Biochemical Genetics, Vol. 14, Nos. 5/6, 1976

Genetic Control of LDH Isozymes in the House Fly, Musca domestica

Takeshi Agatsuma ~ and Takuji Takeuehi ~

Received 24 July 1975--Final 4 Nov. 1975

Electrophoretic variations in lactate dehydrogenase from adult whole body homogenates are described for three laboratory strains of house fly, Musca domestica. Several crosses between different electrophoretic forms provided evidence that the observed variations are due to segregation of alleles at two distinct loci (designated as A and B loci) and that the LDH isozymes of house flies are dimers formed by a random association of subunits controlled by the two loci.

K E Y W O R D S : l a c t a t e d e h y d r o g e n a s e ; h o u s e f ly ; g e n e t i c c o n t r o l .

INTRODUCTION

Lactate dehydrogenase (LDH) of most vertebrates so far examined has been shown to exist in several different molecular forms. Gel electrophoresis of extracts from mammalian tissues has indicated that five isozymes are generally found, each of which is a tetramer formed by the random association of two different protein subunits, designated A and B, in all possible combinations (Markert, 1963).

In invertebrates, there is little information concerning the number of forms and the nature of LDH. According to Pesch (1972), lactate dehydro- genase in the hard clam, Mercenaria mereenaria, occurs as an isozymic pat- tern of either one or two bands with no phenotypic differences between the sexes and has been demonstrated to be controlled by a single autosomal gene. Lauffer (1961) reported one band of LDH activity in an electrophoretic

B i o l o g i c a l I n s t i t u t e , T o h o k u U n i v e r s i t y , A o b a - y a m a , S e n d a i , J a p a n .

441

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442 Agatsuma and Takeuchi

analysis of the blood of the silk moth, Hyalopora cecrop&, and at least three bands of activity in the blood of a related moth, Samia cynthia. Rechesteiner (1970) suggested that although only one electrophoretic form of the LDH can be detected there may well be two forms in Drosophila. Pappas and Rodric (1971) in their electrophoretic studies on LDH of D. melanogaster also indicated the possibility of a dimeric structure for LDH and assumed that two loci were responsible for the synthesis of LDH.

The present article deals with the mode of inheritance of the LDH isozyme patterns in the house fly, Musca domestica.

MATERIALS AND METHODS

Mutant strains Hokota and ge (green) and a wild-type strain Lab of the house fly, Musca domestica, were used in this study. These strains were kindly sup- plied by Dr. T. Hiroyoshi of the Department of Genetics, Osaka University Medical School

Upon emergence, virgins were collected and were mated in cages. Whole flies were homogenized individually in about 0.1 ml distilled water. The homogenate was then centrifuged at 3000 rpm (approximately 1000g) for 1 rain at room temperature, and the supernatant was used for electrophoretic studies.

The procedure for thin-layer acrylamide gel electrophoresis essentially followed the method reported by Moriwaki et al. (1974), except that acryla- mide was dissolved at a final concentration of 5.7% in tris-citrate buffer, pH 8.6. The electrophoresis was conducted at 4 C for 3 hr with a constant-current power supply at 2.0 mA/cm gel width.

LDH assay was performed after electrophoresis by incubating the gel at 37 C in the following standard solution:

0.1 M Tris 1.0 g D,L-Lactate 0.04 g Nitroblue tetrazolium 0.04 g Phenazine methosulfate 0.015 g NAD

500 ml Distilled water

RESULTS AND DISCUSSION

Electrophoretic Variations of LDH Isozymes in Laboratory Strains of House Fly

The results of detection of LDH isozymes in laboratory strains Hokota, ge, and Lab, by thin-layer acrylamide gel electrophoresis are shown in Fig. 1.

Genetic Control of LDH Isozymes in the House Fly 443

+

T m

m

I

m

m

I

m

I I

TYPES I HYBRID(IX]I) TT TTT

Fig. 1. (a) Lactate dehydrogenase zymograms in the laboratory strains Hokota (H), ge (g), and Lab (L) of the house fly. o, Origin. (b) The hybrid pattern (Hy) in the F1 from the cross ge (g) x Hokota (H). (c) Schematic presentation of the LDH isozyme phenotypes observed in the strains. The types I, II, and III represent Hokota, ge, and Lab, respectively. The offspring ob- tained from the cross of Hokota x ge exhibit the hybrid pattern.

Three distinct electrophoretic variations of L D H were observed. The strain Hokota always expressed the type I pattern, ge strain always the type II, and Lab always the type III. Types I and II showed triple bands, but they can be easily distinguished by differences in mobility. Type I I I exhibited only a single band, which corresponded to the slowest band of the other types.

Genetic Control of LDH Isozymes in the House Fly

Some crosses were made between strains in order to test the mode of in- heritance of this enzyme (Tables I, II, and III). Offspring obtained from crosses of Hokota x Hokota, ge x ge, and Lab x Lab showed types I, II, and III , respectively. The Lab x ge mating produced offspring of phenotype II. In F2, phenotypes I I and I I I appeared in a 3 : 1 ratio (Table II). The Lab x

444 Agatsuma and Takeuchi

Table I. Results of the Crosses Hokota × Hokota, ge x ge, and Hokota × ge, and the Backcross

Phenotypes

Crosses I Hybrid II III

Hokota x Hokota 56 0 0 0 ge x ge 0 0 34 0

Hokotax ge 0 33 0 0 F1 x ge 0 50 46 0 (P> 0.5) F1 x FI 23 48 25 0(P> 0.95)

Table II. Results of the Crosses Lab x Lab and Lab x ge

Phenotypes

Crosses I Hybrid II III

Lab x Lab 0 0 0 32 Lab x ge 0 0 35 0 F1 x F1 0 0 50 18 (P>0.7)

Table Ill. Results of the Cross Lab x Hokota

Phenotypes

Crosses I Hybrid I[ III

Lab x Hokota 4l 0 0 0 Fl x F~ 42 0 0 13 (P> 0.7)

H o k o t a mat ing p roduced exclusively offspring o f pheno type I. In F 2, pheno- types I and I I I appeared in a 3 : 1 ra t io (Table I l l ) . In the case o f the g e x

H o k o t a cross, a new type showing a hybr id pa t te rn (Fig. 1) appeared in all F i individuals. The hybr id pa t te rn included all the bands present in the pa ren t strains. In the case o f F 1 x F1, the phenotypes I, hybr id , and II appeared in 1 :2 :1 rat io in the offspring. The rat io o f 46 hybr ids to 50 IIs in the off- spring p roduced f rom the backcross o f hybr id x g e approx ima ted the expected ra t io of 1:1. There was no difference between males and females in the phenotyp ic ra t io p roduced in any type of matings.

Genet ic Control o f L D H Isozymes in the House Fly 445

+

, co oo

o~

Q, 8~

go 11o

lira

41o

. . . . . 1.95

. . . . . 1.75

. . . . . 1.60

. . . . . 1.4 5

. . . . 4 . 3 0

. . . . . | . 0 0

Phenotypes I HYBRID 1"I" ITr

A,o ~,oo f~.oo ;4.0o ~.oo

Genotypes 13195 B1.60 130.00 •

Fig. 2. D iag rammat i c representa t ion o f molecular combina t ion in the L D H isozymes o f the house fly. Symbols : o , subuni t p roduced by the B T M allele; (1), subuni t produced by the B 1"6° allele; o , subuni t produced by the A t ° ° allele.

The triplet-band pattern detected in some of the inbred strains led us to the assumption that the LDH isozymes in house flies are dimers formed by the association of subunits controlled by two distinct loci. The results of the crosses support this assumption. Crosses between type III and either type I or type II indicate that they are variables at a locus and have another common invariant locus. It seems likely that the slowest band represents the molecule controlled by the common locus and that the fast bands are controlled by the variable locus. The intermediate bands are considered to be hybrid molecules. We designate the former locus A, for which we so far have found only one allele A 1"°°. The latter locus, B, on the other hand, presumably possesses two dominant alleles, B 1"6° and B TM, and a null allele, B °'°°. It is also probable that the third allele actually synthesizes a LDH protein, the mobility of which is identical with that ofA 1'°°. In this case, this allele might be designated as B 1"°° instead of B °'°°.

The cross between type I and type II resulted in six LDH bands in the F1; they consisted of five parental bands and one new band. This result is explained only by the dimer model (Fig. 2). If we assume that the LDH isozymes are dimers formed by a random combination of subunits produced by the two loci, then we can expect five parental LDH isozymes and one hybrid molecule of a new type in the F 1 whose genotype is A~'°°/A I"°° B 1"6°

/B TM. Although we assume in our hypothesis that two structural genes are involved in the formation of LDH isozymes, it is also conceivable that the product of one structural gene is posttranscriptionally modified by genetic factors controlled by the other locus, resulting in the same isozyme patterns.

446 Agatsuma and Takeuchi

These alternatives can be discriminated by finding alleles at the A locus and elucidating the interaction between A and B loci.

Sakoyama and Ogita (1969) reported that the L D H isozymes in the house fly occurred as A and B isozyme groups, each of which seemed to be controlled by two loci located on the fifth chromosome and that there existed F and S types in each isozyme group forming no hybrid enzyme between each other. Our results, however, indicate that each L D H isozyme is a dimer, so that hybrid molecules can be formed, and that the B locus seems to be located on the third chromosome because alleles at the B locus always segregate with the ge gene, whose locus is on the third chromosome. The difference may be due to the different methods we used.

According to Long and Kaplan (1968), the invertebrate LDHs studied to date fall into two categories, i.e., L-lactate dehydrogenase and D-lactate dehydrogenase, and no animal possesses lactate dehydrogenase for both stereoisomers. In the arthropods, all the chelicerates (arachnids) studied possess o-lactate enzymes, whereas the mandibulates (crustaceans, myria- pods, and insects) have g-lactate specific dehydrogenases. The polycheate Nereis has a D-lactate specific catalyst, whereas the earthworm and leech have the g-lactate dehydrogenases. Whether the house fly L D H is o-specific remains to be determined.

A C K N O W L E D G M E N T S

We wish to thank Dr. C. R. Shaw of the University of Texas M. D. Anderson Hospital for reading the manuscript and Dr. T. Hiroyoshi of Osaka Univer- sity for supplying us with the strains. We also thank Mr. F. Sato for his technical assistance.

R E F E R E N C E S

Lauffer, H. (1961). Forms of enzymes in insect development. Ann. N. Y. Acad. Sci. 94:825. Long; G. L., and Kaplan, N. O. (1968). D-Lactate specific pyridine nucleotide lactate

dehydrogenase in animals. Science 162:685. Markert, C. (1963). Lactate debydrogenase isozymes: Dissociation and recombination

of subunits. Science 140:1329. Moriwaki, K., Sadate, T., and Hirasawa, S. (1974). Improved method for separation and

identification of serum transferrins: Thin layer acrylamide-gel electrophoresis with acrinol pretreatment. Experientia 30:119.

Pappas, P., and Rodric, G. (1971). An electrophoretic study of lactate dehydrogenase isozymes, protein and lipoprotein of Drosophila melanogaster larvae, pupae and adults. Comp. Biochem. Physiol. 40B:709.

Pesch, G. (1972). Isozymes of lactate dehydrogenase in the hard clam, Mercenaria mer- cenaria. Comp. Bioehem. Physiol. 43B:33.

Rechesteiner, M. (1970). Drosophila lactate dehydrogenase: Partial purification and characterization. J. lnsect PhysioL 16:957.

Sakoyama, Y., and Ogita, Z. (1969). Genetical and biochemical studies on dehydrogenase in house fly. Jpn. J. Genet. 44:407 (abstract in Japanese).