Enzyme patterns in two species of Xenopus and their hybrids

DEVFaLOPMF,NTAL

BIOLOGY

36. 379-390 (1974)

Enzyme Patterns

in Two Species

of Xenopus

and Their Hybrids

DORIS ALYNNE WALL AND ANTONIE W. BLACKLER Genetics, Development

and Physiology,

Plant Science Building, Accepted

Cornell

University,

Ithaca,

New York 14850

October 3, 1973

Differences in the isozyme patterns of Xenopus laevis and Xenopus mulleri have been utilized to examine the expression of alleles of both species in hybrid animals. Mitochondrial MDH and tetrazolium oxidase phenotypes were examined during the development of non-hybrid embryos of each species and of reciprocal hybrids. Early stages of the hybrids resemble the enzyme phenotype of the maternal parent. Appearance of paternal enzyme takes place just prior to the active feeding tadpole stage for both mitochondrial MDH and oxidase. The maternal effect disappears shortly thereafter in early feeding tadpoles, at which point reciprocal hybrids have identical isozyme patterns. There is no evidence for a predominance of one species over the other. Examination of feeding tadpoles and adult toads indicates that both laeuis and mulleri expression is stable. The appearance of paternal mitochondrial MDH does not correspond to the time when other mitochondrial components begin to increase in Xenopus. Multiple bands of MDH in both species and of oxidase in Zueois are probably not due to the aggregation of subunits produced by different alleles at the same locus. There is no evidence for the formation of “hybrid” molecules consisting of subunits of both species. INTRODUCTION

The unfertilized amphibian egg contains a large amount of material which supports early development. Embryogenesis does not require transcription of the embryo’s own genes for these stored products until maternal supplies are exhausted (Davidson, 1968). It is therefore possible to obtain information regarding the temporal pattern of activation of the embryonic genome in crosses in which maternal and paternal molecules can be distinguished. One system which has been utilized for this type of investigation is the hybrid between animals which display variations in the electrophoretic mobility of their enzymes. This approach has been applied to the analysis of gene activity during early development of Rana hybrids (Wright and Moyer, 1966, 1968; Wright and Subtelny, 1971; Johnson, 1971; Johnson and Chapman, 1971a) and Pleurodeles hybrids (Gallien et al., 1973) as well as to hybrids of trout (Hitzeroth, et al., 1968; Klose, et al., 1969; Goldberg, et al., 1969) and chicken-quail hybrids (Castro-sierra and Ohno, 1968; Ohno, et al., 1968). Hybrid systems can also be used to

examine the efficiency with which alleles of one species are expressed in the background of the other. An interesting example of allelic exclusion is found in hybrids between the African toads Xenopus laevis and Xenopus mulleri. Interspecific matings yield viable hybrids of which the females are sometimes fertile (Blackler and Gecking, 1972a,b). Examination of nucleolar number in these Xenopus hybrids has shown that only one of the expected two full sized nucleoli appears in most cells at the late neurula stage (Blackler and Gecking, 1972b). Amplified rDNA from the oocytes of hybrid females is predominately or exclusively laevis (Brown and Blackler, 1972). A similar, but not absolute, predominance of laevis rDNA transcription in liver has been found by utilizing differences in the rDNA transcripts of the two species (Honjo and Reeder, 1973). Xenopus laevis and Xenopus mulleri are largely allopatric (Blackler and Gecking, 1972a), and their reproductive isolation has been sufficient to allow for considerable divergence of mitochondrial DNA sequences (Dawid, 1972) and spacer sequences of rDNA (Brown, et al., 1972). Some differences in 879

380

DEVELOPMENTAL BIOLOGY

the electrophoretic mobility of their isozymes would therefore not be surprising. An asymmetry of gene function analogous to the nucleolar situation should be detectable if the phenomenon extends to the loci coding for the enzymes studied. Our survey of various enzyme systems in Xenopus has revealed several useful differences in enzyme mobility between the two species. In this paper we discuss the expression of malate dehydrogenase (MDH) and tetrazolium oxidase (revealed as achromatic regions in tetrazolium stained gels). The changes in isozyme patterns during early development of intraspecific and interspecific matings are discussed, and these results are followed by an examination of isozyme patterns in older feeding tadpoles and selected adult tissues. Some comments on the mode of inheritance of oxidase and MDH will be made on the basis of data obtained during the course of this investigation. MATERIALS

AND

METHODS

The sources of animals and procedures for mating of Xenopus laevis and Xenopus mulleri are given in Blackler and Gecking (1972a,b). The embryos were raised in charcoal filtered water at a room temperature of 21-23°C or at a constant temperature of 19°C. Staging was determined in accord with the normal table of Nieuwkoop and Faber (1956). Jelly coats were removed from unfertilized eggs and pre-hatching embryos by treatment with 2% cysteine-HCl and 0.2% crude papain (Sigma) dissolved in calcium and magnesium free Niu and Twitty saline (Hamburger, 1960) buffered to pH 7.8 with Tris. Treatment for two to three minutes at room temperature was sufficient to remove the jelly coats. Dejellied material was washed four times in deionized water before storage at -90°C. Material of stage 48 or earlier was frozen in groups of lo-20 in one volume of deionized water, while older tadpoles and adult organs were frozen individually, with or without added water.

VOLUME 36,1974

Enzyme phenotypes were examined by horizontal starch gel electrophoresis using apparatus similar to that described by Smithies (1955). All samples were thawed prior to homogenization and the homogenate centrifuged at 4°C for 12 minutes in an International micro-capillary centrifuge fitted with a 5905 head to take 6 x 50 mm culture tubes. Different material was prepared in varying ways. Eggs and early embryos (up to stage 19) were broken apart by drawing the material several times into a Pasteur pipette of about 1 mm tip opening. All older material was ground by hand in a small glass Ten Broeck or Dual1 homogenizer (Kontes Glass Co.). Tadpoles from stage 50 to metamorphosis and all adult organs except liver were minced with scissors prior to homogenization. Coils of the gut were removed from large tadpoles before grinding. The supernates were applied to the gels in small (6 x 6 mm) pieces of Whatman 3MM paper, excess liquid was blotted off, and the papers were aligned in a slit cut into the starch gel. Electrophoresis for 4 to 6 hours at 10 volts/cm was performed in a 4°C coldroom with a fan directed horizontally across the gel trays. Gels contained 10.7 grams of Electrostarch (Otto Hiller, Madison, Wisconsin) per 100 ml of gel buffer. The gels were sliced into halves prior to staining. Details of the staining mixtures, buffers, and running times are given in Table 1. Gels were fixed in 50% ethanol. Control gels were incubated without substrate to ensure that the bands of MDH activity were not due to artifacts, such as the “nothing dehydrogenase” (Shaw and Koen, 1965). Mitochondrial pellets were obtained by the method of Dawid (1966) with the following modifications: homogenization was done in one volume rather than ten volumes of buffered sucrose, and the mitochondrial pellet obtained after the second 10,000 rpm spin was resuspended in about 0.1 ml deionized water. Samples of the supernates from the 2,000 rpm spin, the

Hybrid

WALL AND BLACKLER

TABLE

1

PROCEDURES USED FOR ELECTROPHORESIS

Buffers

Enzyme MDH

Bridge: pH 7.0 0.167 M K,HPO, (anh) 0.027 M citric acid. H,O

Bridge: pH 8.0 0.5 M Tris 0.65 M boric acid 0.016 M Na,EDTA Gel: 1: 10 dilution of bridge (modified from Shaw and Prasad, 1970)

10,000 rpm spin, and the resuspended mitochondrial pellet were each adsorbed to chromatography paper and analyzed as described above.

AND STAINING

Duration of run

Stain

4 hrs

15 ml 0.5 M Tris-HCl, pH 7.2 50 mg NAD 30 mg NBT 3 mg PMS 10 ml 1 M Na-L-malate, pH 7.0 24.5 mg NaCN 75 ml deionized H,O (Shaw and Prasad, 1970)

4-6 hrs

15 ml 0.5 M Tris-HCl, 60 mg NBT 5 mg PMS 85 ml deionized H,O (expose to light)

Gel: 0.0095 M K,HPO, (anh) 0.0012 M citric acid. H,O (modified from Shaw & Prasad, 1970) Oxidase

381

Xenopus Isozymes

pH 8.0

LXL

MDH

+

RESULTS

The findings described here were seen in at least two matings of different animals. Developmental patterns were determined with groups of animals taken at various times from the offspring of a single mating. All samples were run at least in duplicate to ensure that the results were reproducible. Matings are designated by listing the female parent first and the male parent second. Malate

Dehydrogenase

Before an analysis of the hybrid patterns of enzyme expression is possible, their ontogeny in the progeny of intraspecific matings of the two species must be examined. The MDH pattern in Xenopus laevis can be divided into two portions; a rapidly migrating group of four to five bands, and a group of two or three bands found closer to the origin (Fig. 1). By analogy with the situation in most animals (Shaw, 1969), it is reasonable to speculate that one of these

’

47M

46

45

41

36

27

21

FIG. 1. Malate dehydrogenase patterns of offspring from laeuis matings. L = laeuis; M = mulleri; numbers indicate Nieuwkoop-Faber stages. Material which is not derived from the mating designated at the top right of the illustration is so indicated by a letter following the stage number. s = supernatant MDH bands; mt = mitochondrial MDH bands.

groups represents mitochondrial MDH (mtMDH) and the other a soluble form of the enzyme. When mitochondria from laevis embryos are isolated by differential centrifugation the slow group of bands are found in association with the mitochondrial pellet, while the fast group can be recovered from the supernate. Two bands of mitochondrial MDH occur during the early developmental stages of laeuis embryos (Fig. 1). A third light band

382

DEVELOPMENTALBIOLOGY

of activity can be discerned above the two prominent ones at stage 46; a faint suggestion of this third area of activity can sometimes be seen in stage 45 embryos. This top band persists through late tadpole stages (stages 50 to 58) and is found in some adult tissues. The leftmost channel in figure 1 presents the MDH pattern of mulleri for comparison to that of laevis. The mulleri bands are also found in two groups, and clearcut differences in mobility are seen only in the slowly migrating proteins. Isolation of mullet-i mitochondria confirms that the fast bands are soluble and the slow mitochondrial enzyme. We will confine our attention to the organelle enzyme for the remainder of our discussion. Examination of MDH during mulleri development reveals temporal changes in pattern (Fig. 2). Three bands of mitochondrial enzyme persist through early stages, but the relative strength of the bottom band changes. Beginning in the mid 40’s, the slowest band begins to lighten noticeably in contrast to the top and middle bands. Tadpoles of stages 49 to 56 show the same pattern as the stage 47 embryos seen here. However, the slowest band is not found in some adult tissues. Once intraspecific ontogenies were es-

+

tablished, the hybrids were examined. Matings of laevis females by mulleri males showed a maternal pattern of mtMDH during early stages (Fig. 3). A faint suggestion of activity can be seen just above the laevis pair of bands at stage 45, but evidence of paternal gene product is not obvious until stage 46, when two bands of mulleri mobility appear. As can be seen in figure 4, the early stages of development in the reciprocal hybrid are also characterized by maternal MDH +

LxM

-, S j

mt j 0 F‘lc. 3. Malate dehydrogenase patterns of hybrid embryos from the mating of a laeuis female by a mulleri male. The mulleri bands in the righthand channel did not migrate as rapidly as the corresponding bands in the middle channels. This is an artifact; samples on the edge of a starch gel often do not run as fast as the same sample in the middle. L = laeuis; M = mullet-i; numbers indicate Nieuwkoop-Faber stages. s = supernatant MDH bands; mt = mitochondrial MDH bands.

+

MxM

MDH

VOLUME 36,19’i4

MDH

MxL

) S

mt .‘ 0

47L

47

46

45

41

36

27

i

Fro. 2. Malate dehydrogenase patterns of offspring from mulleri matings. L = laeuis; M = mulleri; numbers indicate Nieuwkoop-Faber stages. s = supernatant MDH bands; mt = mitochondrial MDH bands.

”

n

l)

iz

35 41 45 46 46L 47 46M E‘lc. 4. Malate dehydrogenase patterns of hybrid embryos from the mating of a mulleri female by a laeuis male. L = laeuis; M = mulleri; numbers indicate Nieuwkoop-Faber stages. s = supernatant MDH bands; mt = mitochondrial MDH bands.

WALL

AND BLACKLER

type enzyme. This situation persists until stage 45, when a faint band of laevis mobility is seen. At stage 46 and 47, a band of mtMDH activity corresponding to the slowest laevis band can be found. The results agree with the L x M findings: paternal gene product is suggested at stage 45 and definitely present by stage 46. A comparison of the two hybrids at stage 46 demonstrates that the maternal effect persists to this stage, since the strength of maternal bands is relatively greater than the strength of paternal bands. In contrast, stage 47 tadpoles appear to have identical phenotypes. The expression of paternal alleles appears to be stable. Heart, liver, and skeletal muscle from both hybrids show laevis and mulleri activity. The patterns from reciprocal hybrids are indistinguishable (Fig. 5); the example from heart shown is representative of that seen for all three tissues. The slowest of the three mulleri mtMDH bands is not detectable in heart material, although skeletal muscle and liver show this band, and it is very promi-

MDH

HEART

383

Hybrid Xenopus Isozymes

nent in unfertilized eggs produced by fertile hybrids (Fig. 6). Tetrazolium

Oxidase

Light bands on a dark tetrazolium background are frequently seen in isozyme investigations of both plant and animal material (Brewer, 1970). The tetrazolium oxidase activity is substantial in unfertilized eggs and early embryos of both Xenopus species, where it is found in the supernate of mitochondrial preparations. Its function in this organism is unknown. We will refer to this enzyme as oxidase in this paper. Oxidase displays a striking species difference (Fig. 7); the single band of activity found in mulleri material (right hand channel) migrates less rapidly than the 2 to 4 laevis bands. As development proceeds, oxidase activity becomes more and more difficult to detect, suggesting that the amount of active enzyme per ml of sample decreases with age. Although the amount of protein per channel has not been strictly standardized for these runs, a comparison of the strength of oxidase activity relative to the strength of MDH activity for the

MDH

+

UNE EGGS

mt 0

FIG. 5. Malate

dehydrogenase patterns of heart muscle from adult laevis (L), mulleri (M), and their hybrids. Hybrids are designated by giving the species of the female parent first and the male parent underneath. s = supernatant MDH bands; mt = mitochondrial MDH bands.

FIG. 6. Malate dehydrogenase patterns of unfertilized eggs from laevis CL), mulleri (M), and their hybrids. Hybrids are designated by giving the species of the female parent first and the male parent underneath. This gel was run for a shorter time than those of previous figures. s = supernatant MDH bands; mt = mitochondrial MDH bands.

384

DEVELOPMENTALBIOLOGY

VOLUME 36,1974

OXIDASE

LXL

+

w

30

FIG. 7. Oxidase Datterns Nieuwkoop-Faber stages.

39

of offsorine .

45 - from he&

same samples shows that oxidase fades out markedly in the later stage 40’s while MDH activity remains roughly constant. This fading is minimized in the illustrations because samples of late stage material were ground with less water to make visualization of the faint bands easier. The progressive fading results in the disappearance of the topmost band of the four banded pattern of early embryos and almost complete absence of the bottom band as the activity of enzyme falls below the limit of our ability to detect it. Nevertheless, older la&s tadpoles ranging from stage 53 to 58 have three bands of oxidase activity; the top band is missing, but the two middle and bottom ones are present. The slow band in these tadpoles is weaker than the other two, a relationship which seems to hold for all material examined, regardless of age. Adult organs also do not show the top band despite very strong activity of the other three (Fig. 11, righthand channel). Xenopus mulleri matings exhibit a single band throughout development which fades in later stages as did the laevis oxidase (Fig. 8). As in laevis, the activity never fades completely, and can be found in feeding tadpoles of older stages as well as in adult tissues.

46 matings.

47

48

47M

L = laeuis; M = mulleri;

numbers

indicate

The mulleri by laeuis cross (Fig. 9) illustrates the familiar maternal effect until stage 45, when a band of paternal activity appears just above the maternal form. A second laeuis band is found at stage 47, when the mulleri and laevis bands are of approximately equal strength. Comparison of these paternal bands with the laevis pattern indicates that paternal activity is due to the synthesis of the two middle bands of the laevis pattern. The reciprocal hybrid is more difficult to analyze because the single mulleri band and the slowest laevis band migrate anodally at almost the same speed and tend to run together as a single broad band on gels. Activity of the paternal allele in laevis by mulleri matings is detectable only as a broadening of the slow laeuis band (Fig. 10) first clearly seen at stage 46. Earlier evidence for paternal enzyme would be difficult to detect even if present. Stage 48 hybrid embryos possess three oxidase bands of approximately equal strength in contrast to the same stage laevis embryos which display a weaker slow band (compare laevis ontogeny in figure 7 with the hybrid ontogeny of figure 10). At stage 46 a comparison of the reciprocal hybrids shows that the maternal effect persists to this point in development;

WALL ANDBLACKLER

Hybrid

OXIDASE

27

37

MxM

41

FIG. 8. Oxidase patterns of offspring eggs; numbers indicate Nieuwkoop-Faber

45

from mulleri stages.

46

27

35

47

48

matings. L = laeuis; M = mulleri;

U.E.L U.E. = unfertilized

MxL

OXIDASE

u

385

Xenopus Isozymes

41

45

46

U.E.M 47

U.E.L

FIG. 9. Oxidase patterns of hybrid embryos from the matings of mulleri female by a laeuis male. Only three bands are seen in the laeuis unfertilized laeuis; M = mulleri; U.E. = unfertilized

egg sample because it was diluted more than in previous gels. L = eggs; numbers indicate Nieuwkoop-Faber stages.

mulleri activity is relatively stronger in the former and laeuis bands are relatively more prominent in the latter. Both hybrids appear identical by stage 48. Both laevis and mulleri enzyme is found in adult organs. Reciprocal hybrids are indistinguishable (Fig. 11). Oxidase activity is particularly strong in liver and in unfertilized eggs laid by fertile hybrid females (Fig. 12). The example of oxidase pattern in liver is representative of the findings for heart and skeletal muscle.

DISCUSSION

The expression of mulleri alleles in a laevis background and vice versa indicates that the recognition of these presumably allelic genes for transcription in hybrids is taking place normally. Both species’ enzymes are active in hybrid cells; the contribution of one parent does not predominate. Reciprocal crosses yield offspring of identical phenotype after disappearance of the maternal effect. Clearly, the failure of ribosomal cistron expression seen in inves-

386

DEVELOPMENTALBIOLOGY

VOLUME 36,1974

LxM

OXIDASE

”

30

36

41

45

46

47

47L

48

47M

FIro. 10. Oxidase patterns of hybrid embryos from the mating of a laevis female by a mulleri male. L = eggs; numbers indicate Nieuwkoop-Faber stages. laevi is; M = mulleri; U.E. = unfertilized

OXIDASE

LIVER

0 FIG. 11. Oxidase patterns of liver from adult laevis (L), mulleri (M), and their hybrids. Note the presence of only three bands in laeuis and hybrid material. Hybrids are designated by giving the species of the female parent first and the male parent underneath.

tigations of the nucleolar organizer region does not extend to all loci, but is either specific to the nucleolar organizer region or covers a restricted number of loci not including those for mtMDH and oxidase. The appearance of paternal forms of these enzymes takes place quite late in development (stage 45). Transcription of

the embryo’s alleles for mtMDH and oxidase may take place much earlier than the time when functional gene product appears. Thus, the time when the paternal product is first detected as active enzyme represents the latest possible time when transcription of the genome could have taken place. An investigation of lactate dehydrogenase in developing Xenopus laevis presents evidence favoring the transcription of a stable mRNA coding for LDH prior to the time when active enzyme is detected (Claycomb and Villee, 1971). Reciprocal hybrids bear a stronger resemblance to the maternal enzyme phenotype until st. 46 or 47, after which they are indistinguishable. Possible mechanisms for this effect include activation of only maternally derived alleles, storage of enzyme mRNA in the unfertilized egg, or storage of the protein itself. Evidence from haploid hybrids in Rana eliminates preferential transcription of maternal genes and favors the hypothesis that enzymes are stored as proteins, not mRNA (Wright and Subtelny, 1971). If this hypothesis applies to Xenopus, embryos do not require newly synthesized mtMDH or oxidase until at least stage 45. Ribosomes are known to be stored in the Xenopus egg, enabling em-

WALL AND BLXKLER

387

Xenopus Isozymes

UNF. EGGS

OXIDASE

0

Hybrid

L

k M

y L

M

FIG. 12. Oxidase patterns of unfertilized eggs from laeuis (L), mulleri (M), and their hybrids. designated by giving the species of the female parent first and the male parent underneath.

bryos lacking functional nucleolar organizer regions to survive until the early 40’s (Brown and Gurdon, 1964). Mitochondria are apparently stockpiled in a similar manner (Chase and Dawid, 1972). Since anucleolate embryos die at a stage when paternal enzyme is not yet present in substantial amounts, stored enzyme or mRNA present in the unfertilized egg apparently supports development without requiring new synthesis for an even longer time than stored ribosomes. Dawid and Blackler (1972) have shown that the mitochondrial DNA of Xenopus hybrids is inherited maternally and cytoplasmically. The appearance of mtMDH of the paternal type in these hybrids must mean that the organelle enzyme is coded by nuclear genes. A similar situation obtains in Neurospora (Munkres, et al., 1965), maize (Long0 and Scandalios, 1969), Rana (Wright and Subtelny, 1971), man (Davidson and Cortner, 1967), the mouse (Shows, et al., 1970), and probably the rat (Roodyn, et al., 1962). Work of Chase and

Hybrids

are

Dawid (1972) indicates that the amount of mitochondrial DNA per embryo in Xenopus laevis is constant until stage 40, then increases until doubled in amount by stage 45. These authors also assayed cytochrome oxidase activity and total mitochondrial protein during laevis development, finding that the amount of this enzyme per organism and total protein both begin to increase at stage 38 and are doubled by stage 45. Another mitochondrial enzyme, monoamine oxidase, has been assayed in this species by Baker (1966). The activity shows a sharp increase at stage 35-36 and continues rising to stage 46. Furthermore, Wright and Subtelny (1971) have demonstrated paternal mtMDH appearance at the heartbeat stage of Rana, which corresponds to approximately stage 33-34 in Xenopus. These findings are in contrast to our results in which new synthesis of mitochondrial MDH is not detected until stage 45 at the earliest. The biogenesis of mitochondria in the developing embryo of the late stage 30’s

388

DEVELOPMENTALBIOLOGY

may result from a synthesis of new components which are assembled into more mitochondria to meet developmental requirements. The apparently asynchronous activation of mtMDH genes is therefore a somewhat surprising observation. It is possible that the onset of paternal allele activation is delayed in this hybrid system since these “foreign” alleles may not be recognized by the transcriptional machinery of another species. (See Castro-Sierra and Ohno, 1968 and Hitzeroth, et al., 1968 for examples of this phenomenon.) However, no evidence suggests difficulty of gene recognition in our system. Changes in the amount of an enzyme per organism may take place without new transcription, for example, by stimulation of translation of pre-existing mRNA stockpiles or by decreased rates of degradation of the enzyme made from stable mRNA templates. As noted above, stored mRNA is unlikely, at least in Rana (Wright and Subtelny, 1971). Alternatively, an excess of mtMDH stored in mitochondria of unfertilized eggs and early embryos may obviate new synthesis until stage 45. The latter may be the case with mitochondrial tRNAs (Chase and Dawid, 1972). The observed pattern of three evenly spaced bands of laevis and mulleri mtMDH suggests that this enzyme may take a multimeric form consisting of two or more different subunits aggregating into a dimer of the forms AA, AB, and BB. Genetic evidence in favor of such an enzyme structure for mitochondrial MDH has been obtained in man (Davidson and Cortner, 1967), in the mouse (Shows, et al., 1970), and in fish (Whitt, 1970). In all of these cases, the subunits are coded by different alleles at the same locus, resulting in a three banded pattern only when the animals are heterozygous. Homozygotes have only one band of either fast (BB) or slow (AA) mobility. It seems unlikely that this obtains in Xenopus. All individual animals examined for the adult tissue sur-

VOLUME 36.1974

vey and all unfertilized eggs or early embryos grouped together for homogenization show the multi-banded pattern predicted for a heterozygote by this mode of inheritance. No instances of a homozygous phenotype have been seen, either for mtMDH, or soluble MDH and laevis oxidase. Definitive conclusions await the results of an examination of individual sibs from single matings, a type of analysis that has revealed an allelic polymorphism in the glutamate-oxaloacetate transaminase patterns of Xenopus laeuis (Johnson, 1972). The hybrid patterns are essentially additive; that is, one sees the number of bands predicted if all laevis and all mulleri isozymes are synthesized. No evidence of “hybrid” molecules consisting of both species’ subunits is found. Hybrid molecules would be predicted to have intermediate mobility, running to a position between the normal laevis and mulleri bands. The failure to detect such forms of these proteins may have several explanations. These enzymes may not exhibit subunit assembly construction. If they do, “hybrid” enzymes may fail to form because mulleri and laevis subunits are not sufficiently homologous for recognition and association to take place. Even if formed, such enzymes might not have sufficient activity to be detected. A satisfactory explanation of the lack of new isozymes in Xenopus hybrids will require a greater understanding of the molecular basis for the three mtMDH bands. Such understanding may also illuminate the nature of changes in the relative strengths of mtMDH bands during and on the differences in development, mtMDH patterns in different adult tissues. The authors thank Ms. Donna Cassidy for helpful discussions during the course of the investigation and during preparation of this paper. We thank Drs. A. M. Srb and J. M. Calvo for their comments on the manuscript, and Dr. R. J. MacIntyre for assistance with the electrophoresis technique and for the use of equipment. The senior author was supported by a traineeship under Grant Tl GM 1035 from the National Institute of General Medical Sciences, USPHS.

WALL AND BLACKLER REFERENCES BAKER, P. C. (1966). Monoamine oxidase in the eye, brain, and whole embryo of developing Xenopus Levis. Develop. Biol. 14, 267-217. BLACKLER, A. W., and GECKING, C. A. (1972a). Transmission of sex cells of one species through the body of a second species in the genus Xenopus I. Intraspecific matings. Deuelop. Biol. 27, 376-384. BLACKLER, A. W., and GECKING, C. A. (1972b). Transmission of sex cells of one species through the body of a second species in the genus Xenopus II. Interspecific matings. Deuelop. Biol. 27, 385-394. BREWER, G. J. (1970). “An Introduction to Isozyme Techniques.” Academic Press, New York. BROWN, D. D., WENSINCK, P. C., and JORDAN, E. (1972). A comparison of the ribosomal DNA’s of Xenopus laevis and Xenopus mulleri: the evolution of tandem genes. J. Mol. Biol. 63, 57773. BROWN, D. D., and BLACKLER, A. W. (1972). Gene amplification proceeds by a chromosome copy mechanism. J. Mol. Biol. 63, 75-83. BROWN, D. D., and GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laeuis. Proc. Nat. Acad. Sci. U.S. 51, 139-146. CASTRO-SIERRA, E., and OHNO, S. (1972). Allelic inhibition at the autosomally inherited locus for liver alcohol dehydrogenase in chicken quail hybrids. Biochem. Genet. 1, 323-335. CHASE, J. W., and DAWID, I. B. (1972). Biogenesis of mitochondria during Xenopus laeuis development. Deuelop. Biol. 27, 504-518. CLAYCOMB, W. C., and VILLEE, C. A. (1971). Lactate dehydrogenase isozymes of Xenopus laeuis: factors affecting their appearance during early development. Deuelop. Biol. 24, 413-427. DAVIDSON, E. H. (1968). “Gene Activity in Early Development.” Academic Press, New York. DAVIDSON, R. G., and CORTNER, J. A. (1967). Mitochondrial malate dehydrogenase: new genetic polymorphism in man. Science 157:1569-1571. DAWID, I. B. (1966). Evidence for the mitochondrial origin of frog egg cytoplasmic DNA. Proc. Nat. Acad. Sci. U.S. 56, 269-276. DAWID, I. B. (1972). Evolution of mitochondrial DNA sequences in Xenopus. Deuelop. Biol. 29, 139-151. DAWID, I. B., and BLACKLER, A. W. (1972). Maternal and cytoplasmic inheritance of mitochondrial DNA in Xenopus. Deuelop. Biol. 29, 152-161. GALLIEN, C. AIMAR, C., and GUILLET, F. (1973). Nucleocytoplasmic interactions during ontogenesis in individuals obtained by intra- and interspecific nuclear transplantation in the genus Pleurodeles (urodele amphibian). Morphology, analysis of two enzymatic systems (LDH and MDH) and immunity reactions. Deuelop. Biol. 33, 154-170. GOLDBERG, E., CUERRIER, J. P., and WARD, J. C.

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