o u g h o u t d e v e l o p m e n t , w i t h liver and heart displaying the most intense staining of the M-AAT-A isozyme. Nakata

et al.

(1964) have e x a m i n e d the o n t o g e n y of rat A A T isozymes. They

lsozyme Expressions During Chicken Ontogeny

281

noted that the ratio of AAT-A2 (presumably S-AAT-S) to AAT-B2 (presumably M-AAT-A) activity in the rat fetus was approximately 10:1. After birth AAT-Az activity increased, while AAT-B2 remained constant.

Creatine Kinase (EC 2.7.3.2). Most terrestrial vertebrates appear to have two CK isozymes, CK-A and CK-C, both of which are cytoplasmic (Fisher et al., 1980). Eppenberger et al. (1967) have examined the evolution of CK isozymes in birds and suggested that there are at least two loci encoding CK in birds. In the present study, two G. gallus CK isozymes were detected (Table II). We did not discern a third mitochondrial isozyme which has been reported previously in birds (Jacobs et al., 1964). In m. pectoralis, CK-C activity was found in the 10-day embryo only; CK-A was found in m. pectoralis of the other three ontogenetic stages investigated. CK-A activity was not detected in any other G. gallus tissue investigated. CK-C displayed an increasing trend in activity in liver, gizzard, and heart. Intensely staining bands of mobility intermediate between CK-A and CK-C isozymes were evident in gizzard and heart, indicating a possible CK-AC interlocus heterodimer. However, this view is problematic since CK-A activity is not indicated by visible staining in either of these tissues. The phenomenon of interlocus heterodimer formation between CK isozymes is well documented for a variety of taxa (Harris and Hopkinson, 1976; Buth et al., 1985). Glycerol-6-phosphate Isomerase (EC 5.3.1.9). A single GPI locus has been observed for most land vertebrates (De Lorenzo and Ruddle, 1969; Fisher et al., 1980). GPI-A activity remained at fairly constant levels during development, in liver and gizzard (Table II). However, in heart and m. pectoralis there was an overall increase in activity from embryo to 7-month-posthatch individuals. Glycerol-3-phosphate Dehydrogenase (EC 1.1.l.8). Hopkinson et al. (1974) suggest that alpha and beta subunits of G3PDH in mammals are determined by separate autosomal loci. White and Kaplan (1969) and Ballas et al. (1984) have proposed that two distinct G3PDH isozymes have evolved in the chicken. However, Fisher et al. (1980) could consistently resolve only a single isozyme in higher vertebrates. In the present study, a single skeletal muscle specific isozyme could be clearly resolved (Table II). Following the suggestion of Fisher et al. (1980), we have designated this isozyme G3PDH-A. Although there were some background bands visualized on gels stained for G3PDH, they could not unequivocally be identified as G3PDH isozymes. Hexokinase (EC 2.7.1.1). Ureta (1982) has compared the structural, kinetic, and regulatory attributes of HK isozymes found in various vertebrates. Four hexokinases have been observed in 10-day chicken embryo livers using DEAE-cellulose chromotography (Ureta, 1982). However, it appears

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that HK-A and HK-D isozymes which are found in mammals are not identical to those found in Aves (Ureta, 1982). Lagos and Ureta (1982) suggest that the four observed HK isozymes in birds be designated HK-1 through HK-4, until such time when further research has clarified homologies. In the present study, a total of three G. gallus HK isozymes (labeled from anode to cathode as HK-1, HK-2, and HK-3; Fig. 1) was visualized. A single isozyme was detected in 10-day chicken embryo liver, contrary to the findings of Ureta (1982), who suggested that the liver isozymes labeled HK-1 and HK-4 decrease in activity and are absent at hatching. In the present study, HK-1 activity in liver did decrease with age, while HK-3 increased. HK-3 was found in liver only (Fig. 1). HK-2 was m. pectoralis specific and displayed an increasing trend in activity with age. Gizzard and heart demonstrated relatively constant HK-1 staining intensities throughout development; no other HK isozyme activity was detected in these two tissues.

Isoeitrate Dehydrogenase (EC 1.1.1.42). Birds typically possess ICDH isozymes encoded by two loci (Avise et al., 1980a, b). This situation is similar to that which exists in many other vertebrates where both a cytoplasmic locus (S-ICDH) and a mitochondrial locus (M-ICDH) are found (Harris and Hopkinson, 1976; Buth, 1983; Murphy and Crabtree, 1985). In G. gallus, both loci are expressed in all four tissues examined (Table II). However, there are age-related trends of expression. Lactate Dehydrogenase (EC 1.1.1.27). LDH isozymes in birds are encoded by three loci, a skeletal muscle-specific locus (LDH-A), a heartspecific locus (LDH-B), and a testis-specific locus (LDH-X) (Zinkham et aI., 1969; Murphy and Crabtree, 1985). Testis was not examined in the present study, thus the spatial and temporal trends of only LDH-A and LDH-B were examined (Table II). LDH-A activity decreased in liver with increased developmental stage, remained relatively constant in gizzard and heart, and increased in m. pectoralis. An increasing trend in level of activity was noted for LDH-B in liver and m. pectoralis. Moderate LDH-B activity was detected in gizzard of 1-day-posthatch individuals (Table II). Malate Dehydrogenase (EC 1.1.1.37). Two MDH isozymes are active in birds, a cytoplasmic form (S-MDH-A) and a mitochondrial form (M-MDH-A) (Avise et al., 1980a, b). Both S-MDH-A and M-MDH-A activity was detected in all tissues for every ontogenetic stage examined (Table II). The levels of activity of both isozymes remained fairly constant in all tissues throughout development (Fig. 2). M-MDH-A activity levels demonstrated a trend toward greater activity with increased ontogenetic stage in heart muscle. NADP-Dependent Malate Dehydrogenase (EC 1.1.1.40). Two ME isozymes have been identified in many vertebrates (e.g., Buth, 1983; Murphy

IsozymeExpressionsDuringChickenOntogeny

283

and Crabtree, 1985). Avise et al. (1980a) found several zones of activity for "malic enzyme" in thrushes but could reliably score only two isozymes. In the present study only one zone of activity could be detected (ME-A in Table II), this being found in liver of 7-day- and 7-month-posthatch individuals.

Peptidase (EC 3.4.1. l or 3.4.1.3). There are a number of peptidases with different substrate requirements in vertebrates (Harris and Hopkinson, 1976). The substrates used to stain PEP in the present study are listed in Table I. The isozymes are identified numerically as PEP-1 and PEP-2 and were not identified with respect to homology with the peptidase isozymes of other vertebrates. PEP-1 activity was detected in all tissues for every ontogenetic stage examined (Table II, Fig. 3). In liver, gizzard, and heart, the level of activity remained fairly constant throughout development. However, in m. pectoralis, PEP-1 demonstrated a decreasing trend in activity. PEP-2 activity was restricted to the liver and gizzard of later developmental stages. Phosphoglucomutase (EC 5.4.2.2). The number of PGM isozymes which have been reported for birds have varied from between one and three: one zone of activity scored in sparrows (Avise et al., 1980c), one of two bands of activity scored in thrushes (Avise et al., 1980a), and three PGM isozymes scored in parulids (Barrowclough and Corbin, 1978). In the present study, two zones of PGM activity were detected. PGM-A activity was detected in liver only and remained fairly constant throughout development (Table II). PGM-B increased in activity levels in m. pectoralis during development. PGM-B in liver, gizzard, and heart demonstrated little or no activity in the 10-day embryo and remained fairly constant in the other three developmental stages. Phosphogluconate Dehydrogenase (EC 1.1.1.44). One PGDH isozyme has been reported for many bird species (Avise et al., 1980a, b, c; Barrowclough and Corbin, 1978). In G. gallus PGDH-A was active in all examined tissues. PGDH-A was constant and relatively intensely stained in liver, gizzard, and heart in all developmental stages. In m. pectoralis, PGDH-A was most active in the 10-day embryo, gradually decreasing to minimal activity in 7-month-posthatch individuals. The pectoralis muscle of the chicken has been the subject of recent investigations which have shown that several components of the structural and contractile proteins of the muscle are expressed as different isoforms in the embryo, neonate, and adult, in both the slow- and the fast-contracting muscle fibers (Lowey et al., 1983; Bandman, 1985; see also Perry, 1985). We did not detect any differences in either the staining intensities or the loci expressed between the areas of the pectoralis containing entirely fast-twitch and up to 30% slow-twitch muscle fiber isozymes in the 7-month-posthatch birds. Furthermore, of the 15 enzyme systems presently described in the pectoralis

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muscle, only 1 demonstrates an age-dependent change in the isozyme expressed: CK-C is present only in the embryo, and CK-A is restricted to the other three stages studied. Eppenberger et al. (1970) obtained the same results for CK in developing chicken pectoralis. G3PDH-A, ALD-A, CK-A, and PGM-B, the former three observed only in the pectoralis during this study, all show an increase in activity in the muscle during development. This concurs with quantitative increases reported for other enzymes of energy metabolism in developing skeletal muscle (see Baldwin, 1984), although our results for P G D H - A run contrary to this trend. PEP-1 also decreased in the pectoralis muscle during development, and PEP-2 was not resolved in this muscle. This is in agreement with quantitative studies on developing muscle which have shown a decrease in the enzymes of protein catabolism during periods of protein synthesis and growth (Goldspink and Lewis, 1985). The present study demonstrates that several chicken isozymes are clearly tissue specific. In addition, several isozymes are expressed only at certain times during development. In these respects, our findings reaffirm and strengthen those of earlier studies which examined fewer loci (Eppenberger et al., 1970; Leung and Haley, 1974; Meyerhof and Haley, 1975). Our results emphasize the need to use similar ontogenetic stages and tissue samples in comparative electrophoretic studies of class Aves and may serve as a reference for those wishing to initiate similar studies of natural avian populations. ACKNOWLEDGMENTS

We are grateful to Dr. J. P. Bogart of the Department of Zoology, University of Guelph, for his encouragement and valuable criticisms and the use of his laboratory facilities. Birds for this study were kindly supplied by the Arkell Poultry Research Farm, Department of Animal and Poultry Science, University of Guelph. We appreciate the efforts of R. Lougheed in preparing the photographs for the figures. REFERENCES

Avise, J. C., Patton, J. C., and Aquadro, C. F. (1980a). Evolutionary genetics of birds. I. Relationships among North American thrushes and allies. A u k 97:135. Arise, J. C., Patton, J. C., and Aquadro, C. F. (1980b). Evolutionary genetics of birds. Comparativemolecularevolutionin New World warblers and rodents. J. Hered. 71:303. Avise, J. C., Patton, J. C., and Aquadro, C. F. (1980c). Evolutionarygenetics of birds. II. Conservative protein evolution in North American sparrows and relatives. Syst. Zool. 29:323. Baldwin, K. M. (1984). Muscledevelopment:neonatal to adult. Exer. Sport. Sci. Rev. 12:1. Ballas, R. A., Garavelli,J. S., and White, H. B., III (1984). Estimation of the rate of glycerol 3-phosphatedehydrogenaseevolutionin highervertebrates. Evolution 38:658. Bandman, E. (1985). Continued expressionof neonatal myosin heavy chain in adult dystrophic skeletal muscle. Science 227:780.

lsozyme Expressions DuringChickenOntogeny

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Barrowclough, G. F., and Corbin, K. W. (1978). Genetic variation and differentiation in the parulidae. Auk 95:691. Bogart, J. P. (1982). Ploidy and genetic diversity in Ontario salamanders of the Ambystoma jeffersonianum complex revealed through an electrophoretic examination of larvae. Can. J. Zool. 60:848. Bogart, J. P., Licht, L. E., Oldham, M. J., and Darbyshire, S. J. (1985). Electrophoretic identification of Ambystoma laterale and Ambystoma texanum as well as their diploid and triploid interspecific hybrids (Amphibia: Caudata) on Pelee Island, Ontario. (1985). Can. J. Zool. 63:340. Buth, D. G. (t983). Duplicate isozyme loci in fishes: Origins, distribution, phyletic consequences, and locus nomenclature. In Rattazzi, M. C., Scandalios, J, G, and Whitt, G. S. (eds.), lsozymes: Current Topics in Biological and Medical Research, Alan R. Liss, New York, Vol. 10, pp. 381-400. Buth, D. G. (1984). The application of electrophoretic data in systematic studies. Annu. Rev Ecol. Syst. 15:501. Buth, D. G., Murphy, R. W., Miyamoto, M. M., and Lieb, C. S. (1985). Creatine kinases of amphibians and reptiles: Evolutionary and systematic aspects of gene expression. Copeia 1985:279. Clayton, J. W., and Tretiak, D. N. (1972). Amine-citrate buffers for pH control in starch gel electrophoresis. J. Fish. Res. Board Can. 29:1169! De Lorenzo, D. L., and Ruddle, F. H. (1969). Genetic control of two electrophoretic variants of glucosephosphate isomerase in the mouse. Bioehem. Genet. 3:151. Eppenberger, M. E., Eppenberger, H. M., and Kaplan, N., O. (1967). Evolution of creatine kinase. Nature 214:239. Eppenberger, H. M., Eppenberger, M. E., and Seholl, A. (1970). Comparative aspects of creatine kinase isoenzymes. In Shugar, D. (ed.), Enzymes and Isoenzymes, FEBS Symposium, Vol. 18, pp. 269-279. Fisher, S. E., Shaklee, J. B., Ferris, S. D., and Whitt, G. S. (1980). Evolution of five multilocus isozyme systems in the chordates. Genetica 52/53:73. Goldspink, D. F., and Lewis, S. E. M. (1985). Age- and activity-related changes in three proteinase enzymes of rat skeletal muscle. Biochem. J. 230:833. Harris, H., and Hopkinson, D. A. (1976). Handbook o f Enzyme Electrophoresis in Human Genetics, North-Holland, Amsterdam. Hopkinson, D. A., Peters, S., and Harris, H. (1974). Rare electrophoretic variants of glycerol3-phosphate dehydrogenase, evidence for two structural gene loci (GPD 1 and GPD2). Ann. Hum. Genet. 37:477. Jacobs, H., Heldt, H., and Klingbergen, M. (1964). High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isozyme of creatine kinase. Biochem. Biophys. Res. Comm. 16:516. Lagos, R., and Ureta, T. (1982). Ontogeny of chick liver hexokinase isozymes. Arch. Bul. Med. Exp. 14:331. Leung, E. S., and Haley, L. E. (1974). The ontogeny of phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in Japanese quails and chicken-quail hybrids. Biochem. Genet. 11:221. Lowey, S., Benfield, P. A., LeBlanc, D. D., and Waller, G. S. (1983). Myosin isozymes in avian skeletal muscles. I. sequential expression of myosin isozymes in developing chicken pectoralis muscle. J. Muse. Res. Cell Motil. 4:695. Masters, C. J., and Holmes, R. S. (1972). Isozymes and ontogeny. Biol. Rev. 47:309. Matson, R. H. (1984). Applications of electrophoretic data in avian systematics. Auk 101:717. Matsuda, R., Bandman, E., and Strohman, R. C. (1983). Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle. Dev. Biol. 95:484. Meyerhof, P. G., and Haley, L. E. (1975). Ontogeny of lactate dehydrogenase isozymes in chicken-quail hybrid embryos. Biochem. Genet. 13:7. Murphy, R. W., and Crabtree, C. B. (1985). Evolutionary aspects of isozyme patterns, number of

286

Lougheed and Rosser

loci, and tissue-specific gene expression in the prarie rattlesnake, Crotalus viridis viridis. Herpetologica 41:451. Nakata, N., Suematsu, T., and Sakamato, Y. (1964). Transaminase activities in some rapidly growing tissues. Biochem. J. (Tokyo) 55:199. Nomenclature Committee of the International Union of Biochemistry (1984). Enzyme Nomenclature, Academic Press, New York. Perry, S. V. (1985). Properties of the muscle proteins: A comparative approach. J. Exp. Biol. 115:31.

Selander, R. K., Smith, M. H., Yang, S. Y., Johnson, W. E., and Gentry, J. B. (1971). Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old-field mouse (Peromyscus polionotus). Univ. Texas Publ. No. 7013, pp. 49-90. Shaw, C. R., and Prasad, R. (1970). Starch gel electrophoresis of enzymes--a compilation of recipes. Biochem. Genet. 4:297. Ureta, T. (1982). The comparative isozymology of vertebrate hexokinases. Comp. Biochem. Physiol. 71B:549. White, H. B, and Kaplan, N. O. (1969). Purification and properties of two types of DPN-linked glycerol 3-phosphate dehydrogenases from chicken breast muscle and chicken liver. J. Biol. Chem. 244:6031. Wittenberger, C., and Coprean, D. (1981 ). Posteclosiomal development of phosphorylase activity in chick pectoral muscle. J. Comp. Physiol. B 141:439. Zinkham, W. H., Isensee, H., and Renwick, J. H. (1969). Linkage of lactate dehydrogenase B and C loci in pigeons. Science 164:185.