And URSPRUNG(1966)

GENETICS OF OCTANOL DEHYDROGENASE IN DROSOPHILA METZIP+

SARAH BEDICHEK PIPKIN Homrd Uniuersity, Washington, D.C.20001 Received October 11, 1967

CTANOL dehydrogenase (ODH) was studied in Drosophila melanogaster first by URSPRUNGand LEONE(1965) and distinguished from alcohol dehydrogenase by COURTRIGHT,IMBERSKI, and URSPRUNG(1966). The neotropical species Drosophila metzii is polymorphic for complex octanol dehydrogenase patterns which will be shown to depend on two distinct structural genes, ODH,, apparently homologous with the locus studied by COURTRIGHTet al. (1966), and ODH,, responsible for a more slowly migrating isozyme. The ODH loci are unlinked, and variants display unifactorial inheritance. The ODH molecule is considered at least a dimer but probably a tetramer. Isozyme formation can involve combinations of polypeptides produced by either or both of the two structural genes. Genetic evidence will be presented indicating that egg or embryonic and imaginal ODH isozyme patterns depend on the same structural genes.

MATERIALS AND METHODS

Single flies were assayed in crude homogenates with 1-octanol as substrate, using the agar gel electrophoresis method, with formazan staining according to the method of DR.H. URSPRUNG (1x5). Modifications of the method for the present work included grinding of single flies in a drop of glass distilled water in specially prepared small homogenizers and allowing the electrophoresis to proceed at 25Ov for forty minutes instead of half an hour. Enzyme assays for the genetic analysis were made on single females aged 4 to 6 days. The smaller males do not provide sufficient enzyme for reliable single fly analysis. Flies for experimental crosses were reared in pair matings on a medium of corn meal-Karo-Brewer’s yeast #2019 (Standard Brands). Isozyme patterns of extracted lines were also studied in flies reared on the usual medium enriched by the addition of Kellogg K breakfast food. ODH variants were isolated from strains of D. metzii from Trinidad, West Indies; Barro Colorado Island, Canal Zone; Turrialba, Costa Rica; Darien, Panama; and Almirante, Panama. Eggs were collected by allowing fertilized females to lay for thirty hours in small Petri plates containing a solution of Fleischmann’s yeast sprinkled with dilute acetic acid. Contents of the Petri plates were washed onto a Buchner funnel lined with a black cloth overlaid with cheesecloth. The latter strained out drowned flies and debris. Eggs were taken from the black cloth after repeated washing. These were ground in small homogenizers with a drop of glass distilled water.

RESULTS Upon inbreeding with pair matings, lines with true breeding isozyme patterns

This work was supported by the Division of General Medical Sciences, National Institutes of Health, Bethesda, blaryland, RG-12018 to the Johns Hopkins University, Baltimore, Maryland, and biM-14937 to Howard University, Washington, D.C. + This paper is dedicated to the memory of Dr. WIXSONS. STONE,the Genetics Foundation, the University of Texas, Austin, Texas.

Genetics 60: 81-92 September 1968. a2 SARAH BEDICHEK PIPKIN

+a b C

FIGURE1 .-Zymograms of ODH isozymes of single adult females of (a) a pattern 5 extracted line (from left to right, first two slits); (b) a pattern 3 extracted line (from left, third and fourth slits); (c) a pattern 1, or pattern 1, extracted line (from left, fifth through eleventh slits). were extracted: (1) those showing a strong band at position 5 and sometimes weak bands at positions 3 and 4, with sometimes also a strong band at position 6 and/or 7 (called pattern 5 and shown in Figure la); (2) those showing a single formazan band at position 3 (called pattern 3 and shown in Figure lb); (3) those OCTANOL DEHYDROGENASE IN D.nzetzii 83 showing a single fomazan band at position 1 (called pattern 1 and shown in Figure IC).The pattern 5 line could be extracted from the Turrialba, Almirante, and Trinidad strains, but most of the breeding work was done with the line extracted from the Turrialba strain in two generations of inbreeding. Without selection the Barro Colorado Island strain was true breeding for pattern 3, al- though this strain originated from 8 females collected in nature (PIPKIN1968). Pattern 3 lines could be extracted in one generation of inbreeding from the Darien strain and in two generations from the Trinidad strain. An almost true breeding line with isozymes in positions 1, 2, 3, 4, 5, and sometimes 6 and 7 (called pattern 1-5) was isolated in two generations from the Trinidad strain and used in early breeding work. Occasionally an individual with isozymes only at positions 1, 2, and 3 would be seen, and in the course of a year, the pattern 1-5 line showed just one isozyme at position 1 (Figure IC).A photograph from single females of the pattern 1-5 line is given in PIPKIN(1968). Sublines from the original pattern 1-5 line were maintained which subsequently showed only a single isozyme at position 1 but exhibited different breeding behavior in crosses with individuals of the pattern 3 line as will be described later. Most of the breeding work was carried out using ordinary corn meal-Brewer's yeast medium. When cultured on medium enriched by Kellogg K breakfast food, isozyme patterns of the Barro Colorado Island pattern 3 line, the Turrialba pattern 5 line, and the Trinidad pattern 1 line, which were used for most of the breeding work, remained unchanged except for stronger staining indicating a higher enzyme level. The pattern 1-5 line had become the pattern 1 line by the time the enriched medium was being used. On the other hand, progeny of a single female of the Trinidad pattern 3 line cultured on the enriched medium showed four isozymes at positions 3,4, 5, and 6 in eight individuals and at position 3 only in three individuals. This was taken at first as a sign of heterogeneity in the line, but efforts to isolate a pattern 3 line by inbreeding further single females failed 'when the enriched medium was used. An explanation will be offered after the breeding results are presented. Males of the freshly extracted pattern 1-5 line were mated singly with pattern 3 females of the Barro Colorado Island strain. Twelve F, females assayed showed isozymes at positions 1, 2, 3, 4, 5, and sometimes 6 and 7. The F, males were backcrossed individually to females of the Barro Colorado Island pattern 3 line. Fifteen backcross progenies appeared to be segregating for the two pairs of alleles because they consisted of four different isozyme patterns in equal numbers as shown in Table 1 (see also diagram, Figure 2). A photograph of these segregating progeny appeared in a preliminary research note (PIPKIN1968). The four patterns occurred in a 1: 1: 1: 1 ratio, and frequencies of the four patterns in separate sibships were homogeneous. The four isozyme patterns were found in similar backcross progenies (cultured on ordinary corn meal medium), in which the pattern 3 PI parent had been extracted from the Trinidad strain. Thus, with either a similar or diverse genetic background, backcross sibships were segregating for four isozyme patterns. There is no possibility that any of the ODH isozymes seen here are caused by 84 SARAH EEDICHEK PIPKIN TABLE 1 Inheritance of ODH isozyme patterns: backcrosses and F, X F,

Patterns of Patterns of progeny Chi parental lines 1 3 1.3.5.5 3.4.5 1-5 5 1-3 square P 1-5/3 X 3 .. 48 .. . . 45 57 43 2.378 0.5>P>0.3 3/5 x 3/5 . . 11 . . 24 . . 8 . . 0.999 0.7>P>0.5 1B/3 X 1B/3 9 9 ...... , . 15 0.272 0.9>P>0.8 1A/5 X 1A/5 Trinidad/Turrialba 15 . . 54 . . . . 17 . . 3.22 0.2>P>O.I Trinidad/Trinidad 16 .. 46 .. . . 22 . . 1.61 O.S>P>O.3 IA/5 x 5 .. .. 21 .. .. 14 . . 1.40 0.3>P>0.2 lA/5 x 3 .. .. 21 19 .. . . 0.10 0.8>P>O.9 the alcohol dehydrogenase locus. PIPKIN,in COURTRIGHT,IMEERSKI, and UR- SPRUNG 1966, reported that Drosophila metzii and other members of the tri- punctata species group as well as members of the related cardini and calloptera species groups show little ADH activity using ethanol, primary hexanol, heptanol, or octanol as substrates. A faint ADH band is not uncommonly seen in D. metzii gels four centimeters cathodal to the fastest ODH band. A graduate student, Mr. WILLIAMDOUGLAS, has confirmed the observation that in D. metzii there is

FIGURE2.-Segregating progeny of the backcross pattern 1-5/ pattern 3 F, male x pattern 3 female. Assuming a tetramer subunit structure of ODH, the genotypes of parents of the backcross we ODHF,/ODHS,; ODHF,/ODHS, males x ODHs,/ODHS,; ODHs2/ODHs2,where the fast alleles, ODHF, and ODHF,, either produce subunits earlier in development or produce more subunits than the slow alleles, ODHS, and ODHS,. Four classes of segregating progeny of the backcross include: pattern 1-5 individuals (genotype ODHF,/ODH",; ODHF,/ODHs2); pattern 3,4,5 individuals (genotype ODHF1/ODHsl; ODHS2/ODHs,) ; pattern 3 individuals (genotype ODHs,/ODHs,; ODHs,/ODHs2); pattern 1-3 individuals (genotype ODHs,/ODHs,; ODHFz/ ODHS,). From left to right, the first three slits contain single female backcross progeny of pattern 3,4,5; the fourth, fifth, seventh, eighth, and ninth slits, single females of pattern 1-3; the sixth slit, a single female of pattern 3, and the tenth and eleventh slits, single females of pattern 1-5. OCTANOL DEHYDROGENASE IN D. mtzii 85 weak or undetectable ADH formazan staining since he has used the species as a control in testing substrate specificity of ODH and ADH isozymes in a number of neotropical species, using a variety of primary and secondary alcohols (including 2-butanol) as a substrate. Even if the ADH and ODH portions of the D. metzii gels are cut apart after electrophoresis and stained separately, either one or two faint ADH isozymes are seen or these are undetectable. The four classes of isozyme patterns seen in the backcross progeny of (pattern 1-5 x pattern 3) F, X pattern 3 individuals (Table 1) , suggest that these isozyme patterns depend on two distinct structural genes for ODH. If the strongly staining isozymes sometimes present at positions 6 and 7 could be ignored, the backcross results could be interpreted on the basis of a dimer structure of the enzyme, with slow and fast alleles at each ODH locus. On this hypothesis the genotypes of parents of the backcross would be ODH,"/ODHIS; ODH,F/ODH," X ODHls/ ODHi"; ODH,"/ODH,". The subunit composition of ODH isozymes expected on the basis of two gene loci and a dimer structure appears in Table 2. The dimer hypothesis further requires that several of the subunit combinations migrate to the same positions upon electrophoresis, as Table 2 indicates. An alternate hypothesis which has the merit of accounting for the presence of the isozymes which are sometimes seen at positions 6 and 7 supposes that the ODH molecule is a tetramer. This hypothesis will be discussed after the remaining breeding results and information on changes of ODH isozyme patterns during development are presented. Hybrid enzyme formation involving subunits coded by each ODH structural gene could be detected in the Fl progeny of the cross of Turrialba pattern 5 individuals with those of Turrialba pattern I*. The latter strain showed a single isozyme at position 1 and was derived from the old pattern 1-5 line. 94 F, progeny assayed had isozymes at positions 1, 3.5, 5, and sometimes at 6 and/or 7 (Figure 3).The intermediate isozyme clearly ran to a position nearer the cathode than the midpoint between positions 1 and 5. A graduate student, Miss CAROL MAJOR,working on a strain originating from this cross, has shmthat the pattern 1, 3.5, 5 can be seen in homogenates of adult ovaries, intestines, Mal- pighian tubules, and fatty tissue, assayed separately. Since crude homogenates of mixed pattern 5 and pattern 1 flies showed only isozymes at positions 1 and 5, it is believed that the isozyme at position 3.5 represents a hybrid enzyme com-

TABLE 2

Subunit composition of adult ODH isozymes: dimer hypothesis

Isozyme position Isozyme subunits 1 2 3 3.5 4 5 86 SARAH BEDICHEK PIPKIN

5 0000 3.5 0 0 0 0

I @a@@ Origin - - - -

FIGURE3.-Zymograms of F, female progeny of the cross pattern 5 x pattern 1,. According to the tetramer hypothesis, the isozymes at both positions 3.5 and 5 are hybrid enzymes and the F, hybrid females have the genotype ODHF,/ODHF,; ODHFJODHS,. Pattern 1, parents are considered hom3zygous for the strong ODHF, allele (assuming a tetramer subunit structure) since pattern l,/pattern 3 hybrids exhibit isozymes at positions 1,2,3,4,5, and sometimes 6. In pattern 1, and patterns 1, lines, isozymes having subunits produced by ODHF, do not result in formazan staining probably owing to reasons other than productivity of this allele.

- 50 3.5

1 0 Origin+ -

FIGURE4.-Segregating female progeny of the cross of pattern 5/pattern 1, F, male x F, female. From left to right, the first two slits contain pattern 5 progeny (genotype on the tetramer hypothesis, ODHF,/ODHF',; ODHS,/ODHS,) ; third slit, pattern 1, individual (genotype, ODHF,/ODHF,; ODHF,/ODHF,) ; fourth and" fifth slits, pattern 1, 3.5, 5 (genotype ODHF,/ ODHF,; ODHF,/ODHS,). On the tetramer hypothesis the fast alleles, ODHF, and ODHF,, either produce subunits earlier in development or produce more subunits than the slow alleles. OCTANOL DEHYDROGENASE IN D.metzii 87

FIGURE5.--Segregating female progeny of the backcross pattern 5/pattem 1, F, males X pattern 5 females. From left to right, the first four slits contain pattern 5 individuals (genotype on the tetramer hypothesis, ODHF,/ODHF,; ODHS,/ODHS,) ; the last three slits contained pattern 1, 3.5, 5 individuals (genotype ODHF,/ODHF,; ODHF,/ODHS,). Pattern 5 individuals occasionally show bands at positions 3 and 4 as well as at 5.

- 500 400 30s. 2 I

FIGURE6.--Segregating F, progeny of the cross (pattern S/pattern 3) F, males x F, females. From left to right, the first two slits contained pattern 3,4,5 individuals (genotype on the tetramer hypothesis, ODHF,/ODHS,; ODHS,/ODHS,) ; the third slit, a pattern 3 individual (genotype, ODHS,/ODHS,; ODHS,/ODHS,) ; and the fourth slit, a pattern 5 individual (genotype ODH”,/ODHF,; ODHSJODHS,). 88 SARAH BEDICHEK PIPKIN

- 5 ooo** ***ooo 4 o***ooo 3***00000 *** 2000 000 1000 8&***000 o**ooo Origin ,- ~-,~~,-~-,,-.-, ~,--,V3’3r -,- - +o bcd ef 9 hij k I

FIGURE7.-ODH isozyme patterns in different developmental stages of various extracted OCTANOL DEHYDROGENASE IN D.metzii 89 posed of subunits contributed by each structural locus. Inbreeding these F, hybrids yielded a 1: 2: 1 ratio of pattern 5, pattern 1, 3.5,5, and pattern 1 individuals in the F, generation, as shown in Figure 4 and Table 1. Similar results were obtained in the F, progeny where the P, parents came from the Trinidad pattern 5 and Trinidad pattern 1, strains, respectively (Table 1). Backcrosses of F, hybrids of composition pattern 5/ pattern 1, to pattern 5 individuals resulted in a 1: 1 ratio of progeny with pattern 1,3.5,5 and progeny with isozymes at 3,4,5 and sometimes 6 (Figure 5, Table 1). Hybrids obtained by crossing the Turrialba pattern 5 and the Barro Colorado Island pattern 3 lines uniformly possessed three isozymes at positions 3,4, and 5 with the strongest staining at position 4. Upon inbreeding F, hybrids, a 1:2:1 ratio of pattern 5, pattern 3, 4, 5, and pattern 3 types was observed (Figure 6, Table 1). Similarly, pattern 3 flies crossed with pattern 1, gave all F, hybrids with 3 isozymes at positions 1, 2 and 3. F, progeny showed a 1:2:1 ratio of pattern 3; pattern 1, 2, 3; and pattern 1 individuals. The pattern 1, line, like the pattern 1, line, has a single isozyme at position 1, and both lines were derived from the same old pattern 1-5 line. The two pattern 1 lines differ in their F, hybrids with the Barro Colorado Island pattern 3 line. Pattern l,/pattern 3 hybrids have isozymes in positions 1,2,3,4, 5, and sometimes 6, while (pattern lB/pattern 3) hybrids have isozymes detectable only at positions 1,2 and 3. Studies of ODH isozymes in mass homogenates of fertilized eggs up to 30 hours old (Figure 7a) of a pattern 3 line revealed isozymes at positions 1 and 2 in addition to the isozyme at position 3 characteristic of the third instar lama and adult of this line. The embryonic isozymes at positions 1 and 2 of the pattern 3 line were faint in the first instar larva and absent in the third instar larva (Figure 7b) and in 4 to 6 day adult females (Figure 7c). In third instar larvae a faint band was sometimes seen at position 4 in addition to the expected band at position 3. The band at position 4 has been seen only twice in several hundred pattern 3 adults. In embryos of the pattern I, line (Figure 7d), isozymes were present at positions 2 and 3, in addition to position 1, where the single isozyme characteristic of third instar larvae (Figure 7e) and adults (Figure 7f) of this line is found. In a pattern 5 line, embryonic ODH isozymes at positions 1,2, and 3 (Figure 7g) were observed in addition to the isozyme at position 5 typical of the adult pattern. The bands at positions 1 and 2 disappeared in third instar larvae (Figure 7h) and adult females (Figure 7i) which always have a strong band at position 5 and sometimes also bands at positions 3, 4, 6, and 7. In a line segregating for lines: (a) embryonic isozymes of the pattern 3 line, including the leftmost three slits; (b) pattern 3 third instar larva, fourth and fifth slits; (c) pattern 3 adult female, sixth slit; (d) embryonic isozymes of the pattern 1, line, seventh and eighth slits; (e) third instar larvae of pattern 1, line, ninth and tenth slits; (f) adult of pattern 1, line, eleventh slit; (g) embryonic isozymes of a pattern 5 line, twelfth, thirteenth, and fourteenth slits; (h) third instar larva of a pattern 5 line; fifteenth slit; (i) adult female of a pattern 5 line, sixteenth slit; (j) adult female of a pattern 3 line, seventeenth slit; (k) embryonic isozymes of a line segregating for the pattern 1, 3.5, 5; pattern l,, and pattern 5; slits eighteen through twenty; (1) adult females of the previous line, slits 21 through W. 90 SARAH BEDICHEK PIPKIN patterns 1, 3.5, 5; 1; and 5, in adult females (Figure 71; Figure 4), the same pattern 1,3.5,5 pattern was seen in egg homogenates (Figure 7k). This is strong genetic evidence that egg or embryo and adult structural genes determining ODH isozymes are the same. The isozyme at position 3 in Figure 7j is from a single pattern 3 adult female, run as a control on the same gel as the mass egg homogenates with pattern 1,3.5,5.

DISCUSSION Facultative hybrid enzyme formation involving subunits coded by distinct structural genes has been described for mammalian lactate dehydrogenase where two loci, A and B, contribute peptides that are combined at random in groups of four to form the LDH tetramer (MARKERT1963). Later ZINKHAM,BLANCO, and KUPCHYKshowed that three non-allelic genes, A, B, and C, could produce subunits found in LDH isozymes of mammalian testis (1963) and pigeon testis (1964). Subunits coded by the C gene were not aggregated completely at random with those produced by the A and B genes. The egg or embryonic and adult ODH patterns of D.metzii are believed to depend on the same structural genes since the peculiar 1, 3.5, 5 pattern is seen in both fertilized eggs and adult tissues of hybrids of certain lines. If this is true, the disappearance of isozymes, present in eggs, at positions 1 and 2 from adult tissues of pattern 3 and pattern 5 lines requires an explanation. These findings can be interpreted in the light of in vitro work of URSPRUNGand CARLIN(in press, 1967) , SOFERand URSPRUNG(1967), JACOBSON(1968) on alcohol dehydrogenase in D.melanogmter. URSPRUNGand CARLIN(1967) concluded that vary- ing amounts of the cofactor, nicotinamide adenine dinucleotide, NAD, accounted for the presence of three isozymes in either homozygous slow or homozygous fast alcohol dehydrogenase variants. SOFERand URSPRUNG,working with the isolated ADH, isozyme, concluded that conformational changes were induced in ADH,, giving rise to ADH, and ADH,, by treatment with either the oxidized or reduced form of the co-factor. JACOBSON(1968) likewise concluded that the cofactor induced a conformational change in ADH,, since he found a greater resistance to heat in isozyme ADH,, induced by incubation of ADH, with NAD than in the untreated ADH,. SOFERand URSPRUNG(1967) found that boiled fly homogenates, presumably containing the cofactor, could also induce the formation of ADH, and ADH, from ADH,. In D.metzii the slowest migrating ODH isozymes present in embryos (fertilized eggs less than 30 hours old) were not detectable in single whole females of the pattern 3 and 5 lines, respectively. The isozymes at 1 and 2 may be first formed in the embryos. Their absence in adults may be interpreted as due to complexing with the cofactor, NAD. Table 3 shows the possible subunit composition of ODH isozymes assuming that they are tetramers dependent on two structural genes, one of which, ODH,, produces S2 subunits which are unbound, and S2’ subunits which are bound to the cofactor. The two slowest migrating isozymes expected to be located anodal to position 1 have not yet been detected in D.metzii but are seen in a sibling species ( PIPKIN,manuscript in preparation). The Barro Golorado Island unselec- OCTANOL DEHYDROGENASE IN D. metzii 91 TABLE 3

Subunit composition of ODH isozymes: tetramer hypothesis

Isozyme uosition Subunits of ODH isozymes

7 Sl Sl Sl Sl 6 SZ'S, s, Sl 5 S,' S,' s, s, 3.5 or 4 S,' S,' S,' s, 3 S2' S,'S,' S,' 2 s, S,' S,' IS, 1 S,' S,' s, s, 0 S,'S, s, s, 0 s, s, s, s, ted pattern 3 strain is considered to have low activity of ODH,, apparently locus characteristic in view of the Mendelian ratios observed in backcrosses and F, x F, crosses using this strain. The pattern 1-5 line originally had high activity of both ODH, and ODH,, but the change of this line into the pattern 1 lines is thought to have been selection of one or more regulatory genes favoring low activity of the ODH, gene, while ODH, activity remained high. The pattern 5 lines are considered to have high activity of ODH, and moderate activity of ODH,. In D. metzii the dimer hypothesis would account for the unifactorial inheritance which distinguishes the pattern 3 and 5 lines, respectively, except for the presence of isozymes sometimes at positions 6 and 7. The latter would require assumption of a third ODH structural locus. The finding that an enriched culture medium causes appearance of four isozymes in a homozygous pattern 3 Trinidad line adds support to the tetramer hypothesis of ODH structure. The fifth isozyme assumed on the basis of random combination of two kinds of subunits into a tetramer could be thought to be undetectable with formazan staining. Actually five isozymes are rarely seen at positions 3, 4, 5, 6, and 7 in flies of the pattern 5 line.

SUMMARY Two unlinked structural genes, ODH, and ODH,, are responsible for complex octanol dehydrogenase isozyme patterns in Drosophila metzii. Hypotheses that the ODH molecule may be a tetramer or a dimer are discussed. ODH isozymes may be composed of subunits coded by the same structural genes or by those of the two distinct ODH genes. Variants of each ODH structural gene display unifactorial inheritance, respectively. These are interpreted on the dimer hypothesis as position electrophoretic variants; on the tetramer hypothesis, as low and high activity variants. ODH isozymes in embryos and adult females are believed from genetic analysis to be determined by the same structural genes. The presence in egg and embryo homogenates of certain extracted homozygous lines of slowly migrating isozymes which disappear in third instar larvae and adults is interpreted as being in agreement with the hypothesis that the subunits of ODH, become bound with NAD. ODH, is thought to begin producing subunits earlier in development than ODH,. 92 SARAH BEDICHEK PIPKIN

LITERATURE CITED

COURTRIGHT,J. B., R. B. IMBERSKI,and H. URSPRUNG,1966 The genetic control of-alcohol dehydrogenase and octanol dehydrogenase isozymes in Drosophila. Genetics 54 : 1251-1260. JACOBSON,K. B., 1968 Alcohol dehydrogenase of Drosophila: interconversion of isozymes. Science 159: 324-325. MARKERT,C. L., 1963 Lactate dehydrogenase isozymes: dissociation and recombination of subunits. Science 14: 1329-1330. PIPKIN,S. B., 1968 Introgression between closely related species of Drosophila in Panama. Evolution 22: 140-156. - 1968 Research Note. Drosophila Information Service 43: 172-1 74. SOFER,W. H., and H. URSPRUNG,1967 Alcohol dehydrogenase: in vitro formation of isozymes. Isozyme Meeting, Sanibel Island, Florida, The Isozyme Bulletin. (in press). URSPRUNG,H., and L. CARLIN,1967 Drosophila alcohol dehydrogenase: In vitro changes of isozyme patterns. Ann. N.Y. Acad. Sci. (in press). URSPRUNG,H., and J. LEONE,1965 Alcohol dehydrogenases: A polymorphism in Drosophila melanogaster. J. Exptl. Zool. 160: 147-154. ZINKHAM,W., A. BUNCO, and L. KUPCHYK,1963 Lactate dehydrogenase in testis: dissociation and recombination of subunits. Science 142: 1303-13M. - 1964 Lactate dehydrogenase in pigeon testis: genetic control by three loci. Science 144: 1353-1354.