SEX-LINKED AUXOTROPHIC AND PUTATIVE AUXOTROPHIC MUTANTS OF DROSOPHILA MELANOGASTER

D. R. FALK* AND DAVID NASH

Department of Genetics, University of Alberta, Edmonton, Alberla, Cunadu

Manuscript received October 15, 1973

ABSTRACT

Thirty-two mutants with improved growth response on a yeast-sucrose compared with a defined medium have been characterized with respect to ribonucleoside supplementability. Twenty mutants respond to either py- rimidine ribonucleoside. Four mutants respond to one or both purine ribo- nucleosides. Eight mutants (“putative” auxotrophs) do not respond to dietary RNA supplementation. Mapping and complementation studies suggest that eleven loci are represented: one, rudimentary, probably accounts for all py- rimidine requirers; there are three purine loci and seven at which the putative auxotrophs are found.

HE range of biochemically defined mutants in Drosophila melanogaster is Tsomewhat restricted and has been largely dictated by chance. Biochemically defined loci have most commonly been identified as a result of discovery of enzyme polymorphism (see O’BRIEN and MACINTYRE 1971). Other loci have been described because their mutants yield visible phenotypes (for example, BEADLE and EPHRUSSI 1937; FORREST, GLASSMAN and MITCHELL 1956). More recently, specific techniques for identification of mutants in specific known or postulated loci have been devised, either using metabolic inhibitors ( SHERALD and WRIGHT 1972; SOFER and HATKOFF 1972) or segmental aneuploids in con- junction with enzyme assays (LINDSLEY et al. 1972; O’BRIEN and GETHMANN 1973). Molecular hybridization techniques have been used for localization of the genes controlling ribosomal-RNA (PARDUE et aZ. 1970; WIMBER and STEF- FENSEN 1970), transfer-RNA (STEFFENSEN and WIMBER 1971) and histone messenger-RNA ( PARDUE et a1 1972) synthesis.

The classic technique for isolation of biochemically defined mutants, selection of auxotrophs (BEADLE and TATUM 1941 ) , has not, however, been employed on a large scale. Although the absence of a wide range of de novo synthetic path- ways partly accounts for this omission, the technical difficulties involved in handling flies in defined, axenic culture have probably been the critical deterrent. The work to date has been reviewed recently (FALK and NASH 1972). One mutant (1308) was isolated in a screen for nutritionally supplementable lethals (VYSE and NASH 1969) and shown to possess a double requirement for adenosine and a pyrimidine nucleoside (VYSE and SANG 1971). An RNA-requiring mutant (FNC22) was described by FALK and NASH (1 972) and later proved to require

* Present address: Department of Zoology, University of Bribsh Columbia, Vancouver, B C , Canada.

Genetics 76: 755-706 April 1974.

756 D. R. FALK A N D D. N A S H

pyrimidine nucleosides. FNC22, under the pseudonym ( r - ) will be discussed further in this paper.

NORBY (1970) has described pyrimidine requirements in preexisting rudi- mentary wing mutants and ascribed the auxotrophic phenotype to a demon- strable lsck of aspartate transcarbamylase activity in flies possessing some I alleles (NORBY 1973), He suggests that other r- alleles may lack carbamyl phos- phate synthetase, although no biochemical evidence is yet available. Thus, it is now established that bona fide auxotrophic mutants exist in Drosophila.

The present paper is concerned with the question as to the frequency of occurrence of loci capable of yielding auxotrophic mutants and with the types of such mutants which they yield. The study is confined to X-linked mutants which show close-to-normal survival and development rate on yeast-sucrose medium, but which are lethals, temperature-sensitive lethals, or are delayed in development on the defined medium of SANG ( 1956).

Thirty-two mutants are described. Mutants at four loci show distinctive responses to dietary nucleosides. Mutants at seven other loci remain undefined with respect to their nutritional requirements.

MATERIALS A N D METHODS

Stock and breeding scheme: The mutagenesis scheme used was described by FALK and NASH (1972). Compound-X females are mated to EMS-treated (LEWIS and BACHER 1968) males and single F, male progeny used to establish a stock by further crosses to compound-X females. The males used in the original cross are from an inbred line (stock 1, Amherst College, Drosophila Information Service, 1968) and in the females the compound-X chromosome, C(I )RM, y2 wa S U - W ~ bb, is accompanied by an autosomal complement derived from six successive backcross generations to the inbred line.

The compound-X stocks produced were screened for auxotrophic mutants, carried on male x chromosomes, using the segregating non-mutant females as internal controls.

It has been assumed that use of an inbred line (brother-sister matings for 500 generations) and its derivatives in these experiments limits incidental genetic variation and thereby reduces possible problems associated with genetic modifiers. We have no proof of the correctness of this assumption; however, the mutants described in the paper have fairly stable phenotypes.

A stock with an X chromosome containing the mutants pn, U, m and wy (all EMS-induced in the inbred line X chromosome) and the mutant f was constructed with inbred line autosomes and used for mapping the mutants.

Culture conditions: Flies were reared and crossed axenically, starting from calcium-hypo- chlorite-sterilized embryos. Stock cultures and many crosses were reared on dead yeast-sucrose medium (Table 1). Experimental cultures contained slightly modified SANG (1956) defmed medium with appropriate additives (Table 1).

Experiments on the scale described below would be impracticable without maintenance of sterility over successive generations. We have found that the use of U.V.-irradiated transfer rooms, enclosed sterile hoods for sorting flies and the use of antibiotics is sufficient for this purpose. 3" X 1" shell vials capped with plastic microbiological covers (Kaputs, Bellco Glass Inc.) are most satisfactory as containers for cultures, providing a limited number of larvae is present. In overcrowded cultures larvae, but not flies, escape. When larger numbers of flies are required, they are reared in milk bottles capped with gauze-wrapped cotton, covered with aluminum foil. Microbial infection in defined cultures is usually self-evident, as can be determined by plating samples of medium on nutrient media; dead yeast-sucrose cultures require plating when axenic growth must be assured.

DROSOPHILA AUXOTROPHS

TABLE 1

Media used in screening and testing mutants

75 7

a. Defined medium

Agar (Oxoid No. 3) 3.00 g. Casein (vitamin free) 5.50 g. Sucrose 750 mg. Cholesterol 30 mg. Lecithin 400 mg. Thiamine .2mg. Riboflavin .I mg. Nicotinic acid 1.2mg. Ca pantothenate 1.6 mg. Pyridoxine .25 mg.

Biotin Folic acid NaHCO, (anhydrous) KH,PO, (anhydrous) K,HPO, (anhydrous) MgSO, (anhydrous) Streptomycin Penicillin* Water RNA (when added) Ribonucleosides (when added)

0.16 mg. .3 mg.

140 mg. 183 mg. 189 mg. 62 mg. 17 mg.

25,000 I.U. to 100 ml.

400 mg. 100 mg.

b. Dead yeast-sucrose medium

Brewers yeast 12.5 g. Penicillin* 25,WO I.U.

Granulated agar 2.0 g . Water 90 ml. Streptomycin 25 mg.

Sucrose 10.0 g. Propionic acid* 1.0ml.

* Added after autoclaving.

The mutant screen. 5,655 stocks containing males with potentially mutant X chromosomes were tested for the presence of mutations affecting their ability to grow on SANG'S defined me- dium. Intially, approximately twenty flies of each sex were transferred from dead yeast-sucrose cultures to vials containing the defined medium and removed within 48 hours. Eclosion from the new cultures was monitored. Cultures which yielded less than fifteen females during the first four days of eclosion were rejected if a single male was produced. One male was tolerated if fifteen or more females were produced. Five mutants described in this paper were retained after earlier, less stringent, selection (FALK and NASH 1972), of which one (adel-lad) would have been discarded had it arisen in later experiments.

One thousand six hundred and ninety-four strains were tested at 25". The coincidental dis- covery of a temperature-sensitive auxotroph, and the theoretical consideration of the possible problems arising from the fact that embryonic and pupal phases of development are closed nu- tritional systems, led to the modification of this regime to select, additionally, for temperature- sensitive auxotrophs. The remaining 3961 strains were tested with larvae at 29" and embryos and pupae at either 20" or 25".

Prospective mutant strains were retested several times under similar conditions. Approxi- mately 75% of strains were rejected during retesting or because males grown in permissive con- ditions (dead-yeast sucrose medium with an equivalent temperature regime) proved substantially subnormal.

The mutant strains finally retained contained males which were either lethal (less than 5% normal viability) on defined medium either at 25" or at 29", but were of essentially normal viability on dead yeast-sucrose medium at 25" and, in the case of temperature-sensitive auxo- trophs, at 29". Three mutants which are slow in developing on defined medium but which de- velop at normal rate on yeast sucrose medium were alm obtained and have h e n characterized.

Characterization of the mutants: a) Nutrition: Mutants were tested for growth on defined medium with RNA; those with sub-

stantial responses to RNA were tested on each of the four ribonucleosides. Concentrations used are shown i n Table 1.

758 D. R. F A L K A N D D. N A S H

b) Temperature sensitiuity: Strains were tested for temperature sensitivity on restrictive and permissive media. Temperatures used were 18" or 20", 25" and 29".

Nutritional and temperature sensitivity tests followed the same protocol as was described for the initial screening.

c) Genetic mapping: Females heterozygous for the X chromosome from mutant strain and the multimarked X chromosome described above ( p n U m w y f ) were mated to pn U m w y f males. Their progeny were reared under restrictive conditions. Deviations in the frequency of rarious recombinant classes of males from these frequencies in a control cross were used to identify the map interval within which a mutant was located. The relative frequency of recipro- cal recombinants within that interval, combined with published data on the map position of the markers involved (LINDSLEY and GRELL 1968), was used to yield a map position for each mu- tant.

It should be noted that the map positions shown in Table 9 require refinement, since the method employed is not, in itself, particularly accurate and the recombinant frequencies found in controls show deviations from published data. The latter effect is not the result of a cyto- logically identifiable chromosomal aberration. The mapping studies confirm the sex linkage of the mutants, indicate a rough map position and also rule out the possibility of major X-chromo- soma1 rearrangements in the mutant stocks.

d) Complementation studies: Crosses designed to yield double heterozygotes for certain com- binations of mutants with similar locations and phenotypes were grown in restrictive conditions. Criteria used for determining lack of complementation are described in the caption to Table 8.

Mutant designations: We have adopted a modified system of microbial designations for the mutants. Each mutant is named for its nutritional requirement (yea = yeast; gua = guanosine; pur = purine nucleosides; ade = adenosine), except for pyrimidine nucleoside-requiring mu- tants, which are probably all rudimentary alleles, and have been designated rpvr or ( r )mr , depending upon whether they show abnormal wings or not. Numerical suffixes (e.g., 1-1) indi- cate a locus and an allele number, respectively, in order of discovery. "ts" and "sd" are affixed to an allele number when its phenotype in restrictive conditions involves temperature sensitivity or slow development.

RESULTS A N D DISCUSSION

Thirty-two mutants have been isolated. Their characteristics are broken down in Table 2. Data substantiating the attributions shown in Table 2 are found in Tables 3-8.

Table 3 describes four typical examples from the twenty pyrimidine auxo- trophs; complete characterization of these mutants will be included in a later

TABLE 2

The spectrum of mutants produced in the screen for auxotrophs

Requirement

Nucleosides

Purine nucleosides Either pyrimidine

Phenotype* nucleoside Adenosine Guanosine Either Yeast

Lethal (25")t 16 0 0 2% 3 Lethal (29")t 4 0 1 0 3 Slow developments 0 1 0 0 2

* On restrictive medium. t Less than 5% survival. 2 One mutant shows approximately 7% survival.

Greater than three days delay.

DROSOPHILA AUXOTROPHS 759

TABLE 3

The effects of nucleoside supplementation on four typical pyrimidine-requiring mutants

Supplement*

Adenmine Guanmine Uridine Cytidine -- None

Mutant+ Temperature d $? d ? d ? a ? a ? ,.pyrl-l9 25 ' 0 621 0 27 0 22 26 53 47 123 pyrl-17 25" 0 332 0 40 0 118 72 30 104 82 (r) pyrl-1 25 6 240 17 212 21 270 305 278 154 155

29 1 141 0 57 1 201 121 102 87 90 _ _ _ - - - rJ ~ ~ r l - 7 Is 25" 151 651 - -

* Added to defined medium at 0.1 % concentration. + Males carry the mutant X chromosome; females are non-mutant X X / Y siblings.

TABLE 4

Nutritional responses of purine supplementable mutants purl-1, purl-2, gual-lts and adel-lsd

(a) Viability

Supplement*

None Adenosine Guanosine Undine Cytidine - __-

Mutanti Temperature d 0 d ? d ? a ? d ? ~

purl-I 25 27 390 94 163 188 263 279 20 241 purl-2 25" 9 283 55 25 142 163 1 181 2 240

29 6 152 1 49 69 71 0 34 5 64 _ _ - - _ - gual-1 t s 25" 112 217 - -

(b) Developmental rates (days delayed compared with non-mutant sisters) at 25"

Supplement*

Mutant+ hone Adenosme Guanosine Undine Cytidine

purl-l purl-2 adel-lad

- 2.763 - -0.32 -

3.12$ 0.22

- - 0.15

2.453 2.28$ 2.243 - - -0.37

* Added to defined medium at 0.1 % concentration. + Males carry the mutant X chromosome; females are non-mutant X X / Y siblings. Significantly different at .02 level; remaining differences non-significant.

TABLE 5 Surviual of three putatiue auxotrophs on defined medium (with or without RNA) and

on yeast-sucrose medium at 18", 25" and 29"

Culture conditions

Yeast-sucrose medium _ _ _ ~ Defined medium at 25'

- RNA + RNA* 250 18' 29 - Mutanti o " ? a ? d ? d ? d ? yeal-l 0 287 11 128 128 116 3 68 many many yea3-l 1 427 0 80 68 67 many many 2 34 yea5-2 0 40 0 74 20 18 many many 0 94

* Added a t 0.4% concentration. -f Males carry the mutant X chromosome; females are non-mutant X X / Y siblings

760 D. R. FALK AND D. NASH

TABLE 6

Survival of three putative temperature-sensitive auxotroph at 20" and 29" on defined medium and at 29" on RNA-supplemented medium and yeast-sucrose medium

Culture conditions

Defined medium (- RNA) 29' - Defined medium (4- RNA)* Yeast-sucrose ~ _ _ _____ 20" 290

-.

Mutant: d ? d ? a ? a ? yea2-I ts 88 117 8 377 1 37 107 158 yea4-2ts 24 125 25 1047 19 233 65 53 yea6-I t s 131 184 5 147 7 68 148 205

* Added at 0.4% concentration. + Males carry the mutant X chromosome; females are non-mutant X X / Y siblings

TABLE 7

Development rates (days delayed* compared with non-mutant f e d e siblings) of two slow-developing putative auxotroph

Defined medium (- RNA) Defined medium (+ RNA)+ Yeast-sucrose medium Defined medium (- RNA)

29 4.65 4.37 29" 3.37 4.75 29 0.78$ 0.61 $ 25" 3.02 2.44 (20")s

* In all cases, except on yeast-sucrose medium, the delays shown are minimal estimates since males emerging mare than six days after the f i females were counted as emerging on day seven. All females, and males grown on yeast-sucrose medium, emerge within this six-day period.

Jr Added at 0.4% concentratiosn. $ Not significant; remaining differences significant at .Ct2 level.

Large scale experiments not performed on yea7-ISd at 25".

TABLE 8

Complementation tests on three purine-requiring mutants (purl-1, purl-2 and gual-Its) and t h e e putative auxotroph (yeaklsd, yea42ts and yea3-I)

Genotype Double heterozygotes Single heterozygotes

purl-l/purl-2 purl-l/gual-lt~ purl-Z/gual-I ts

yea4-1sd/yea4-2ts * yea4-Isd/yea3-1+ yea4-2ts/ yea3-1

0 55 19 0

34 37

223 74 19 55 44 53

Larval development was at 29" on unsupplemented, defiied medium. Data show the summation of progeny from two reciprocal crosses; for mutants "a" and "b", the crosses were a p M 7 0 x b/Y 8 and b F M 7 0 x a/Y 8 . "Single heterozygotes" are both FM7/a 0 and FM/b 0 , not neces- sarily in equal proporticms.

* Data taken from the first three days of eclosion (yea4-lsd is a slow-developing mutant).

DROSOPHILA AUXOTROPHS 76 1

paper, in which we will also discuss complementation studies on these and pre- viously described rudimentary alleles. The four mutants chosen illustrate four classes into which the twenty mutants can, somewhat arbitrarily, be sub-divided: rudimentary mutants with a less-than-normal survival on 0.1 % pyrimidine nucleosides (rpg71-19) ; rudimentary mutants with normal survival on 0.1 % pyrimidine nucleosides ( r p ~ ~ ~ - ~ ~ ) ; slightly “leaky”, normal-winged mutants ( ( r ) wrl-‘) ; and temperature-sensitive, normal-winged mutants ( (r)pgr1-7ts). Table 4 describes four mutants which respond to either or both purine nucleo- sides: adel-Isd7 gual-lts7 purl-l and purl-2.

Table 5 describes the growth of the three putative auxotrophic lethal mutants on defined media and indicates that these mutants are temperature-sensitive on permissive medium. yeal-l is a cold-sensitive lethal; yea3-l and yea5-l are both heat-sensitive lethals. Table 6 describes the temperature-sensitive putative auxotrophs yea2-lt5, ~ e a 4 - 2 ~ ~ and yea6--ltS. Table 7 shows the developmental delays associated with two slow-developing putative auxotrophs, yea4-lsd and ~ e a 7 - l ” ~ . “Putative” auxotrophs have not been characterized nutritionally beyond the demonstration of their requirement for yeast-sucrose medium.

Table 8 shows complementation tests between mutants with adjacent map positions and similar phenotypes.

TABLE 9

Known auxotrophic and putative auxotrophic loci on the X chromosome of Drosophila melanogaster

Phenotype Approximate -

Locus Alleles* location Nutritional requirement Other

yea? 1 0.8 Yeast Heat-sensitive yea4 1sd,2ts 3-5 Yeast . . . . . . . . 1308 t s 1 4 Adenosine plus pyrimidine . . . . . . . . yea2 1 ts 16’ Yeast . . . . . . . . purl 1 3 2 30-33 Either purine nucleoside purl-1 slow-developing

gual 1 t S 31 Guanosine . . . . . . . . yea1 1 3 7 Yeast Cdd-sensitive, 0 sterile yea6 1 ts 45 Yeast Heat-sensitive 11523 t 46 Yeast (Female sterile) $ yea7 1 sd 53 Yeast . . . . . . . . rpyrl,(ripyrl many 52-56(54.5) Either pyrimidine nucleoside VaricrustJ adel 1d 5 7 Adenosine Guanosine-sensitive yea5 1 66 Yeast Heat-sensitive

* The designations sd (slow development) and ts (temperature-sensitive) refer to the auxo-

f See VYSE and NASH (1969) . $ Never tested for independent female sterile mutation; lost. s S e e V ~ s ~ a n d SANG (1971) .

See CARISON (1971) and FALK and NASH (in preparation). All map positions were obtained from crosses to pn (0.8), U (33.0) , m (36.1) , wy (41.9) ,

f (56.7) . Those to the right of f were rechecked with a y+ duplication on the right end of a y sc U f chromosome.

with adenosine

trophic phenotype.

762 D. R. FALK A N D D. NASH

Table 9 summarizes the characteristics of all mutants described in detail in this paper and includes tentative map positions ascribed to them. Full mapping data is available in FALK (1973).

The pyrimidine cluster: Twenty mutants with varying degrees of dependence upon dietary pyrimidine were isolated. The mutants all map close to the known pyrimidine locus, rudimentary (NORBY 1970). Eleven are, indeed, mutants of rudimentary wing phenotype with a range of expressivity and penetrance similar to that described by GREEN (1963). We designate such mutants r since all give rudimentary wing phenotypes in double heterozygotes with the mutant rA5, which does not complement with any previously tested rudimentary allele (CARLSON 1971). These new alleles of rudimentary are designated r*yr*-8 through

The remaining nine mutants have normal wings. We believe that these mutants are rudimentary isoalleles. Two of them, ( r ) Wn-4 and ( r ) wT1-16, produce flies with rudimentary wings in the double heterozygote with rh5. (r)Pyr1--16 shares with all the rwr mutants complete dependence upon dietary pyrimidine. (r)pyr1-4 is slightly leaky (< 0.1 % survival without suppkmentation) but less so than all other (r)Wr mutants. Since the two (r)pgr mutants are demonstrably rudimentary isoalleles, it seems likely that the remaining seven mutants are also.

We suggest that pyrimidine auxotrophy at the rudimentary locus is sometimes exhibited by mutants which may have considerable capacity for pyrimidine synthesis, because developing flies are dependent upon a level of supply of pyri- midines close to the wild-type capacity for endogenous synthesis. This suggestion is supported by the finding that rwr and ( r )wr mutants are partially dominant auxotrophs; some mutant heterozygotes show less than 10% wild-type viability. However, dominance is en obstruction to complementation tests and we must employ less direct evidence to argue that the non-rudimentary mutants are indeed isoalleles of rudimentary. A full exposition of this argument will be pub- lished in a later paper.

The large number of rWr mutants is no surprise. FAHMY and FAHMY (1959) and GREEN (1963) have reported that the locus is a mutational “hot-spot7’. Six or seven rwr mutants would certainly have been discovered as morphological mutants. The use of auxotrophic screening further increases the apparent muta- tion rate at the locus, even discounting the temperature-sensitive alleles. It is possible that high sensitivity of the fly to pyrimidine deficiency is the cause of this high rate of recovery of mutants. Even slight deficiencies in pyrimidine supply caused by mutants with small effects upon enzyme levels or activity would then yield recoverable auxotrophs.

Purine-supplementable mutants: Although the four mutants which respond to purine nucleosides are qualitatively different (Table 4), three map close together (30-33) and two of these (purl-I and purl-2) do not complement (Table 8). These two mutants respond differentially to adenosine supplementation; purl-2 develops more slowly than does purl-2. On the other hand, guanosine restores normal development rates to both mutants. The third mutant in this map region, gual-It8, responds only to guanosine. adel-lsa maps at 57 and responds to

,-w71--15 and W S l - 1 7 &rough rPpYrl-i9.

DROSOPHILA AUXOTROPHS 763

II * IMP pJh

adel

FIGURE 1 .--Simplified diagram of purine biosynthesis, showing the possible positions of blocks produced by pur l , adel and gual mutants.

adenosine, but actually is somewhat sensitive to guanosine, showing approxi- mately a 50% reduction in viability.

It is possible to interpret the behavior of these four mutants primarily as classical auxotrophs: The purl locus mediates a step in the biosynthesis of ino- sinic acid (IMP) (see Figure 1 ) ; gual and adel mediate steps in guanylic acid (GMP) and adenylic acid (AMP) synthesis from IMP. Preliminary supple- mentation studies with inosine suggest that this interpretation may be correct; the purl mutants respond to inosine; neither adel-lsd nor gual-l t5 does so.

The above explanation has a certain predictive value, particularly with respect to the behavior of double mutants, which have not yet been constructed. How- ever, we consider other explanations possible. Confirmation of these predictions will have to await enzymological study for which "stronger" alleles of adel-lSd and gual- l t5 would probably be desirable.

Undefined mutants: A total of eight mutants with, as yet, undefined require- ments was obtained. Three mutants are lethal on defined medium at 25"; three mutants are lethal on defined medium at 29"; two mutants are slow developing on defined medium at 25". All develop normally on yeast-sucrose medium at appropriate temperatures (25" , or 29" for the temperature-sensitive putative auxotrophs). These eight mutants occupy seven different loci. The one case of apparent allelism involves a temperature-sensitive auxotroph and a slow-devel- oping mutant at the yea4 locus.

It is curious that all five mutants which are not themselves temperature-sensi- tive auxotrophs are, in fact, temperature-sensitive in some other way (Tables 5 and 7). In contrast, none of the nucleoside requirers, apart from the temper- ature-sensitive auxotrophs, behaves in this manner. This finding suggests some special property o l yeast-supplementable mutants, although attempts to explain it are, we consider, premature.

We have not attempted to define the requirements of these mutants; indeed, we do not know whether any of them will prove to be auxotrophs, in the oper- ational sense that their abnormal growth characteristics on unsupplemented, defined medium will be revertable by the addition of one, or even several, specific metabolic supplements. Alternative explanations are possible. For example, it

764 D. R. F A L K A N D D. N A S H

has been found that nutrient “balance” is critical to normal development (SANG 1962; GEER 1963) and that use of truly minimal amino-acid medium (GEER 1965) brings about a requirement for nucleoside supplementation. We would not be surprised to find mutants which changed the optimal balance requirements sufficiently to produce lethal mutants on SANG’S defined medium. If this explana- tion, or some other, should prove correct, there is no reason to suppose that the mutants cannot be properly characterized and useful as genetic tools. On the other hand, a number of metabolic transitions other than those in nucleoside synthesis can be postulated as sources of auxotrophs-including carbohydrate, steroid and fatty acid metabdism-and these possibilities are worth exploring. Slow development as an auxotrophic phenotype: FALK and NASH (1972) have

shown that, in most semi-lethal putative auxotrophic strains, flies surviving on restrictive medium develop slowly. It is reasonable to suggest that this effect could result from an incomplete block in synthesis of an essential metabolite, arising from a “leaky” mutation or from the presence of alternative, but less efficient, synthetic pathways. Hence, strikingly slow-developing mutants were retained in this study. Several results presented above encourage us to believe this rationalization is correct. One slow-developing mutant ( yea4-ISd) has an allele which is a temperature-sensitive putative auxotrophic lethal (yea4-P) . A second slow developer (adel-1“) is corrected completely by adenosine and, furthermore, proved significantly sensitive to guanosine. suggesting that purine metabolism is fairly directly involved in its phenotype. One aspect of the phenotype of purl-2 is its slow development on adenosine. These observations indicate that slow development is probably a sensitive measure of metabolic disturbance.

Temperature-sensitive auxotrophy: Eight heat-sensitive auxotrophs or putative auxotrophs were described, mostly from screens in which the phenotype was specifically selected. Since the embryonic and pupal stages of development are nutritionally closed, it had been supposed that the relevant endogenous synthetic capacities might have been utilized in embryogenesis or metamorphosis, SO the temperature-sensitive screen selected, specifically, for larval temperature-sensi- tive mutants (see MATERIALS AND METHODS). However, all eight temperature- sensitive mutants survive the duration of development at high temperatures on permissive medium. Hence, there is no evidence suggesting the existence of genes which are required during the embryonic and pupal phases, but whose action can be substituted by nutrients during larval life.

CONCLUSIONS

The result described in this paper places beyond reasonable doubt that a fairly large number of loci which are capable of yielding auxotroph-like mutants exist in Drosophila melanogaster. Since only one of the eleven loci identified has more than two alleles and eight have only one, it is clear that we are unlikely to have found all the possible sites on the X chromosome. However, even if these eleven loci represent the maximum number to be found on the X chromosome, assuming that the X chromosome is one-fifth of the total genome and that it is not atypical of the genome as a whole, an estimate of fifty-five loci is obtained.

With the exception of rPYrl, the mutation rate at other loci is about .0002, which

DROSOPHILA AUXOTROPHS 765

seems rather low. In the course of screening 5655 chromosomes, with average sex-linked recessive lethal mutation rates exceeding 40% (NASH, in preparation), more than 3750 sex-linked recessive lethals would have been produced and lost in the F, of the mutation screen. This would, presumably, have produced an average of more than 3 alleles at each of a probable maximum of 1000 vital loci (extrapolated from JUDD, SHEN and KAUFMAN 1972) on the X chromosome. It is entirely possible that our screening technique is, in some way, inefficient. However, it is also possible that the loci have genuinely low mutation rates, or that not all mutations in the loci which we identified give auxotrophic pheno- types, other alleles being either non-conditionally lethal or less extreme.

The number of loci which we suggest may yield auxotrophic mutants is not inordinately high. The complexity of the biosynthetic pathways of vital sub- stances which could be substituted by a yeast-sucrose diet is probably not exten- sive. Nonetheless, de nouo nucleoside synthesis, for example, involves about twenty steps, excluding the phosphorylation reactions. If the rmr locus mediates two steps in pyrimidine synthesis (NORBY 1973) and if we have described three further loci in purine biosynthesis, then known X-chromosome mutants account for a quarter of nucleoside biosynthesis. Since the precise biochemical and nutri- tional situations with respect to other pathways are less well known, concrete calculations are not possible, but it does not seen unreasonable to suggest that upwards of thirty-five enzymatic steps are involved in synthesis of vital carbo- hydrates, fatty acids, steroids and vitamins, for examples, which are also avail- able in our permissive medium.

Finally, our results to date indicate that it is possible that we may be able to identify many of the genetic loci involved in at least two major biosynthetic pathways, those of nucleotide metabolism. Some of the loci in these pathways which are unlikely to yield auxotrophs can be identified with metabolic inhibitors and double mutant selective techniques. We consider extension of current studies so as to saturate the genetic map with mutants in nucleotide metabolism will provide invaluable tools for studies of gene action in higher eukaryotes.

Killam Foundation Scholarships. We wish to thank DR. 3. H. SANG for invaluable advice. This work was supported by NRC Grant A3269 to D. N. D. R. F. was recipient of NRC and

LITERATURE CITED

BEADLE, G. W. and B. EPHRUSSI, 1937 Development of eye colors in Drosophila: diffusable sub- stances and their interrelations. Genetics 22 : 76-86.

BEADLE, G. W. and E. L. TATUM, 1941 Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci., U.S. 27: 499-506.

CARLSON, P. W., 1971 A genetic analysis of the rudimentary locus of Drosophila melanogmter. Genet. Res. 17: 53-81.

FAHMY, 0. and M. FAHMY, 1959 Di€ferential gene response to mutagens in Drosophila melano- gaster. Nature 184: 1927-1929.

FALK, D. R., 1973 Sex-linked auxotrophic mutants in Drosophila melanogaster. Ph.D. thesis, University of Alberta.

FALK, D. R. and D. NASH, 1972 The search for auxotrophic mutants in Drosophila melano- gaster. pp. 19-31. In: Insect and Mite Nutrition. Edited by J. G. RODRIQUEZ. North Holland, Amsterdam.

766 D. R. FALK A N D D. N A S H

FORREST, H. S., E. GLASSMAN and H. K. MITCHELL, 1956 Conversion of 2-amino-4-hydroxy- pteridine to isoxanthopterin in D. melanogaster. Science 124: 725.

GEER, B. W., 1963 A ribonucleic acid-protein relationship in Drosophila nutrition. J. Exp. Zool. 154: 353-364. -, 1965 A new synthetic medium for Drosophila. Drosophila Inform. Serv. 40: 96.

GREEN, M. M., 1963 Interallelic complementation and recombination at the rudimentary wing locus in Drosophila melanogaster. Genetica 34 : 242-253.

JUDD, B. H., M. W. SHEN and T. C. KAUFMAN, 1972 The anatomy and function of a segment of the X-chromosome of Drosophila melanogaster. Genetics 71 : 139-156.

LEWIS, E. B. and F. BACHER, 1968 Method of feeding ethyl methanesulfonate (EMS) to Dro- sophila males. Drosophila Inform. Serv. 43: 193.

LINDSLEY, D. L. and E. H. GRELL, 1968 Genetic Variations of Drosophila melanogaster. Car- negie Inst. of Wash. Publ. 627.

LINDSLEY, D. L., L. SANDLER, B. S. BAKER, A. T. C. CARPENTER, R. E. DENELL, J. C. HALL, P. A. JACOBS, G. L. G. MIRLOS, B. K.DAVIS, R. C. GETHMANN, R. W. HARDY, A. HESSLER, S. M. MILLER, H. NOZAWA, D. M. PARRY and M. GOULD-SOMERO, 1972 Segmental aneuploidy and the genetic gross structure of Drosophila genome. Genetics 71: 157-184.

A specific requirement for pyrimidines in rudimentary mutants for Dro- sophila melanogaster. Hereditas 66: 205-214. - , 1973 The biochemical genetics of rudimentary mutants of Drosophila melanogaster, I. Aspartate carbamoyl-transferase levels in complementing and non-complementing strains. Hereditas 73 : 11-1 6.

O’BRIEN, S. J. and R. C. GETHMANN, 1973 Segmental aneuploidy as a probe for structural genes in Drosophila: mitochondrial membrane enzymes. Genetics 75: 155-167.

O’BRIEN, S. J. and R. J. MACINTYRE, 1971 A biochemical genetic map of Drosophila melano- gaster. Drosophila Inform. Serv. 56 : 89-93.

PARDUE, M. L., S. A. GERBI, R. A. ECKHARDT and J. G. GALL, 1970 Cytological localization of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera. Chromosoma 29: 268-290.

PARDUE, M. L., E. WEINBERG, L. H. KEDES and M. L. BIRNSTEIL, 1972 Localization of se- quences coding for histone messenger RNA in the chromosomes of Drosophila melanogaster. J. Cell. Biol. 55: 199a (Abst.)

The quantitative nutritional requirements of Drosophila melanogaster. J. Exptl. Bid. 33: 45-72. - , 1962 Relationships between protein supplies and B vita- min requirements in axenically cultured Drosophila. J. Nutr. 77: 355-368.

aMethyl Dopa is a screening agent for mutants of Drosophila melanogaster with elevated Dopa decarboxylase activity. Genetics 71 : ~ 5 8

Chemical selection of alcohol dehydrogenase negative

Localization of t-RNA genes in the salivary chro-

Nutritional conditional mutants of Drosophila melanogaster.

A purine and pyrimidine requiring mutant of Drosophila

Localization of 5SRNA genes on Drosophila

Corresponding editor: B. H. JUDD

NORBY, S., 1970

SANG, J. H., 1956

SHERALD, A. F. and T. R. F. WRIGHT, 1972

(Abst.)

SOFER, W. H. and M. A. HATKOFF, 1972 mutants in Drosophila. Genetics 72: 545-549.

STEFFENSEN, D. M. and D. E. WIMBER, 1971 mosomes of Drosophila by DNA:RNA hybridization. Genetics 69 : 163-178.

VYSE, E. R. and D. NASH, 1969 Genet. Res. 13: 281-287.

VYSE, E. R. and J. H. SANG, 1971 melanogaster. Genet. Res. 18: 117-121.

WIMBER, D. E. and D. M. STEFFENSEN, 1970 chromosomes by RNA-DNA hybridization. Science 170: 639-641.