DEVELOPMENTAL BIOLOGY 68, 334-339 (1979)

Hybrids of Xenopus laevis and Xenopus borealis Express Proteins from Both Parents

EDDY M. DE ROBERTIS AND PHILLIPPA BLACK

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England

Received July 14, 1978; accepted July 18, 1978

Xenopus laevis and X. borealis oocytes were compared by two-dimensional electrophoresis of radioactive proteins. At least one-third of the major newly synthesized proteins differ in their electrophoretic mobility. Protein-coding genes from both parents are expressed in interspecific hybrids, thereby providing useful genetic markers for a variety of embryological studies.

INTRODUCTION

Xenopus laevis and X. borealis (previ- ously called X. mulleri, see Brown et al., 1977) are two closely related species which can interbreed (Blackler, 1970) and which have been used extensively in studies of amphibian development. Some gene prod- ucts are known to be distinguishable be- tween both species. These include rRNA precursors (Brown and Blackler, 1972; Honjo and Reeder, 1973), 5 S RNA (Ford and Brown, 1976), a protein of mitochon- drial ribosomes (Leister and Dawid, 1975), mitochondrial malate dehydrogenase (Wall and Blackler, 1974), and histone Hl (Cas- sidy and Blackler, 1978). In the case of rRNA, it has been shown that in hybrid frogs the laevis genes are expressed in pref- erence to the borealis genes (Brown and Blackler, 1972; Honjo and Reeder, 1973). Under appropriate experimental conditions X. borealis/X. laevis gene differences could be used as a substitute for the very few mutants that are available in Xenopus (Gurdon and Woodland, 1975). A simple method of detecting multiple gene markers would be especially valuable in studies on the expression of individual genes, provided that the expression of some alleles is not excluded as with rRNA.

cellular proteins. Secondly, utilizing those proteins which differ, we ask whether the hybrid frogs express protein-coding genes of both parents, or if one genome is used preferentially, as in the case of rRNA. We have analyzed a large number of radioactive oocyte proteins using two-dimensional elec- trophoresis (2D gels; O’Farrell, 1975). We find that many of the proteins of X. laevis and X. borealis oocytes differ in their elec- trophoretic mobility and that both genomes are expressed in hybrid frogs, thereby pro- viding genetic markers which could be use- ful for a variety of embryological studies.

MATERIALS AND METHODS

Here we ask initially whether these two closely related species differ in their overall

Hybrid frogs were obtained from a mat- ing of a borealis male with a small laevis female (Blackler, 1972). Ovaries were taken from young immature frogs (5 months old) and labeled for 18 hr in modified Barth solution (Gurdon, 1976) containing [35S]me- thionine at 0.5 mCi/ml (800 Ci/mmole, Amersham) or 14C-labeled amino acid mix- ture at 250 @i/ml (57 mCi/mAtom, Amer- sham). All oocytes were at stage 1 (Du- mont, 1972). Proteins were extracted, elec- trophoresed in two dimensions, and fluo- rographed as described previously (De Robertis et al., 1977). The differences ob- served between Fig. 1A and our previously published 2D gel patterns of X. laevis pro-

334 0012-1606/79/010001-15$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

BRIEF NOTES 335

teins are due to the use of oocytes at a different stage and amino acids of a differ- ent kind.

RESULTS AND DISCUSSION

Figs. la and lb show the 2D gel patterns of X. laevis and X. borealis radioactive proteins synthesized in oocytes. Many dif- ferences can be seen; in fact, so many are observed that initially it is difficult to detect the similarities between these two gels. However, when X-ray films are compared closely, many proteins common to both spe- cies become apparent. To test whether these proteins migrate precisely in the same position, laevis and borealis radioactive proteins were mixed and electrophoresed within the same gel, as shown in Fig. lc. From the results shown in Figs. la-c, it is clear that many of the major oocyte pro- teins of laevis and borealis are common to both species. The most prominent of the common proteins are indicated in Fig. 2a. Some of the proteins which differ between species are indicated in Fig. 2b. We estimate that at least one-third of the major oocyte proteins differ in 2D gel mobility when the two species are compared.’

The oocytes from hybrid frogs express the proteins of both parents, as shown in Fig. Id. The pattern of the hybrid ovary is almost indistinguishable from that of a mix- ture of equal amounts of Eaevis and borealis proteins, as expected if both genomes were expressed. To help comparison, some lae- vis-specific proteins are indicated (down- ward arrows) in Fig. la and some borealis-

’ This is a minimal estimate, based on visual ex- amination of X-ray ffims. We find that a precise quan- titation of total numbers of spots in 2D gels is difficult to make for several reasons: (1) Some proteins overlap with other spots; (2) some regions of the gels are more difficult to compare (due to the lack of clear reference spots); (3) when the weaker spots are taken into ac- count, the classification becomes less objective, vary- ing considerably with the observer. In any event, al- though accurate quantitation is difficult, it is clear from Fig. 1 that many unambiguous protein differ- ences can be detected by 2D gels between X. laeuis and X. borealis.

specific proteins (upward arrows) in Fig. lb. All of these proteins are expressed in hybrid frogs (Fig. Id). Spots absent in the hybrids but present in one of the parents are very rare (only two spots), and it is conceivable that they could become detectable upon longer exposure of the gels. Clearly, the phenomenon of allelic exclusion that is ob- served with rRNA does not apply to most protein-coding genes.

The intensity of the protein spots in the hybrid oocytes varies according to the num- ber of genes present. In other words, those genes which comigrate in both species (such as actin and tubulin) show spots of the same relative intensity as in Fig. la or lb, while those spots which are present only in one species are about half as intense in the hybrids.

Eventually it would be useful to know which pairs of spots represent electropho- retically different products of the same gene. Since small changes in charge are more probable than changes in overall size of the proteins, pairs of spots which have small displacements in the horizontal (iso- electric focusing) direction can be provi- sionally assumed to be alleles of the same gene. Although this applies to several pairs of spots (some of which are indicated in Fig. Zb), in other cases it is very difficult to assign a possible allele. To unambiguously show that two spots correspond to a similar protein would require comparison of the peptides of both proteins. This should be technically feasible, since the peptides of minute amounts of protein in 2D gels can be analyzed using the methods devised by us (Gurdon et al. 1976a; De Robertis and Gurdon, 1977) and others (Cleveland et al., 1977).

Although Xenopus offers many advan- tages as an experimental animal (availabil- ity of large number of eggs and possibilities for micromanipulation), very few mutants are available (Gurdon and Woodland, 1975). The use of borealis and laevis radio- active proteins provides gene markers po- tentially useful for studying the expression

FIG. 1. Fluorographs of two-dimensional gels of oocyte proteins labeled with [35S]methionine. (a) Xenopus laeuis oocytes; (b) Xenopus borealis oocytes; (c) mixture of samples a and b electrophoresed in the same gel; (d) oocytes from an interspecies hybrid frog. Downward arrows indicate 15 of the Zaeuis-specific proteins, and upward arrows indicate 15 of the borealis-specific proteins. Note that all 30 proteins from both parents are seen in c and are expressed in hybrid frogs.

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BRIEF NOTES 337

Mixture

FIG. 1. Continued.

0” 2a

0

0 0

0

O0

0

0 0

a

0 o/1

t * o tubulin

tactin O o.

0

0

0

0

a 0 00 O6

0

Corn mon protei ns

2b

0 Yi 0

Species specific

*0

FIG. 2. Tracings of some of the proteins that can be most easily distinguished by comparing the gels in Fig. 1. (a), proteins that comigrate in Zaeuis and borealis. (b), proteins that differ in the two species; open circles indicate laeuis-specific proteins, filled spots indicate borealis-specific proteins. The arrows indicate pairs of spots which could be products of alleles of the same gene.

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BRIEF NOTES 339

of individual genes (see below). In view of the results presented here, it seems possible that the mechanisms that regulate gene expression in one species can operate in the other, at least for protein-coding genes.

These multiple gene markers could be very useful in two types of experiments. It has been shown that genes microinjected into laevis oocytes in the form of somatic nuclei (Gurdon et al., 197613; De Robertis et al., 1977; De Robertis and Gurdon, 1977) or of purified DNAs (Mertz and Gurdon, 1977; Brown and Gurdon, 1977) are transcribed and sometimes translated into protein (De Robertis and Mertz, 1977). Microinjected Xenopus borealis genes (as somatic nuclei or as cloned DNA segments) could be used to study the expression of protein-coding genes in very closely related species. Sec- ondly, more embryological questions could be approached, such as how much of the mRNA used in the synthesis of individual proteins during early development is ma- ternally inherited, by analyzing hybrid or nuclear transplant embryos.

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