Copyright 0 1983 by the Genetics Society of America

TWO SEX-LINKED LOCI IN THE LEOPARD FROG, RANA PIPIENS

DAVID A. WRIGHT

Department of Genetics, The University of Texas, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030

CHRISTINA M. RICHARDS

Department of Biology, Wayne State University, Detroit, Michigan 48202

Manuscript received April 7, 1982 Revised copy accepted September 27, 1982

ABSTRACT

Crosses involving one heterozygous parent were performed to test the inher- itance of enzymes in the leopard frog, Rana pipiens. After metamorphosis, offspring were sexed and tissue extracts from them were analyzed by gel electrophoresis. Enzyme genotype and sex showed independent assortment for 10 of 12 enzymes heterozygous in the male parent. However, among the offspring of males heterozygous for peptidase C (Pep-C) or superoxide dismu- tase 1 (SOD-I), male progeny tend to inherit one allele, whereas female progeny tend to inherit the other. Data from several different crosses yield recombination frequencies of 8.6% between sex and SOD-1, 6.9% between SOD-1 and Pep-C and 12.1% between sex and Pep-C. When the female parent is heterozygous for these enzymes no significant difference is seen, in the offspring, between male and female homozygotes and heterozygotes. These results confirm that males are the heterogametic sex in R. pipiens and suggest that sex is determined by a small number of genes on otherwise identical X and Y chromosomes.

HE breeding of sex-reversed animals has established +hat the male is T heterogametic (XU) and the female homogametic (XX) in several species in the genus Rana (KAWAMURA and YOKOTA 1959; GALLIEN 1974). Genetic female R. pipiens made into phenotypic males by testosterone treatment and crossed with normal females produce predominantly female offspring (RICHARDS and NACE 1978). Furthermore, it has also been demonstrated that male R. pipiens (WACHTEL, KOO and BOYCE 1975) and other species of Rana (ENGEL and SCHMID 1981) have the H-Y (male) antigen on their cell surfaces, whereas this antigen is lacking on female cells.

Despite the evidence for an XX-XY sex-determining mechanism, no cytoge- netic differentiation of the X or Y chromosomes has been detected. Each of the 13 homologous pairs of chromosomes is morphologically identical in R. pipiens males and females (DIBERARDINO 1962). This indicates that in Rana the sex- determining mechanism differs from that of mammals, in that there is no large X chromosome bearing many genes nor a small Y chromosome bearing primarily male-determining genes.

One would predict that in R. pipiens sex-linked genes could be heterozygous or homozygous in either sex. Since the female is homogametic, sex linkage

Genetics 103 249-261 February, 1983

250 D. A. WRIGHT AND C. M. RICHARDS

should be undetectable in crosses involving heterozygous females. No sex linkage was found for 15 different loci in a series of such crosses (WRIGHT, RICHARDS and NACE 1980). If it is the male that is heterogametic, then the offspring of such males heterozygous for a sex-linked gene should show a preponderance of males with one allele (Y-linked) and females with the other allele (X-linked). In fact, ELINSON (1981) has shown this is indeed the case in backcross hybrids of R. catesbeiana female X R. clamitans - R. cutesbeianu hybrid male heterozygous for lactate dehydrogenase B variants.

In this paper we present evidence for the independent assortment of the loci for ten enzymes with respect to sex and the sex linkage of peptidase C (previously designated Pep LA 2, WRIGHT, RICHARDS and NACE 1980) and superoxide dismutase 1 loci in intraspecific crosses of northern R. pipiens.

METHODS

Adult R. pipiens from wild populations were obtained from commercial sources (J. M. HAZEN, Alburg, VT; Nasco, Fort Atkinson WI and Graska, Oshkosh, WI). These animals were screened for electrophoretic variants of several enzymes from an amputated toe, as described previously (WRIGHT, RICHARDS and NACE 1980). Crosses involving one heterozygous parent were set up using standard procedures of artificial fertilization (RUGH 1962). The resulting embryos were reared through metamorphosis (NACE et al. 1974). Sex was determined on the newly metamorphosed animals by visual examination of the gonads. Tissues were removed and homogenized, and enzymes were analyzed by starch gel electrophoresis (SICILIANO and SHAW 1976) as previously described (WRIGHT, RICHARDS and NACE 1980). Data were tabulated separately for males and females in each cross, and contingency x2 values were calculated (CAMPBELL 1974) to test the association of sex with genotype at each locus. Crosses 7, 8 and 9 are backcrosses of an F, hybrid R. palustris x R. pipiens male to R. pipiens females. The original R. palustris female was obtained from M. TOLLEY (Nashville, TN).

RESULTS

Crosses involving R. pipiens males heterozygous at loci controlling several different enzymes were examined for possible sex linkage (Table 1). The enzyme loci considered in Table 1 are aconitase 2 (Acon-a), acid phosphatase 1 (AP-I), adenosine deaminase (ADA), esterase 1 (Est-I), esterase 5 (Est-5), glyox- alase (GJy), mannosephosphate isomerase (MPI), peptidase A (Pep-A), peptidase D (Pep-D) and phosphoglucomutase 1 (PGM-I). Electrophoretic patterns of the variants of each of these have been described elsewhere (WRIGHT, RICHARDS and NACE 1980, WRIGHT and RICHARDS 1982). When linkage between locus pairs was tested for the loci mentioned in Table 1, the Gly-AP-Est-5 linkage (WRIGHT, RICHARDS and NACE 1980) was confirmed. Other testable locus pairs showed independent assortment.

Data from individual crosses or data pooled from several crosses having the same heterozygous male parent are tabulated for each of the ten enzyme loci. When x2 values are calculated with an expected 1:1:1:1 ratio for each of the combinations of sex and enzyme genotype there are a few clear cases of deviation from the expected in the data shown in Table 1. Crosses 10 and 11 (Acon-2) have an overall deficiency of males, 11 males and 26 females (2 = 6.08, P < 0.02). The data on Est-I show an apparent deficiency of males of the Est a/c genotype in crosses 12 and 13 (x2 = 5.46, P =: 0.02). There is an apparent

FROG SEX-LINKED ENZYMES 251

TABLE 1

Inheritance of nonsex-linked loci in crosses with heterozygous male parents

Parents' genotypes Offspring

Locus Cross Female Male Genotype

Acon-2

A P-1

ADA

Est-1

Est-5

GlY

M PI

Pep-A

Pep-D

PGM-1

1 0 , l l

12, 14, 16

13,16

1, 2

3

1, 2

12,13

13

14

12, 13, 14, 15, 16

12, 13, 14

7, 8 , 9

1

19

1

3, 4

Male Female x2

6 5

77 82

45 55

30 18

18 26

27 21

33 17

25 16

27 40

112 116

80 86

8 4

11 10

4 2

5 19

23 20

15 11

79 78

60 58

29 17

17 20

26 22

29 30

30 24

23 32

117 127

89 81

9 6

10 10

5 4

10 12

21 14

0.03

0.11

0.74

0.003

0.21

0.04

3.09

0.28

0.03

0.06

0.58

0.13

0.02

0.19

3.17

0.33

P > 0.8

P > 0.7

P > 0.35

P > 0.9

P > 0.6

P > 0.8

P > 0.07

P > 0.5

P > 0.8

P < 0.7

P > 0.4

P > 0.6

P > 0.8

P > 0.65

P > 0.07

P > 0.5

deficiency in heterozygous PGM-1 b/a genotype but only in cross 1. Likewise, there is a deficiency in AP-1 b/b homozygotes in crosses 1 and 2 (xz = 6.13 P < 0.02), whereas no significant deviation from expected values is seen in AP-1 genotypes in crosses 13, 16 or 3.

252 D. A. WRIGHT AND C. M. RICHARDS

Contingency 2 values, which test the association of sex with genotype, are listed on Table 1. These values indicate that none of the ten loci are linked to sex and that none of the deviations from the expected ratio of genotype or sex are cases of sex linkage.

In contrast to these ten loci the data for two other enzyme loci peptidase C (Pep-C) and superoxide dismutase 1 (SOD-1) indicate sex linkage and will be considered in detail. In frogs, Pep-C is found in most tissues with the exception of eggs and early embryos. It is revealed along with Pep-A when the substrate L-leucyl-alanine is used in the staining mixture. For this reason Pep-A and Pep- C were originally referred to as Pep LA I and Pep LA 2, respectively. Subsequent characterization (WRIGHT and RICHARDS 1982) has demonstrated homologies of frog and mammalian Pep-A and Pep-C isozymes. Electrophoretic variants of Pep-C are relatively frequent among R. pipiens. Allelic forms are identified by their electrophoretic mobilities and assigned a letter of the alphabet in general in decreasing order of mobility. Pep-C has a monomer structure, so that heterozygotes have a two-band electrophoretic pattern (Figure 1). A comparison of Pep-C to other frog peptidases has been published elsewhere (WRIGHT and RICHARDS 1982).

+

PEPA

PEPC

FIGURE I.-Photograph of a starch gel slice stained for peptidase isozymes using the substrate L- leucyl-alanine. Samples are liver extracts of offspring of cross 17. Pep-A is invariant in this cross. Pep-C shows either single band (b /b) or double band (b/c) patterns characteristic of the mother and father, respectively. in this cross. The two-band pattern of heterozygotes is typical of a monomeric enzyme. The female offspring are predominantly of the single band (b/b) type and male offspring are predominantly of the double band (b /c ) type. Two recombinants are indicated by stars. The apparent difference between the position of homozygous Pep-C b/b band in the 5th channel (male *) and that of the corresponding band in the 4th channel (female *) is due to the inexact piecing together of the left hand and right hand side of the same starch gel slice before photography to eliminate irrelevant intervening samples.

FROG SEX-LINKED ENZYMES 253

Several different crosses involving males heterozygous at the Pep-C locus were analyzed. In these cases male offspring tend to inherit one paternal allele, whereas female offspring tend to inherit the other paternal allele. Crosses 1 and 2 (Table 2) illustrate the results of crosses of the same male with two different females. In cross 1, involving a Pep-C b/b female with the a /b male, 21 of the male offspring were a /b heterozygotes and 19 of the females are homozygous. These are parental types. Only three male offspring are homozygous and two female offspring heterozygous. These are the recombinant types. In cross 2, involving a Pep-C a/a female and the same a /b male as in cross 1, most of the male offspring (24) are a/a homozygotes, whereas most of the females (18) are a/b heterozygotes, with only two male heterozygotes and four female homo- zygotes. Although this seems to be a reversal of the pattern seen in cross 1 in terms of which sex is heterozygous, in both cases maleness is associated with the a allele. The two a /b males and the four a/a females are the recombinants in cross 2. The possible chromosomal basis of the inheritance of Pep-C is illustrated in Figure 2, using cross 1 as an example.

In crosses 5 and 6 (Table 2), involving a different male parent than crosses 1 and 2, maleness is associated with the b allele. As in cross 2, most of the males are homozygous and the females heterozygous. In cross 17, maleness seems to be associated with the c allele. Most males are b/c and most females b/b. The electrophoretic patterns of the offspring of cross 17 are illustrated in Figure 1.

The same male parent used in cross 17 was also used in cross 18 (Table 2). This is a more difficult cross to analyze since the female was also heterozygous, but, as in cross 17, maleness is associated with the c allele. There is an excess of females (21 males to 46 females) and an excess of those inheriting the b allele of the father (23 c to 44 b). These results suggest some differential viability of male and female offspring of cross 18 that is not apparent in cross 17, in which a different female parent was used.

Crosses 7, 8 and 9 involved homozygous R. pipiens females crossed to an FI hybrid male produced by crossing a R. palustris female with a R. pipiens male. Although the peptidase type of the original R. pipiens and R. palustris parents were not determined beforehand, the FI hybrid was a b/c heterozygote. Back- cross hybrid offspring showed predominantly Pep-C b/b males and b/c females, indicating that maleness is linked to the b allele (Table 2). This suggests that the b allele came from the original R. pipiens male that supplied the Y chromosome.

In crosses in which the female parent is heterozygous for Pep-C and the male parent homozygous no association of sex and genotype is seen in the offspring. In crosses 14 and 16 (Table 2) there are approximately equal numbers of homozygotes and heterozygotes among the male and female offspring. This confirms that the mode of inheritance of Pep-C variants is sex linkage and that the male is the heterogametic sex.

Superoxide dismutase (SOD) is revealed after electrophoresis by reason of its inhibition of light-induced reduction of tetrazolium dyes. The zymogram shows pale bands on a blue background. Two isozymes are seen, an anodal SOD-l and a slightly cathodal SOD-2 when electrophoresis is performed at pH 8 (Figure 3). The SOD-1 pattern in homozygotes shows a primary band plus a

254 D. A. WRIGHT AND C. M. RICHARDS

TABLE 2

Inheritance of the sex-linked Pep-C locus in crosses involving heterozygous moles or females

Cross

1

2

5, 6

17

18

7, 8, 9

14

16

Parents' Pep-C genotype

Female Male

b/b a/b'

a/a a/b'

b/b o/b

a/b b/b

Offspring

Pep-C" genotype Male Female

21 2 b/a b/b 3 19

a /b 2 18 o/a 24 4

b/a 2 16 b/b 18 3

bc 12 4 dc 6 1 bb 3 22 db 0 19

b/c 2 18 b/b 12 2

a/b 36 24 b/b 41 38

a/b 24 3 1 b/b 29 25

X2

27.25

26.94

21.59

7.02

35.85

19.49

0.91

1.11

P c o.oO01

P < o.Ooo1

P 4 0.0001

P < 0.01

P c 0.0001

P < 0 . m 1

P > 0.3

P > 0.25

P:Rb __ 40:5

42:6

34:5

1 2 2

59:8

30:4

65:74

60:49

a In listing the genotype of the offspring, the allele of the mother is listed first.

e The same male was used in crosses 1 and 2. " The same male was used in crosses 17 and 18.

P:R is the parental to recombinant ratio.

weaker satellite band. This has been described for the homologous SOD locus in other organisms and is apparently the result of postranslational modification of the product of the SOD-2 locus (CROSTI 1978). Animals heterozygous for electrophoretic variants at the SOD-2 locus have a pattern in the SOD-1 region that contains the three bands predicted for the dimer structure of the enzyme that make up the primary bands, plus a fourth band that corresponds to the satellite band of the most anodal primary band. The satellite forms of the other SOD-2s that are predicted by this interpretation are not resolved in Figure 3 but have been detected in other frog SOD-2 heterozygotes where the allelic forms show greater charge differences. Genetic linkage of the loci for SOD-2 and Pep- C has been previously reported (WRIGHT and RICHARDS 1982).

Analysis of the offspring of males heterozygous for the SOD-2 locus reveals that this enzyme is also sex linked. The same pattern of inheritance is seen for SOD-2 as for Pep-C. Figure 3 and Table 3 shows the results of the crosses

FROG SEX-LINKED ENZYMES 255

The Possible Chromosome Constitutions of Animals in a Cross Showing Sex Linkage of

Pep C Cross No. 1

h

Y

eachtype 19 Number of

cr 21

cr 3

0 2

\ / . /

Paren tals Recombinants FIGURE 2.-Hypothetical explanation of inheritance of Pep-C and sex determination in R. pipiens

using cross 1 as an example. The sex-determining region of the chromosome is shown as either X or Y some distance from the Pep-C locus. Recombinants must have resulted from crossing over between the Pep-C locus and the sex-determining region.

involving males heterozygous for SOD-2. Male offspring tend to be heterozygous b/c, whereas females are predominantly homozygous b/b in cross 1. In cross 2 both parents were SOD-2 b/c heterozygotes. In this case there are approximately equal numbers of male and female b/c heterozygous offspring, whereas most c/c individuals are males and b/b individuals are females. The father was the same in crosses 1 and 2, and in both crosses the c allele was associated with maleness.

In crosses 7 , 8 and 9 the male parent was an F, hybrid and an a /b heterozygote at the SOD-1 locus. Pooled data from backcrosses to b/b homozygous R. pipiens females (Table 3) suggest that maleness is linked to the b allele in these crosses. This argues for the location of the b allele on the Y chromosome of the original R. pipiens male used to produce the hybrid.

The data from several crosses on sex linkage of Pep-C and SOD-2 and of linkage between the loci for the two enzymes are summarized in Table 4. In each case, the number of parentals and recombinants is significantly different from the 1:1 ratio expected for unlinked characters. In crosses 1, 2, 7, 8 and 9 both Pep-C and SOD-1 were heterozygous, and since males are the heteroga- metic sex these crosses are true three-point linkage crosses. The recombinant

256 D. A. WRIGHT AND C. M. RICHARDS

S O D 1 t

FIGURE %-Photograph of a starch gel slice stained for superoxide dismutase. Samples are liver extracts from offspring of cross 1. SOD-1 shows either the heterozygous (b/c) or homozygous (b/b) patterns characteristic of the father and mother, respectively, in this cross. All but one of the male offspring in this cross are heterozygous (b/c). All of the females shown here are homozygous (b/b). The single male b/b indicated by the star is a recombinant. The hybrid pattern of the b/c heterozygotes with a strong intermediate band between the primary homozygous forms is typical of a dimer enzyme structure. The presence of an intermediate band indicates that both allelic products are present in the same cell to allow formation of the heterodimer. Heterozygous female recombinants (not shown) have an identical pattern with the heterozygous males.

frequencies between pairs of the three genetic “loci” Pep-C, SOD-1 and sex can be used to establish a genetic map. The data presented in Table 4 indicate the gene order and relative distances illubtrated in Figure 4. Data from crosses 5,6, 17 and 18 are included in estimating the Pep-C-sex distance since the overall value (12.1%) is probably more accurate than the value (13.4%) obtained if only the crosses involving all three loci are used. The linkage of Pep-C and SOD-I (6.9% recombination) has also been reported elsewhere (WRIGHT and RICHARDS 1982) where sex was not considered. The recombination between Pep-C and sex (12.1%) is greater than the recombination between SOD-I and sex (8.6%). This indicates that SOD-I is closer to the sex-determining genes than Pep-C and that SOD-I is between Pep-C and sex.

DISCUSSION

Our results demonstrate that Pep-C and SOD-I are linked to sex in R. pipiens. The mode of inheritance of these sex-linked genes is quite different from that found in mammals in several respects. Among the progeny of females hetero- zygous for the sex-linked gene (e.g., Pep-C) there are no significant differences

FROG SEX-LINKED ENZYMES 257

TABLE 3

Sex-linkage of SOD-1 in crosses involving heterozygous males

Parents' SOD 1 genotype Offspring

SOD-1 Cross Female Male genotype Male Female x2 P:R

1 b/b b/c b/c 22 3 28.17 P < 0.0001 41:5 b/b 2 19

2 b/c b/c c/c 11 1 21.28 P < 0.0001 24:l 13 10

b/c b/b 0 13

7,8,9 b/b a/b b/b 12 1 22.72 P < O.OOO1 31:3 b/a 2 19

TABLE 4

Summary of crosses

Recombi- Locus pair Cross Parental nant X2

Recombi- nant fre- quency

Pep-C-sex 1 40 5 2 42 6 5, 6 34 5 17 12 2 18 59 8 7, 8, 9 - 30 - 4

Total 217 30 141.6 P < 0.0001 0.121

SOD-I-sex 1 41 5 2 24 I

3 9 - 31 7, 8, 9 -

Total 96 9 72.1 P < 0.0001 0.086

Pep-C-S OD-1 1 43 4 2 25 1

6 - 7, 8, 9 - 79 Total 147 11 117.1 P < 0.001 0.069

in the number of heterozygotes vs. homozygotes between males and females. The progeny of males heterozygous for a sex-linked gene consist predominantly of males that inherit one allele and females that inherit the other allele. This finding supports other evidence that the male is the heterogametic sex in R. pipiens (RICHARDS and NACE 1978; WACHTEL, Koo and BOYCE 1975). It is consistent with the mode of inheritance of the sex-linked gene LDH-B, in R. catesbeiana - R. clamitans backcross hybrids described by ELINSON (1981). It is also consistent with the mode of inheritance of a sex-linked peptidase in the

258 D. A. WRIGHT AND C. M . RICHARDS

Three Point Linkage Showing the Relative Arrangement of Genes for Pep C,

SOD 1 and Sex in Rana pipiens

PEP C SOD 1 Sex 1 I 1 I I I

+% Recombination d FIGURE 4.-Diagrammatic representation of the genetic linkage map including the loci for Pep-C,

SOD-1 and sex in R. pipiens.

salamander, Pleurodeles, where females are the heterogametic sex (FERRIER et al. 1980).

The pattern inheritance where linkage to sex is detected in the offspring of a heterozygous parent of the heterogametic sex has also been found in certain species of fish (AIDA 1921; GORDAN 1947; WINGE and DITLEVSEN 1947; YAMAMOTO 1961), brine shrimp (BOWEN 1963) and Culex mosquitos (GILCHRIST and HALDANE 1947; PASTEUR 1975). This type of sex linkage is sometimes referred to as partial sex linkage (HALDANE 1941; BOWEN 1965; OHNO 1967) to distinguish it from classical sex linkage. In classical sex linkage, the genes are exclusively X linked. In Rana pipiens and those other species mentioned before, sex-linked genes are both X- and Y-linked. This probably reflects the primitive state of sex chro- mosome evolution in these species but clearly indicates genetic linkage to a chromosomal region controlling sex.

The same or homologous genes are not sex-linked in the amphibian species for which sex linkage has been reported. The peptidase that is sex-linked in Pleurodeles is not homologous with Pep-C in the frog. Based on its substrate preference, the Pleurodeles sex-linked Pep-I (FERRIER et al. 1980) is similar to Pep-A in R. pipiens. Pep-A is not sex-linked and not linked to Pep-C in the frog (WRIGHT and RICHARDS 1982). More surprising, because of the remarkable similarity of chromosome number, size and shape within the genus (HAERTEL, OWCZARZAK and STORM 1974; SCHMID 1978), is the lack of sex linkage correlation between different species of Rana. In Rana clamitans - R. catesbeiana backcross hybrids, LDH-B is sex-linked (apparently to the R. catesbeiana Y chromosome) (ELINSON 1981). Recently, ELINSON (1982) has shown that in pure R. clamitans crosses, Acon-I is sex-linked, whereas LDH-B and MPI are closely linked to each other but not linked to sex. In R. pipiens, preliminary data indicate that LDH-B and MPI are closely linked (WRIGHT and RICHARDS 1980). Data in Table

FROG SEX-LINKED ENZYMES 259

1 indicate that MPI is not sex-linked in R. pipiens. Sex linkage of Acon-I has not been tested directly in R. pipiens crosses, but Acon-I is not linked to Pep-C (WRIGHT, RICHARDS and NACE 1980).

The occurrence of recombinant types with regard to sex and enzyme allele enables us to map the location of the sex-determining genes with respect to Pep-C and SOD-I in R. pipiens. This ability to map sex-determining genes supports our hypothesis (Figure 2) that most of the so-called X or Y chromo- somes of R. pipiens have nothing to do with the sex-determining mechanism. Perhaps only a small region (or locus) controls sex. This phenomenon could account for the fact that no sex chromosome has yet been identified in R. pipiens, even though some claims to sex chromosome observation have been published for other species of Rana. One of these suggested that a chromosome identified by C banding was heteromorphic in female R. clamitans (MENGDEN 1981). Another report, using late replication (BrdU) banding, identified a late replicating region of one chromosome 4 of males but not females in R. esculenta (SCHEMPP and SCHMID 1981). This kind of pattern would fit our hypothesis.

Another difference from mammals seen in the sex chromosomes of R. pipiens is that the sex-linked genes on both chromosomes are fully expressed. The SOD-I heterozygotes (Figure 3), both male and female, have a hybrid pattern suggesting that SOD-I is a dimer and that in the cell each chromosome expresses its allelic protein subunit. Otherwise, the heterodimer would be absent, as is the case for the sex-linked human enzyme glucose-6-phosphate dehydrogenase (GGPD) in heterozygous females (HARRIS and HOPKINSON 1976).

With the identification of sex-linked genes, the chromosomal basis of sex determination and evolution of sex chromosomes in amphibians should now be approachable. Our hypothesis suggests that YY frogs could be viable and that haploid frog cells carrying a Y chromosome might grow as well as those carrying an X chromosome.

There are now five reported linkage groups in R. pipiens (WRIGHT, RICHARDS and NACE 1980; WRIGHT and RICHARDS 1982). With this report, placing sex with Pep-C and SOD-I, there are three linkage groups containing three or more loci.

The authors are indebted to DR. RICHARD ELINSON of the University of Toronto, who pointed out how to recognize sex linkage in our data. The technical assistance of MS. ANNE CAMOZZI, Ms. BARBARA KUNZ and Ms. RETA HAYNES is greatly appreciated. This work was supported by grant PCM8003774 from the National Science Foundation.

LITERATURE CITED

AIDA, T., 1921 On the inheritance of color in a fresh water fish Aploeheilus latipes Temmick and

The genetics of Artemia salina. 11. White, a sex-linked mutation. Biol. Bull.

The genetics of Artemia salina. V. Crossing-over between the X and Y

Schlegel, with special reference to the sex linked inheritance. Genetics 6 554-573.

BOWEN, S. T., 1963 124: 17-23.

BOWEN, S. T., 1965 chromosomes. Genetics 52: 695-710.

CAMPBELL, R. C., 1974 CROSTI, N., 1978

Statistics for Biologists, Ed. 2. Cambridge University Press, London.

On the two electrophoretic forms of the human superoxide dismutase A in the homozygote SODA 1. Biochem. Genet. 16 739-742.

260 D. A. WRIGHT AND C. M. RICHARDS

DIBERARDINO, M. A., 1962 The karyotype of Rono pipiens and investigation of its stability during

ELINSON, R. P., 1981 Genetic analysis of developmental arrest in an amphibian hybrid Rana

ELINSON, R. P., 1982 Inheritance and expression of a sex-linked enzyme in the frog, Rono clamitons. Biochem Genet. In press.

ENGEL, W. and M. SCHMID, 1981 H-Y antigen as a tool for the determination of the heterogametic sex in amphibia. Cytogenet. Cell Genet. 30: 130-136.

FERMER, V., A. JAYLET, C. CAYROL, F. GASSER and J. J. BUISAN, 1980 Electrophoretic study on red blood cell peptidases in the newt Pleurodeles woltlii: evidence for a sex-linked locus. C. R. Acad. Sci. (Paris) 290: 571-574.

Intersexuality. pp. 523-549. In: Physiology of the Amphibia, Vol. 11, Edited by B. LOFTS. Academic Press, New York.

GILCHRIST, B. M. and J. B. S. HALDANE, 1947 Sex linkage and sex determination in a mosquito, Culex molestus. Hereditas 33: 175-190.

GORDON, M., 1947 Genetics of Platypoecilis maculatus. IV. The sex-determining mechanism in two wild populations of the Mexican platyfish. Genetics 32 8-17.

HAERTEL, J. D., A. OWCZARZAK and R. M. STORM, 1974 A comparative study of the chromosomes from five species of the genus Ran0 (Amphibia:Salientia). Copeia 1974 109-114.

HALDANE, J. B. S., 1941 The partial sex linkage of recessive spastic paraplegia. J. Genet. 41

HARRIS, H. and D. A. HOPKINSON, 1976 Handbook of enzyme electrophoresis in human genetics.

KAWAMURA, T. and R. YOKOTA, 1959 The offspring of sex reversed females of Rana japonica

MENGDEN, G. A., 1981 Linear differentiation of the C-band pattern of the W chromosome in

embryonic differentiation. Dev. Biol. 5: 101-126.

cotesbeiana, Rana clamitans. Dev. Biol. 81 187-176.

GALLIEN, L., 1974

141-147.

North Holland Publishing Co., Amsterdam.

Guenther. J. Sci. Hiroshima Univ. Ser B 18: 31-38.

snakes and birds. Chromosoma 83: 275-287.

NACE, G. W., D. D. CULLEY, M. B. EMMONS, E. L. GIBES, V. H. HUTCHISON and R. G. MCKINNEL, 1974 Amphibians: Guidelines for the Breeding, Care and Manogement of Laboratory Animals. Subcommittee on Amphibian Standards ILAR (NAS/NRC), Washington, D.C.

OHNO, S., 1967

PASTEUR, N., 1975

RICHARDS, C. M. and G. W. NACE, 1978

RUGH, R., 1962

SCHEMPP, W. and M. SCHMID, 1981

Sex Chromosomes and Sex-Linked Genes. Springer-Verlag, New York,

Les leucine-amino-peptidases due moustique Culex pipiens: genetique formelle

Gynogenetic and hormonal sex reversal used in tests of

d’un locus chez I’imago. C. R. Acad. Sci. (Paris) 280: 113-116.

the XX-XY hypothesis of sex determination in Ron0 pipiens. Growth 42: 319-331.

Experimental Embryology. Burgess Publishing Co., Minneapolis.

Chromosome banding in Amphibia. VI. BrdU-replication patterns in Anura and demonstration of XX/XY sex chromosomes in Rana esculento. Chro- mosoma (Bed.) 83: 697-710.

Chromosome banding in Amphibia. 11. Constitutive heterochromatin and nu- cleolus organizer regions in Ranidae, Microhylidae and Rhacophoridae. Chromosoma 68:

Separation and localization of enzymes on gels. pp. 184-209. In: Chromatographic and Electrophoretic Techniques, Ed. 4, Vol. 2, Edited by I. SMITH. William Heinemann Medical Books Ltd, London.

Evolutionary conservation of the H-Y (male) antigen. Nature 254: 270-272.

SCHMID, M., 1978

131-148.

SICILIANO, M. 1. and C. R. SHAW, 1976

WACHTEL, S. S., G. C. KOO and E. A. BOYCE, 1975

FROG SEX-LINKED ENZYMES 261

WINGE, 0. and E. DITLEVSEN, 1947. Color inheritance and sex determination in Lebistes. Heredity

WRIGHT, D. A. and C. M. RICHARDS, 1980 Mapping enzyme loci in the frog. Isozyme Bull. 13: 60. WRIGHT, D. A. and C. M. RICHARDS, 1982 Peptidase isozymes of the leopard frog Rana pipiens:

properties and genetics. J. Exp. 2001. 221: 283-293. WRIGHT, D. A., C. M. RICHARDS and G. W. NACE, 1980 Inheritance of enzymes and blood proteins

in the leopard frog, Rana pipiens: three linkage groups established. Biochem. Genet. 18

Progenies of sex-reversal females mated with sex-reversal males in the

Corresponding editor: S . L. ALLEN

165-83.

591-616. YAMAMOTO, T., 1961

medaka, Oryzias latipes. J. Exp. Zool. 146 163-180.