SOMATIC EXPRESSION AND AUTOSOMAL INHERITANCE OF PHOSPHOGLYCERATE KINASE B IN KANGAROOS1

J. L. VANDEBERG'94>5, D. W. COOPERg, G. B. SHARMANZ AND W. E. POOLE3

'School of Biological Sciencev, Macquarie University, North Ryde, A ~ Z U South Wales, Australia 2113

3Diuision of Wildlife Research, CSZRO, P . 0. Box 84, Lyneham, Australian Capital Territory, Australia 2602

Manuscript received November 20, 1978 Revised copy received January 2.2, 1980

ABSTRACT

The PGK-B isozyme, currently known as PGK-2 in the mouse nomencla- ture, is the predominant PGK isozyme in mammalian sperm. In many species it is detectable only in sperm, in spermatogenic testes and in epididymides containing sperm. In this paper, we provide evidence that some kangaroo species express low PGK-B activity in somatic tissues, in addition to high activity in testes. Three kangaroo species, M . rufogrzseus, M . robustus and M . giganteus, exhibit polymorphism of PGK-B. Breeding data support the hypothesis of auto- somal co-domintmt inhcritance, as is the case in mice. Population data for the three polymorphisms are discussed. PGK-B is not detectable in somatic tissues or spermatogenic testis extracts of monotreme mammals, birds or lizards; it is probably restricted to therian mammals.

WO genetically distinct forms of the glycolytic enzyme phosphoglycerate Tkinase (E.C. 2.7.2.3; ATP:3-phospho-D-glycerate 1-phosphotransferase; PGK) have been detected in mammals. The PGK-A isozyme, currently known as PGK-1 in the mouse nomenclature, is defined by two parameters; it is the predominant or sole PGK isozyme in somatic tissues, and it is known to be X - linked in humans, mice and three species of kangaroos (CHEN et al. 1971; NIELSON and CHAPMAN 1977; VANDEBERG, COOPER and SEIARMAN 1973; VANDE- BERG et al. 1977).

The PGK-B isozyme, currently known as PGK-2 in the mouse nomenclature, is restricted to sperm, epididymides and testes in many mammalian species, and it is the predominant isozyme in sperm (VANDEBERG, COOPER and CLOSE 1973). It is autosomally inherited in mice (VANDEBERG, COOPER and CLOSE 1976). Both PGK-A and PGK-B have been purified from mice and rams and subjected to comparative biochemical and biophysical studies (STEWART and SCOPES 19 78; PEGORARO and LEE 1978). Each species exhibits a remarkable degree of similarity

1 This work was supported by grants from the Australian Research Grants Committee and Macquarie University to DWC and GBS and by U.S. Public Health Service grant HD 11200 to WHS and JLV at the University of Wisconsin (Paper No. 2376 of the Laboratory of Genetics). ' Present address: Laboratory of Genetics, Uniyerbity of Wisconsin-Madison, Madison, Wisconsin, USA 53706.

To whom reprint requests should be addressed.

Genetics 95: 413424 June, 1980.

414 J. L. VANDEBERG et al.

in the properties of the two isozymes, although tryptic peptide maps clearly reveal some differences in their primary structure.

Allelic isozymes of PGK-A have been used extensively in studies of the pecu- liar mechanism of sex chromosome dosage compensation in somatic tissues of kangaroos (see review of paternal X inactivation by COOPER et al. 1977). Somatic tissues of some kangaroo species also express a PGK isozyme that stains more faintly after electrophoresis than PGK-A and exhibits the same electrophoretic mobility as the heavily stained PGK-B isozyme in testis extracts (COOPER et al. 1971 ; VANDEBERG, COOPER and CLOSE 1973). In this communication, we describe the use of allelic isozymes of PGK-B in three species of kangaroos to support the hypothesis that the faintly staining somatic PGK isozymes are specified by the same Pgk-B gene that is strongly expressed in spermatogenic testes.

This report presents the first detailed documentation that the PGK-B isozyme is not testis-specific in all mammalian species. Furthermore, it demonstrates that PGK-B is inherited in an autosomal, co-dominant manner in kangaroos, as it is in mice, suggesting that autosomal inheritance is probably characteristic of PGK-B.

MATERIALS AND METHODS

Most of the captive animals used in this study were from our own breeding colonies. Wild animals were shot under permit.

The experimental techniques have been described in detail elsewhere (VANDEBERG et d. 1977; VANDEBERG. COOPER and SEL~RMAX 1977; VANDEBERG and JOHNSTON 1977). Briefly: starch gel electrophoresis was perIormed ovcrnight in a iris-citric buffer system of pH = 7.3. The gels were sliced in half, and 1 slice was stained by a procedure that relied on the backward reaction for PGK coupled with the preceding glycolytic reaction. This results in the conversion of fluorescent NADH to nonfluorescent NAD, as viewed under long-wave UV. To confirm the identity of the nunfluorescat bands as sites of PGK activity, the other s h e of some gels was stained with an identical reaction mixture, except that it lacked the PGK substrate, 3-phosph0- glycerate (3-PG).

RESULTS

No brands of enzyme activity were ever observed when 3-PG was omitted from the stain; thus, all of the brands expressed by the complete strain were PGK isozymes.

Only one PGK isozyme was observed in erythrocytes and other somatic tissues of the following kangaroo species (the numbers in parentheses are the numbers of animals examined) : Aepyprymnus rufescens (1 ) , Lagorchestes hirsutus (6), Macropus irma (1 ) , Petrogale brachyotis ( 5 ) , Petrogale longmani (9), Petrogale penicillata ( 1 ) , Petrogale rothschildi ( 1 ) , Petrogale xanthopus ( lo), Setonix brachyurus (9), Thylogale billardierii (3) and Wallabia bicolor (24). No poly- morphisms were observed in these species, but it is likely that this band is the X-linked PGK-A isozyme. It stained equally intensely as PGK-A in the three kangaroo species that do have X-linked PGK-A polymorphisms, and its mobility in most of these species was identical to that of the common N allelic isozyme

PGK-B IN KANGAROOS 415

of PGK-A in the polymorphic species. Testicular tissue was not available from any of these species.

The following species exhibited a faint somatic PGK-B isozyme in addition to the heavy stained PGK-A band: Bettongia cuniculus ( 5 ) , Bettongia lesueur (3), Macropus agilis (28), Macropus antilopinus (7), Macropus bemardus (4), Macropus dorsalis (9), Macropus eugenii (5 1 ) , Macropus fuliginosus ( 139), Macropus parma (1 12), Macropus parryi (79), Macropus rufus (72), Thylogale stigmatica (5) and Thylogale thetis (22). No intraspecific variation was observed in the electrophoretic mobility of the erythrocyte PGK-B. M . parryi and M . fuli- ginosus are two of the species polymorphic for the heavily stained PGK-A iso- zyme, but the mobility of the less intense PGK-B band was invariant, despite the variation in PGK-A mobility. In adult testis extracts, available from two M. dorsalis and 13 M . parryi, we observed two bands identical in mobility to those in somatic tissues, but their relative activities were reversed, i.e., PGK-A was the more faintly staining (see Figure l a of VANDEBERG, COOPER and CLOSE 1973). The phenotype of aspermatogenic testes from three juvenile M . parryi was not distinguishable from the somatic phenotype in that PGK-A was the more heavily stained isozyme.

Three additional species, the red-necked wallaby ( M . rufogriseus) , the walla- roo or euro ( M . robustus) and the eastern grey kangaroo ( M . giganteus), also exhibited a heavily stained PGK-A isozyme in somatic tissues, and either a fast migrating PGK-B band, a slow migrating PGK-B band or a two-banded PGK-B pattern typical of heterozygotes. M . giganteus is polymorphic for PGK-A, but the variation in PGK-A mobility occurred independently of the variation in the mobility of the faint PGK-B bands. Spermatogenic testes were available from four M. rufogriseus, one PGK-B heterozygote and three homozygotes for the slower migrating isozyme. In every case, testis extracts exhibited bands identical in mobility to those of somatic extracts, but the PGK-A isozyme was more faintly stained and the other one or two bands were more heavily stained. These data demonstrate that PGK-B occurs in both somatic tissues and in the testes. In somatic tissue, it is fainter than PGK-A, but the relative activities are reversed in the testes.

A wide variety of somatic tissues from some individual M . parryi and M. giganteus of both sexes were typed, as were several somatic tissues from indi- viduals of other species (see partial list of tissues in VANDEBERG, COOPER and SHARMAN 1977). If a PGK-B isozyme was expressed in any somatic tissue, it was expressed in all somatic tissues of the same individual. However, activity of the PGK-B isozyme varied from tissue to tissue in respect both to PGK-A activity of the same tissue and to PGK-B activity of other tissues. For example, it is apparent from Figure 2 of VANDEBERG, COOPER and SHARMAN (1977) that M . giganteus leg muscle expresses higher PGK-A activity than, for example, does liver, ventricle or bladder, but lower PGK-B activity than any of the other tissues. These patterns of tissue-specific activity differences were consistent from animal to animal, regardless of sex and age, at least from pouch emergence to adulthood.

41 6 J. L. VANDEBERG et al.

Another difference observed in PGK-B expression in different tissues was the extent of subbanding. While kangaroo PGK-A is not prone to the formation of subbands, PGK-B tends to have a subband of lesser mobility than that of the major PGK-B isozyme. The identity of these bands as PGK-B subbands, rather than PGK-A subbands or some other PGK isozyme, was established in species poly- morphic for PGK-B (described below), where the position of the subband was always dependent on the position of the main PGK-B band. The subband of the faster allelic isozyme migrated to the same position as the main band of the slower allelic isozyme. The intensity of the subband is variable between species and also between tissues of any individual. For example, as shown in Figure 1 of VANDEBERG, COOPER and SHARMAN (1977), while auricle, kidney and ear of M . giganteus express a faint subband, retina is unusual in that it expresses a highly active subband approximately equal in activity to the main PGK-B band.

Three erythrocyte PGK-B phenotypes were observed in a sample of 190 red- necked wallabies, single-banded fast (RNF) , single-banded slow (RNS) and equal activities of fast and slow (RNFIS) (see Figure 2). Population data are given in Table 1 and breeding data in Table 2.

TABLE 1

Population data for the PGK-B polymorphism in Macropus rufogriseus individuals not known to be related

Locality

Gene freq. of the

Lat.(S) Long.(E) F / F F / ? F/S S/? S/S n* F allele

Qld. Injune 25.538 Warwick 28.12

N.S.W. Ewingar 29.21 Tooloom 29.37 Moonpar State

Forest 30.12 Braidwood 35.27 Captain’s Flat 35.38

S.A. Millicent 37.36

Tas. Flinders Is. 40.00 Gladstone 40.57 Launceston 41 2 5 Avoca 41.45 Hobart 42.54 Unknown

148.30 0 1512.00 0

152.32 0 152.26 0

152.41 0 149.50 0 149.28 0

140.22 0

148.00 0 148.01 1 1 147.07 2 147.47 1 147.18 1

2

I

1 4 0 3 1 5 0.33 0 10 10 54 138 0.07

0 1 0 0

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

0 1 0 0 2 J

0 4 0 0 0 12 0 6 5 : ] 0 2 1 0 0.60

0 1 0 0 0 1 0 2 1 0

0.18

a o o o

Unknown 0 2 1 3 1 3

* n is the number of alleles. From each pouch young carried by a homozygous wild-shot mother, it was possible to deduce

one paternal allele (see COOPER 1968). The paternal types derived in this manner are given as F/? and S/?. The symbol n is used to denote the number of aZZe2es. The gene frequencies of the Warwick and Injune populations are highly significantly different in the 2 x 2 contingency table test (xI2 = 10.412, P<O.Ol). The test of the gene frequencies of mainland versus Tasmanian animals also gave a highly significant difference (x12 = 74.879, P<O.01).

PGK-B IN KANGAROOS 41 7

TABLE 2

Breeding data for t h inheritance of PGK-B in Macropus rufogriseus, Macropus robustus and grey kangaroos (M. giganteus and M. fuliginosus)

Phenotypes of parents offspring

Species ? ? dd F F / s S

M . rufogriseus F F 2 I* 0 S F 0 1 0 S S 0 0 10 S ? 0 1 19 ? S 0 0 2 ? F 0 1 0 S F/S I* 0 0 F/S F/S 2 4 I F/S F 1 0 0 F/S ? I 5 1

M . robustus F S 0 I 0 F ? 0 I 0 S S 0 0 12 S ? 0 0 1 F/S S 0 6 4 F/S ? 0 2 0

Grey Kangaroos F F I46 0 0 ? F 12 0 0 F ? 17 2 0 F/S F 4 2 0 F F/S I 0 0 ? F/S 1 e 0

* Inconsistent with the hypothesis of autosomal co-dominant inheritance. It is presumed that these discrepancies were caused by an error such as cross fostering, mistaken coding or decoding of ear marks, mistaken paternity, mislabelling of the samples, or another unascertained mistake.

A detailed breakdown of the species, hybrids and backcrosses involved in each grey kangaroo cross is given in Appendix V of VANDEBERQ (1975). Note that the electrophoretic mobilities of bands designated F and S are different for different species.

Sixty-five euros and wallaroos ( M . robustus) were typed for erythrocyte PGK-B, revealing three phenotypes: fast ( E W ) , slow (EWs) and fast/slow (EWFlS Figures 1 and 2 ) . The EWs type is marginally slower than the RNF type (Figure 2 ) . Three subspecies of M . robustus were tested: M.r. robustus, M.r. erubescens and M.r. woodmrdi. As shown in Table 3, M.r. robustus is monotypically EWs, but the other two subspecies are polymorphic for the two alleles. The breeding data are given in Table 2.

Genetic variation in erythrocyte PGK-B was also observed in the eastern grey kangaroo, M. giganteus, of which 200 were typed. The phenotypes observed are shown in Figures 1 and 2. The common mobility, called EGF, is slightly faster than EWF, but the difference is difficult to resolve. A double-banded pheno- type was occasionally observed; it consisted of EGF and a slower band, EGs, which is slightly slower in mobility than EWs. Failure to observe the puta- tive homozygous EGS type was consistent with the low frequency of the allele. A second variant pattern was observed in two females shot at the same locality.

41 8 J. L. VANDERERG et al.

FIGURE 1 (top) .-Gel showing PGK-R phenotypes of erythrocytes of Mucropus robustus (EW) and Mucropus Riganteus (EG). PGK-A is of the N type in channels 1-5, and of the VE type in channels 6-8 (see VANDEBERG ~t al. 1977n). It is possible. but not very likely, that the band labelled "B?" in the photograph and designated as the EG" mobility of PGK-R could represent paternal expression of a Ppk-A allele. Channel 1, EWs; 2, EI'I'F/s (the PGK-R bands were very faint, hut they were easily seen on the gel itself); 3, EWF; 4, EGF/'/s; 5, EGF; 6, EG'I"; 7, EG'; 8, EGS/'.

PGK-B IN KANGAROOS

TABLE 3

Population data for the PGK-B polymorphism in Macropus robustus individuals not known to be related

419

Genefr uenc of Subspecies Locality F/F F / S S/? S/S n* the? all&

M.r. robustus Qld. Warwick 0 0 0 4 8 Clermont 0 0 0 2 4

N.S.W. Glen Innes 0 0 0 1 2 Coonabarabran 0 0 0 1 2 Bendemeer 0 0 0 1 2 Burrinjuck 0 0 0 1 2 Coombadjha 0 0 0 1 2

Unknown 0 0 0 1 1 2 2 Totals 0 0 0 2 2 4 4 0.00

M.r. erubescens Qld. Morven 0 1 0 0 2 S.A. Fowler’s Gap 0 1 0 1 4

GawlerRanges 0 1 0 0 2 W.A. Unknown 0 1 1 3 9 Unknown 1 0 0 0 2 Totals 1 4 1 4 1 9 0.32

M.r. woodwardi N.T. Arnhem Land 3 1 0 0 8 Totals 3 1 0 0 8 0.88

* n is the number of alleles. The subspecies names used here are those recommended by RICHARDSON and SHARMAN (1976).

See caption of Table 1 for an explanation of the S/? type. The symbol n is used to denote the number of alleles. The gene frequencies in the three subspecies are highly significantly different. The Brandt and Snedecor formula gives x22 = 37.703, P<O.Ol. M.r. erubescens, which has gene frequencies intermediate between those of the other two subspecies, is significantly different from both of them in exact 2 x 2 contingency table tests ( P = 0.023 for M.r. erubescens us. M.r. woodwardi, and P = 0.008 for M.r. erubescens us. M.r . robustus).

It consisted of three PGK bands (Figure 1, channel 8). The fastest migrating band is the VE allelic isozyme of PGK-A (see AN DEB ERG et al. 1977) and the slowest migrating band is PGK-B of the mobility hGF. The third band expresses the same activity as the EGF band and is intermediate in mobility between EGF and VE, precisely in the N position of PGK-A. Two interpretations are possible: (1) the band is a PGK-B allelic isozyme of the same mobility as N of PGK-A; or (2) the band represents faint expression of the paternal Pgk-A allele, known to occur in some tissues of this species (see VANDEBERG, COOPER and SHARMAN 1977b). The first of these possibilities is favored for two reasons: first, faint expression of the paternal Pgk-A allele in blood cells occurs infrequently, and second, the paternally derived PGK-A isozyme exhibited much less enzyme

FIGURE 2. (bottom) .-Gel showing PGK-B phenotypes of erythracytes of Macropus rufo- griseus (RN), Macropus robustus (EW) and Mucropus giganteus (EG). 1, RW; 2, RNFJS; 3, RNF (there is a faint subband of RNF in the position of RNS) ; 4, EWS; 5, EWF/S; 6, EGF./s (the PGK-B bands were very faint, but they were easily seen on the gel itself) ; 7, EGF.

420 J. L. VANDEBERG et al.

activity in blood cells than EGF in cases where it was expressed (VANDEBERG et d. 1977). Therefore, we tentatively designated this PGK-B phenotype as EGNIF. The breeding data are given in Table 2 and the population data in Table 4. One putatively heterozygous male (EGFP) probably transmitted each allele to at least one male (EGFIS) probably transmitted each allele to at least one male and one female progeny, indicating the unlikelihood of partial sex-linkage of PGK-B (Table 5 ) .

The breeding data for PGK-B in kangaroo species have been supplemented with data from species hybrids and their parents (Table 6 ) .

DISCUSSION

Female kangaroos produce, at best, one young per year, and the generation time is several years, Given the small number of captive kangaroos available for research and the large expense incurred in their maintenance, it is not feasible

TABLE 4

Population data for t h PGK-B variation in Macropus giganteus individuals not known io be related

State or territory F/F F/T F/S N/F nr

Qld. U) 3 0 0 4 N.S.W. 33 12 4 2 90 A.C.T. 18 0 0 0 36 Vic. 10 0 0 0 U) S.A. 5 0 1 0 12 Unknown 23 2 2 0 52 Totals 10.9 17 7 2 263

* n is the number of alleles. The detailed data by specific localities are given in Appendix IV of VANDEBERG (1975). See

caption of Table 1 for an explanation of the F/? type. Two of the F / S individuals were not actually tested, but both gave both F / F and F/S progeny when mated to F / F individuals. The symbol n is used to denote the number of alleles. The gene frequency of S is 0.03. Gene frequen- cies for F and N are not given because the EGN mobility is the same as N for PGK-A and would be undetectable in most individuals.

TABLE 5

Family data indicating the unlikelihood of partial sex-linkage of PGK-B in Macropus giganteus

F Offspring

F/S

Phenotype of mother ? ? dd ? ? dd F I 0 0 2 ? 2 1 2 0

The offspring were sired by a single male, G60, mated with eight Werent females. G60 was not tested, but his genotype is inferred as F / S from the family data. He has transmitted the F allele to three females and one male, and has transmitted the S allele to two males. He has prob- ably transmitted the S allele to the two F / S females whose mothers were not tested, since the frequency of S in M. giganteus not known to be related is 0.03.

PGK-B IN KANGAROOS 421

TABLE 6

Breeding data for the inheritance of PGK-B in kangaroo species hybrids

Hybrid ~~~

Female parent

Code SHI SH2 OK1 43 44 RGI R102 -

Sex 0 8 0 0 8 8 8 8

Phenotype EWS/K EWS/K EWS/K EWms/K EW/K RNF/EGF R W / T K=AG

species Phenotype M.r. robustus (EWS) Mar . robustus EWS Mar . robustus EWS M . rujus (K) M . rujus (K)

M . rujus (K)

M . rujogriseus 7 M . rujogriseus RNFP

Male parent

Species Phenotype M . rujus K M . rujus K M . rujus ( K ) M.r. erubescens 3 M.r. erubescens ? M . giganteus 7 M . eugenii (TI M . agilis (AG)

Parental phenotypes in parentheses were inferred from population data. M . = Macropus, M.r. = Macropus robustus. Same of these hybrids were also used in the studies supporting paternal X-inactivation and have been published under the same code numbers (RICHARDSON, CZUPPON and SHARMAN 1971; SHARMAN 1971).

to collect or biopsy testes from enough pedigreed father-son combinations to estab- lish the mode of testicular PGK-B inheritance. However, the co-migration of testicular PGK-B and the faintly expressed PGK isozyme (s) in somatic tissues, even in polymorphic species, strongly supports the hypothesis that the faint somatic isozymes are specified by the same Pgk-B gene locus that is highly expressed in testicular t i s sueno other hypothesis seems plausible.

The PGK-B family and population data derived from kangaroo erythrocytes are consistent with the hypothesis of autosomal co-dominant inheritance. The alternative hypothesis of partial sex-linkage is not disproved, but it is unlikely to be correct, because one putatively heterozygous male M . giganteus probably transmitted each allele to both sexes of offspring. Since the X-linkage group has been highly conserved during mammalian evolution (OHNO 1967) and since the mouse Pgk-2 gene is located on an autosome, it is highly probable that auto- somal inheritance of PGR-B will be characteristic, as is X-linkage of mammalian PGK-A. The mouse Pgk-2 gene is an especially valuable marker for immuno- genetic studies because it is closely linked to the major histocompatibility system (MHS) (EICHER, CHERRY and FLAHERTY 1978; VANDEBERG and KLEIN 1978). It would be extremely interesting to determine if this linkage relationship has been conserved during mammalian evolution.

The population data for M . rufogriseus PGK-B indicate significant differences in gene frequencies between different geographical locations. These differences were highly significant when all the Tasmanian animals were compared with all the mainland animals, and when a comparison was made between the only two mainland populations sufficiently tested to enable comparison. These were populations from near Warwick and Injune, which are approximately 420 k m apart. JOHNSTON (1973) found that the allelic frequencies at several other enzyme loci differ in these two populations. He also observed large gene fre- quency differences between these populations and three populations to the south of Warwick that were much closer to Warwick than to Injune. (The closest was

422 J. L. VANDEBERG et al.

Ewingar, approximately 115 km from Warwick.) The species is continuously distributed between these localities, which exhibit no obvious climatic or habitat differences. It would be interesting to take samplings between localities to deter- mine whether the differences are clinal or are associated with reproductively isolated or semi-isolated demes. The much higher frequency of the RN” allele in Tasmania than anywhere on the mainland is also consistent with large gene- frequency differences between Tasmanian and mainland animals for enzymes studied by JOHNSTON (1973), whose enzyme and morphology results led him to consider the two populations as subspecies (M.r. rufogriseus on Tasmania, King Island and Flinders Island; and M.r. banksianus on the mainland). The two sub- species have probably been geographically isolated at the very least since the land bridge was broken between Tasmania and the mainland approximately 10,000 years ago.

The population data for M . robustus PGK-B provide further evidence for the existence of three subspecies on the mainland, as proposed by RICHARDSON and SHARMAN (1976). The eastern coast population, M.r. robustus, is monotypic for the EWs allele; whereas, M.r. erubescens and M.r. woodwardi express both alleles but with significantly different gene frequencies.

The gene frequency of the EGS allele in M . giganteus is too low in all popula- tions tested for useful comparison, although its absence from M . fuliginosus provides further evidence for the distinctness of the two species, which are sympatric over part of their range (KIRSCH and POOLE 1967,1972).

We conclude that, while PGK-B may be restricted to spermatogenic cells in some mammalian species, it is expressed to a lesser extent than PGK-A in the somatic cells of some kangaroo species. Kangaroos are probably not unique in this regard, however. Fifteen of the 17 species of eutherian mammals tested express, in somatic tissues, a single PGK isozyme, presumably PGK-A, while foxes (Vulpes uulpes) and dogs (Canis familiaris) express two somatic PGK isozymes (VANDEBERG, COOPER and CLOSE 1973). One of these, highly active in all foxes and dogs, was probably PGK-A. The other one, presumably PGK-B, exhibited variable activity in somatic tissues but was always the more highly active PGK isozyme in spermatogenic testis extracts. Of 29 marsupial species tested other than kangaroos, ten exhibited a faint somatic isozyme in addition to the heavily stained putative PGK-A isozyme, and in every case a highly active, co-migrating isozyme was observed in spermatogenic testis extracts (VANDEBERG 1975).

PGK-B appears to be restricted to therian mammals, however, since an exten- sive search revealed no evidence of a second PGK isozyme in somatic tissues or spermatogenic testis extracts of three species of monotreme mammals, three species of birds and ten species of lizards (VANDEBERG 1975). CAM and COOPER (1978) have shown that there is one autosomal locus for PGK in soma and sperm of the domestic chicken (Gallus domesticus), and this is presumably the evolu- tionary analogue of mammalian PGK-A.

The characteristics of PGK-B, then, can be tentatively generalized as follows: (1) It is autosomally inherited (mice, kangaroos), (2) it is the predominant

PGK-B IN KANGAROOS 423

PGK isozyme in sperm (humans, rams, bulls, mice), (3) it is highly active in spermatogenic testis extracts, along with PGK-A (many species) , (4) it is absent from soma of some species and weakly active in soma of others, but never more active than PGK-A, and (5) it is present in marsupial and eutherian mammals, but not in monotreme mammals, birds or reptiles.

VANDEBERG. Part of this work was presented in a Ph.D. thesis to Macquarie University by J. L.

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