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A study of the autonomous behaviour of sex-linked temperature-sensitive lethal mutants in drossophila… Hayashi, Shizu 1969

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A STUDY OF THE AUTONOMOUS BEHAVIOUR OF SEX-LINKED TEMPERATURE-SENSITIVE LETHAL MUTANTS IN DROSOPHILA»MELANOGASTER by SHIZU HAYASHI B.Sc, University of British Columbia, 1 9 6 4 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Genetics in Zoology We accept this thesis as conforming to the required ^ Qndard THE UNIVERSITY OF.BRITISH COLUMBIA July, 1 9 6 9 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head of. my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s thes, is f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f /^yuX^ l^d^tf The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date LJuMiWtt" i i ABSTRACT The autonomous behaviour of sex-linked recessive temperature-sensitive lethal mutants in Drosophila melanogaster could be demonstrated by the pres-ence of mosaic patches of tissue hemizygous for the mutant created by loss of a ring X chromosome in cells at the permissive temperature (21.5°C) and the absence of such patches at the restrictive temperature (29°C), The pres-ence of patches at both temperatures indicated that the mutant was non-auto-nomous. Such non-autonomous behaviour might be attributed to the existence of a substance capable of diffusing from the wild type tissue to supplement the mutant tissue. The experiments carried out showed that the presence or absence of mosaic patches could not be directly interpreted as demonstration of autonomous or non-autonomous properties of the mutant. Other factors such as the time of activity of the ts mutant and the type of tissue undergoing ring X. loss af-fected mosaic tissue production. Therefore, the mere presence of mosaic t i s -sue at 29°C could not be used as a criterion for the non-autonomous behaviour of the ts mutants. However, these mutants can be graded according to the de-gree of autonomy of ts lethality after alterations due to XO survival frequen-cies, lethal periods, and temperature-sensitive periods,have been placed on i i i mosaic frequencies at 29°C. Of the thirteen ts mutants studied, six can he classed as autonomous lethals. The others are equally autonomous as lethals but only in specific tissues, while others do not appear to be as autonomous. In fact, one of these may be considered non-autonomous. iv INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION SUMMARY BIBLIOGRAPHY TABLE OF CONTENTS Page 1 7 22 35 57 58 LIST OF TABLES Table Page Ia General genetic properties of stocks tested for temperature-sensitivity 9 Ib Description of stocks tested for temperature-sensitivity 8 II Number in each class of offspring of the cross J v.. ts. .Add 1 X wvC/dl-49 <M 21 III Ratio of the number of wv^-bearing females and exceptional males to the number of y. .ts. ./dl-4-9 females 23 IV Ratio of the Relative Viability Ratios of wvC-bearing females (21.5°C/29°C) 25 V TSP and LP of the different mutants tested 31 VI Data collected in the scoring of "shift" cultures of y. E25 30 VII Description of y..ts.. mosaics surviving at 29°C 41 y,i. LIST OF FIGURES i Figure Page 1 Crosses made for different aspects of the study 16 2 Crosses carried out for a determination of the autonomy of each temperature-sensitive mutant 15 3 Other crosses made in the investigation of autonomy 14 4 A model of reciprocal shift experiments to determine the TSP based on results obtained with y E25 19 v i i ACKN0WLEDGEI4ENT I would like to thank Dr. D, T. Suzuki for his valuable assistance and advice in preparing this paper. I amualso deeply appreciative of the encouragement and patience of Dr. Leonie Piternick during the course of this work. 1 INTRODUCTION The f u n c t i o n of a gene i s defined as autonomous i f the phenotype devel-oped In a given t i s s u e r e f l e c t s the genetic c o n s t i t u t i o n of that t i s s u e and i s not influenced by the genotypes of neighbouring c e l l s . Non-autonomous genetic behaviour, on the other hand, i s indicated when a c e l l u l a r phenotype does not correspond to the genetic c o n s t i t u t i o n of the c e l l s which produce i t owing to an influence of g e n e t i c a l l y d i f f e r e n t c e l l s (Hadom 1951). These aspects of gene function are generally studied i n higher organisms by surrounding c e l l s or t i s s u e of the genotype under i n v e s t i g a t i o n with c e l l s of a d i f f e r e n t geno-type. The d e f i n i t i o n of autonomy implies that phenotypes can be influenced by the a c t i o n of substances produced by genes of a d i f f e r e n t genotype. The e x i s t -ence of such a substance has been demonstrated by Sussman and Lu (1955) although the mechanism of i t s a c t i o n remains speculative. They showed that a d i f f u s i b l e substance described e a r l i e r by Bonner (19^?) as a c r a s i n , produced by the wild type myxamoeba of the slime mould Dictyostelium discoideum, could pass through an agar membrane and cause aggregationless mutants to c l u s t e r d i r e c t l y opposite wild type centres of aggregation. However, they found that d i f f e r e n t , pairwise combinations of morphogenetically d e f i c i e n t variants, each of which cannot com-2 plete the normal developmental sequence in spore formation alone hut which can do so when mixed in pair combinations, cannot complete development when placed on opposite sides of a membrane. They concluded, therefore, that the exchange of low weight diffusible intermediates was not the only requirement for complete non-autonomous development of those deficient stocks. Early studies on the autonomy of various non-lethal and lethal mutants in Drosophila melanogaster have given ambiguous results, Morgan and Bridges (1919) showed that, in general, the sex and phenotype of thirty different sex-linked characters in gynandromorphs produced by the occasional elimination of one of the X chromosomes from a female cleavage nucleus, were completely auton-omous in development. Later, Sturtevant (1932), studying mosaics produced by chromosome elimination caused by M(l) 2-n (see Lindsley and Grell 1968) found the following exceptions to complete autonomy: l) the Bar phenotype at certain stages of eye development, 2) scute and yellow phenotypes wheniin-;small: patches on the cuticle, and 3) the vermilion-eye phenotype. At that time i t was also noted that specific genes affected certain parts of the body at specific times in development. Demerec (1934) and Ephrussi (1934) studied the autonomy of cells hemizygous for chromosomal deletions of various sizes. Using aberrant somatic segregation and chromosome elimination to produce spots of tissue 3 hemizygous for the deletions surrounded by cells carrying the complete seg-ments, they arrived at different conclusions. Demerec claimed that the lethal expression of deletions was autonomous in development, whereas, Ephrussi recov-ered small spots of tissue hemizygous for the deletions and concluded that the lethal phenotype of the deletion was suppressible by surrounding wild type tissue. Stern (193*0 corroborated Ephrussi's conclusions when he showed that a deficiency lethal was non-autonomous. These interpretations may be ques-tioned since Ephrussi and Stern did not consider the possibility of the genes associated with the deleted segments not being functional in the tissues studied. Poulson (19^5) and Oster and Sobels (1956) studied the autonomous properties of different sex-linked lethals in females carrying the Xc^ chromo-some which is somatically unstable and frequently lost. They concluded that autonomous behaviour varied depending on the tissue affected and on the time of elimination of the wild type allele, Hannah (1953) . using a similar exper-imental procedure, showed thattthe mutant yellow was slightly non-autonomous, -The synthesis of cuticular pigmentation was found to be completely autonomous in the f i r s t four abdominal segments,and the thorax and the head, but non-autonomous in tergites genetically dimorphic for genes affecting pigmentation at the junctionnof wild type female and yellow male tissue. Bristles in the border zone showed complete gradation in colour from yellow (mutant phenotype) through brown to black (wild phenotype) while in other sections large yellow sectors were usually autonomous while small patches may not have been so. From extensive studies on the behaviour of cells carrying different lethals trans-planted" into wild type hosts, Hadorn (1951) bas concluded that the phenotypic behaviour of. the mutants is influenced by the followings l) phase specificity of the mutant, referring to the different times in the development of the or-ganism during which the effect of the mutant can be observed, since different tissues and organs react differently to the lethal constitution; 2) damage due to primary and secondary (because surroundings are abnormal) effects of the lethal; 3) penetrance or expressivity of lethal effects as determined by geno-typic milieu, sex, temperature, nutritional conditions, and other environmental influences, A l l these studies point to the complex nature of cell to cell interactions. Temperature-sensitive (ts) mutants are conditional mutations that survive at permissive temperatures but are lethal at restrictive temperatures (Epstein et a l . 1963). Analyses of temperature-sensitivity in microorganisms have shown thermolability to be a property of the protein product of the mutant gene rather than due to an effect on the actual process of transcription or trans-- 5 lation (Jockusch 1966} Naono and Gros 1967? Sundaram and Flncham I967). In Drosophila.melanogaster, ts mutants with properties of lethality similar to those of microorganisms have been recovered (Suzuki et al. 1967; Baillie, Suzuki and Tarasoff 1968j Suzuki 1969). Several of these mutants have been shown to have delineable temperature-sensitive periods("('TSP)iinddevelppment during which exposure of the organism to restrictive temperatures irrevocably commits the organism to death (Suzuki and Duck 1967; Tarasoff 1968; Suzuki and Procunier 1969) and an effective lethal phase (LP) when the exposure to restrictive temperatures during the TSP is phenotypically manifest in death. The coincidence of the LP and TSP of each mutant varied from concomitance to separation by several days (Suzuki and Procunier 1969). The present study was undertaken in order to detect a non-autonomous temperature-sensitive lethal and to determine whether the non-autonomy might result from the diffusion of a substance produced by the wild type tissue which would supplement the deficiency of the mutant tissue at restrictive temperatures. Recognition of such "supplementable" mutants could result in a bio-assay for the isolation of such a substance with a view to determining the nature of the genetic activity of a locus. Furthermore, once autonomy has been established, survival of cells carrying a ts lethal in certain spe-6 c i f i c regions of a wild type host at restrictive temperatures could separate tissues in which the locus is genetically functional from those in which i t is not required. In order to determine the autonomous behaviour of ts mutants, a scheme similar to that designed by Hannah (1951) was used. The mitotic instability of the ring chromosome, X° 2, in somatic cells (Hinton 1955) is used to generate patches of tissue in which recessive mutants carried on the homologous X chro-mosome are expressed due to the loss of the corresponding wild type alleles when the ring X is eliminated. With the sex-linked ts lethal mutants at per-: missive temperatures, mosaic patches of tissue should occur. However, under restrictive temperatures, mosaic patches should be produced only i f the ts mutant studied is non-autonomous or is not functional ln the tissues observed. It was found that the presence or absence of mosaic patches of tissue was not governed solely by the autonomous or non-autonomous behaviour respectively of the is gene, under restrictive conditions. Other factors such as the time of activity of the ts gene and the time of loss of the wild type allele influ-enced the recovery of mosaic tissue. Thus, the detection of mosaic patches, per se, was not found to be aereliable criterion of the non-autonomy of sex-linked ts lethal mutants. METHODS AND MATERIALS Establishment of y. .ts... stocks Sex^linked ts mutants which have been induced in adult Oregon R males (Suzuki et a l . 1967), localized genetically (Suzuki 1969)» and shown to give no survivors at 29°C, were used in this experiment. They wil be referred to as Hnon-leaky ts mutants" (leaky mutants being defined as those giving a few survivors at 29°C). Some of the chromosomes bearing the ts lethals were marked with different recessive mutations (see Lindsley and Grell 1968, for complete description): x (0»0» yellow body colour), sc (0.0, scute bristle mutant), cv (13.7, crossveins missing), v(33«P» vermilion eye colour), f (56.7, forked bristles), car (62.3, carnation eye colour) in the course of the ge-netic localizations (Suzuki 1969). In the cases in which the ts-bearing X chromosome did not carry other recessive markers, females heterozygous for the lethal and an inversion marked with the dominant mutant. Bar, (ts/FM-6 or ts/M-5) were mated to males with chromosomes marked with j£, sc, cv, y, f and car, females heterozygous for the ts lethal and the multipii-marked X chromosome were test.:crossed at 21.5°C and male progeny carrying the ts lethal, y, and in some instances the other recessive markers, were isolated. Henceforth, these marked ts-bearing chromosomes will be referred to as y..ts.. 8 Table Ib Description of stocks tested for temperature-sensitivity mutant markers inserted i , into ts chromosome stock maintenance E5 y sc cv homozygous E7 y sc homozygous E9 y sc cv car c? E2-5- y homozygous E2_7 y sc f car.... homozygous E34 y sc homozygous E45 y sc cv c? E46 y sc cv v cf E76 y sc < ? • • E82 y v d" E88 y sc cv v E94 yy sc homozygous X8 y homozygous M10 y d> 6IV ES EIII E52, y cv homozygous 9 Table la General genetic properties of stocks, tested for temperature-sensitivity mutant mutagen . viability index* regional , viability at 29°C i21.5°C 29°e localization very slight not EMS homo=30.6 homo=»0.0 y-sn • E2 EMS 1.25 0.0 cv-sn 19.:. EMS homo=73.7 homo=0.0 near cv E25 EMS homo=86.8 homo-0.0 .... cv-sn E27 EMS homp=35«6 homo=0.0 cv-sn 134 EMS 0.98 0.08 at sn E45 EMS homo=60.3 homo»0.0 v-f B46 EMS homo=35»6 homo=0.0 r.car-r.1 E76 3 MS 0.17 0.0 car-1 •S' E82 EMS 0.77 0.0 at cn, E88. EMS (0.54 0.0 at wy E94 EMS 0.92 0.0 cv-sn ... - hot-de.terir .... ... .i. j . lined X8 < -^rays homo=47.0 homo=«0.0 right of y •/ M104 Mitomycin p 0.42 0.0 f-car, 6IV BS EMS 0.13 0.0 g-f not ts EIII E52 EMS 0.75 0.0 V.-1JLY. 1 Viability index - Requency of ts males • > • frequency of heterozygous (ts/EM-6 or M-5) females In the homozygous ts stocks (homo) the figures represent the average number of offspring hatching in cultures set up in an identical man-ner except at different temperatures. 2 Example: v-sn means that the mutant is located betwen.y. and .sn 3 Males not fertile at 29°C 4.. Homozygous females not fertile at 29°C 10 chromosomes. The y..ts.. chromosomes were made homozygous by mating males to a balancer stock, FM-6/y. and backcrossing the y..ts../FM-6 females to y..ts.. males. In cases where homozygous females were sterile or lethal, the y..ts.. chromosomes were maintained in males by mating to females carry-int the compound X chromosome, C(l) RA, marked with _y_ and f. Before the i > determination of autonomy was started, each stock was tested to ensure the presence of non-leaky ts genes. The ts lethals tested and their genetic pro-perties are shown in Tables Ia and Ib (mode of origin, viability indices, > genetic positions, markers on the ts chromosomes). II. Determination of autonomy The unstable ring X chromosome, In (l) X c 2, wvC, (referred to as w^p), was generated by irradiation, with X-rays, of the ring chromosome, R(l)2, containing the inversion, In (l) wvC (Hinton 1955). The resulting chromo-vC some, _w_ t is characterized by variable degrees of instability which produces gynandromorphs, XO males, and dominant lethals in progeny of w^-rbearing females, w_ -bearing males are sterile and the ring is maintained in females balanced over the In (l) dl-49. y w l z s chromosome (referred to as dl-49) Q contributed by fertile males in the stock. The males also carry the sc • Y 11 chromosome which includes the entire Y chromosome and the tip of the X in-cluding l ( l ) J l ' f , j+, and sc +. Virgin wv^/dl-49 females raised at room temperature and less than 3 days old were mated to y..ts... males also raised at room temperature. 20 pairs were allowed to mate and lay eggs for 5 days at 29°C in quarter pint bottles containing standard Drosophila medium. The same procedure was car-: ried out at 21.5°C. The flies were then discarded and the F 1 flies allowed to develop at these temperatures. Al l viable progeny including those that adhered to the medium or remained in the pupal cases were counted and clas-sified. Dead unhatched pupae were classified in the 29°C cultures by dis-secting them to determine the phenotype of the developing imago. Any mor-vG / phologlcal abnormalitiesiin the w_ /y..ts., females, whether they were stuck to the medium, dead, or unhatched were noted. In crosses of w v C/dl-49 $ x y..ts../Y primary non-disjunction in females generated wvC/dl-49/Y females which are phenotypically similar to wvC/y..ts.. females. In order to minimize the number of such females erro-neously classified as w_ /y..ts.., several precautions were taken. Putative vC / w /y..ts.. females were separated into two classes, those displaying external mosaicism and those phenotypically wild type. Mosaic females from the 29°C 12 cultures were tested intensively in the following manner. Those mosaic for y and other recessive markers were classified unequivocally as y..ts.. mosaics. Those displaying only y patches of tissue but having normal female genitalia were tested to determine whether the dl - 4 9 chromosome was in fact carried. If this chromosome was detected, the females were classified as primary excep-tional females. However, most of these females were sterile. They were in-cluded in the y..ts.. mosaic class i f dissection did not reveal the presence of colourless Malpighian tubules or testes due to the presence of the w gene. The majority of y_ mosaic females were gynandromorphs showing mosaicism of : the external genitalia and therefore were sterile. These were dissected likewise and classed as products of primary non-disjunction i f they had unpig-mented Malpighian tubules or testes. Otherwise, these were considered to be y..ts.. mosaics. At 21,5°C mosaic females were classed as y..ts..-bearing females unless patches of w l z s tissue occurred. A l l other non-y females which did not show mosaic patches under a dissection microscope at 25X magni-fication were, at both temperatures, classed as u -bearing non-mosaics as regards external characters. Henceforth, females manifesting external mosa-icism will be referred to as mosaics while those females bearing the wvC chromosome awhich are not mosaic for external^phen6J>ypesv:will..beticalled.:non-13 mosaics. Any y..ts.. males that survived at 29°C were mated to C(l) RA/Y females at room temperature to check their f e r t i l i t y . These adults were transferred to fresh vials and kept at 29 °C to determine by absence of male Fj progeny whether the ts gene had s t i l l been present. In control experiments, wv<Vdl-49 females were mated to y sc cv v f car/Y males and to y cv v f/B S Y y* males in the same manner as described above to measure the survival of non-lethal mosaic and non-mosaic females. They were f also mated to we bfrVB^ Y males to measure these frequencies with a non-ts lethal. The latter test should give results comparable to those obtained with the y..ts.. chromosomes at 29°C In a l l cases, the classification pro-cedure described for the y. .ts.. tests was followed. However:,! since primary non-disjunctional females could be detected in female progeny by the presence of the Bar marker in the last two crosses, the precautions in scoring mosaic and non-mosaic females did not have to be taken. (For a description of a l l the mutants and special chromosomes used above, see Lindsley and Grell 1968). I l l i Determination of the frequency of non-disjunction in the w^ stock Since the wv{Vdl-49/Y females resulting from primary non-disjunction , > 14 Figure 3 Other crosses made in the investigation of autonomy vC A. Control test, of primary non-disjunction in w -bearing y CVJV f/BSY y + eft? x wvC/dl-49 21.5°C and 29 °C score wvC/dl-49/BSY y+ $| ,wVc/y_ cv v f 2; y cv v f/dl-49 $; y cv v f / 0 <? n-ritnaV-u- s B ' ' primary primary exceptions with mosaic patches (mosaic)' wild type.§$ (nonr-mosaic)' exceptions B. Controls to determine regular mosaic and non-mosaic frequencies y sc cv v f car/Y dtf x wvC/dl-49 . . 21.5°C and 29°C score w^v /^y sc cv v f car^ £ y sc cv v f car/dl-49 $ mosaic non-mosaic C.r Test of a non-temperature-sensitive autonomous, lethal we b-b1/BSY dB,..::v . wvC/dl-49 9$ score vC / e , . 1 ,, w /w bb $ 7jion-mosaic) _21.5°C and 29°C we bbVdi-49 9 15 Figure 2 Cross carried out for a determination of the autonomy of each temperature-sensitive mutant y..ts../Y dS1 x wvC/dl-49 9? 21.5°C and 29°C score y. .ts../dl-49 $j w^v /y..ts.. ^; y..ts primary others exceptional 9? $$ with ^tissue '..completely wild type (mosaic) (non-mosaic) 16 Figure 1 Crosses made for different aspects of the study A . Determination of temperature-sensitive lethals ts/2 <? x XX/Y § B. Insertion of markers on the chromosomes carrying, the ts lethal ts/(FM-6) or (H-5) ? JL y sc cv v f car/Y * 21.5°C select y sc cv v f ear/ts $> x X/Y ^ 21.5«C XX/Y $ x select y..ts../Y tf x FM-6/y $ 21.5°C 21.5°C select y.,ts../Y ^ x select y..ts../FM-6 $ 21.5°^ c. Retest of marked stock y.,ts../Y ^ x XX/Y $ select y.,ts../Y ^ x select y..ts../y..ts 29 °C no y..ts../Y ^ 29 °C no survivors 17 mimic wXG/y.»ts.. females, their frequencies of occurrence had to be esti-mated. In those cases where mosaicism involved tissues affected by w and l z s , these females could be recognized. However, this criterion could only be applied in eye tissue, not in the cuticular areas where only v_ could be de-tected. The frequencies of y..ts../0 exceptional males does not give an indica-tion of the rate of primary non-disjunctional female production at permissive temperatures since XO males are produced at higher frequencies than XXY fe-males in crosses involving wvG (Hinton 1955)• Under restrictive temperatures these males should not survive. In order to estimate the rate of primary non-disjunction in females the results of one of the control crosses for the autonomy tests was used. In the cross involving y cv v f/B^ Y y+, the Y-linked Bar marker indicated non-disjunction. The exceptional females carrying Bar were then tested to esti-mate the number resulting from paternal non-disjunction. Summaries of all the crosses made are shown in Figures 1, 2, and 3« IV. Determination of temperature-sensitive period and effective lethal phase Since the assessment of the autonomous or non-autonomous behaviour of a 18 given ts lethal requires knowledge of the time at which temperature affects the viability of the individual carrying the ts mutant (the temperature sen-sitive period or TSP) and also of the time at which exposure to the restric-tive temperature during the TSP manifests itself phenotypieally by death (the lethal period or LP), these periods were determined by the following procedure. Groups of 50 - 100 pairs of flies from each ts stock were placed in empty half pint bottles inverted over petri plates containing standard Drosophila medium and allowed to lay eggs at 21.5°C and 29°C for 1-2 hour periods. Usually, 50 -1100 eggs could be collected within such an interval. These were maintained at the respective laying temperatures and at successive twelve hour intervals, a 29°C culture was shifted to 21.5°C (shift down) and vice versa (shift up) for total periods as long as 240 hours. After sufficient time to allow emergence of adults had elapsed? the cul-tures of homozygous ts lethals were examined for the number of adults, for the presence of pupa cases containing dead fully pigmented imago, for dead early pupae, and for dead larva at various developmental stages as determined by examination of mouth parts and tracheal development (Bodenstein 1950). The onset of the TSP was indicated by the fi r s t culture of a shift down which showed any evidence of death due to exposure to the restrictive temperature ; 19 Figure 4 A model of reciprocal shift experiments to determine the TSP based on results obtained with y E25 20 and the end of the TSP was indicated by the first culture in a shift up which began to yield viable adults (Figure 4 ) . Since loss of the w .?chromosome can occur at any time in development, a l l possible LP's resulting from the exposure of a culture to restrictive temperatures at different times were determined. This was done by noting a l l the stages at which death occurred in the shift cultures. In those stocks in which the ts mutant was maintained in males only, cultures were examined in a manner similar to that outlined above except that certain precautions were taken to differentiate between male and female pro-geny. Fully pigmented pupa were sexed by the presence of sex comb and genital apparatus, whereas the presence of testis or ovary upon dissection was used as the criterion in early unpigmented pupae and third instar larvae. Exten-sive death in the f i r s t and second instar larval stages with the emergence of the expected number of female progeny was assumed to indicate effective lethal phase in the early instars. Controls for these shift experiments were carried out using a y sc cv  v f car stock maintained in the. males. The procedure described for the y.,  ts.. stocks was followed in this case also. 21 Table II Number iri each class of offspring of the cross: y. .ts../Y dtj8 x wvC./dl-49 99 chromosome tested dl-49 y..ts, 9 .non-mosaic y 4 .ts. T • mosaic y. .ts,,. xo d1 1 Excep-tional 9 others •total a y sc cv E5 b c . 835 8 377 79 3 171 65stuck 9 10 94stuck 10 67 5 3 206 7 99 1284 816 a' y sc E7 b c 1760 17 726 127 14 371 103 22 211 22 9 86 3 3 3 386 38 267 2402 1674 a y sc cv E9 v car b c 431 11 336 47 2 146 30 12 13** 29 21 44 1 5 62 6 41 510 706 a y:.E25 b c 956 12 5 4 6 92 10 270. : 52 63 166 4 152 287 4 4 109 1 6 78 1221 1355 a y sc E2? f car. b c 893 6 895 80 6 364 18 14 253 1 149 6 9 163 7 84 1161 1754 a y sc E34 b ... . c 886 9 750 51 9 372 29 48 293 6 104 149 9 7 285 31 110 1276 1680 , a y sc cv E45 b c 2524 2 494 222 6 222 71 8 150 19 11 70 13 5 383 6 73 3233 1014 a y sc cv v E46 >b c 1241 11 852 110 20 384 8 .10 238 3 10 148 5 15 302 16 132 1671 1709 a y sc E76 , b c 874 - 10 656 61 8 307 44 9 187 14 6 70 1 5 126 9 49 1120 1276 a y E82 v , b c 1153 2 620 76 14 290. 37 25 117 •I'dead 49 71 3 12 1 9 5 25 33 1465 1203 a y sc cv v E88 b c 1542 15 494 125 17 222 15 14 179 1 117 5 6 234 18 116.. 1922 1134 a y~sc E 9 4 b c 1423 17: 731 154 7 254 100 13 216 3 3 158 2 229 6 40 1913 1399 a y X8 b c 2209 23 1110 194 28 487 11 30 303 212 15 19 4 4-33 87 326 2865 2444 we bb1/BSY b c 187 4 732 9 . 6 219 - -• 2 24 219 3 98 1073 a y sc cv v f car b c 556 6 562 65 9 216 86 18 210 65 13 75 3 6 8 1 3 6 147 921 1214 a y cv v f/B Y y + b c 1045 18 623 131 13 206 - 96 12 188 106 6 98 . 8 1 31 897 77 560 2284 1708 a at 29°C b' unhatched pupa at 29°C c- at 21.5°C 22 RESULTS After a number of y..ts.. mutants were tested for complete lethality at 29°C and survival at 21.5°C, 16 stocks listed in Table I were selected for ic tests of autonomy. Of these;:,, 3 were subsequently found to be unsuitable for further intensive studyj y cv v E52 was found to be very leaky (i.e. gave large number of survivors at 29°C) when outcrossed to the unstable ring stock; M10 was completely sterile when mated to wv^/dl-49 females at both 21.5°C and 29°G (at both temperatures, a number of white opaque eggs which usually re-present unfertilized eggs or very early embryonic death were recovered; this may be related to the fused wing phenotype of the y.MlO-bearing males); the x third y..ts.. mutant, 6lV BS EIII, was too poorly fertile to merit investiga-tion. The number of individuals in each of the expected phenotypic classes in tests of each lethal at 21.5°C and 29°C, as described in Figure 2, are found in Table II. From these numbers, the relative viability of females in which vC the ts lethal might be unmasked by loss of the w chromosome was indicated by the ratio of the number of wv^/y..ts.. mosaic and non-mosaic females to the number of y..ts../dl-49 females at 21.5°C and 29°C, respectively. The degree of leakiness of the t§_ mutants when outcrossed was estimated by 23 Table III vC Ratio of the number of w_ -bearing females and exceptional males to the number of y..ts.,/dl-49 females (value referred to as relative viability ratio or RVR) chromosome tested mosaic 9* non-nosaic r X0 matro-clin-ous 9 mosaic 0** non-mosaic 0** y" sc cv E5 a .?0?8 , ;P95 . 7006 .078 .091 .169 t .276 .454 .178 ,00£ .259 .429 .688 y sc E? a .058 .072 .01j .002 .056 .068 .124 t .291 .511 .118 .004 .270 .486 .756 y sc cv E9 v car a .087 .138 .085 .00; .086 .134 . .209 t .399 .435 .131 .01f .389 .410 .829 y E25 a .054 .099 .004 .004 .054 .095 .149 t .304 .495 .526 .00? .286 .470 .756 y sc E27 f car a .020 .090 .001 .00? .020 .086 .106 b .283 .407 .166 .010 .268 .382 ;650 y sc E34 a .033 .056 .007 .010 .033 .052 .085 b .391 .496 .199 .009 .375 .471 .846 y sc cv E45 a .028 .088 .008 .005 .028 .084 .112 b .304 .449 .142 .010 .289 .424 .713 y sc cv v E46 a .006 .089 .002 .004 .006 .085 .091 b .279 .451 .174 .018 .272 .426 .698 y sc E76 a .050 .070 .016 .001 .047 .066 .113 b .285 .468 .107 .008 .268 .443 .711 y E82 v a .032 .066 .001 .003 .031 .062 .093 b .264 .468 .115 .019 .258 .443 .701 y sc cv v E88 a .010 .081 .001 .003 .009 .077 .086 b .362 ...449 .237 .001 .338 .424 .762 y sc E94 a .070 .108 .002 .001 .O67 .104 .171 b .295 .347 .216 - .270 .422 .692 y x 8 a .005 .088 - .007 .005 .084 .089 we bbVB sY b .273 .439 .191 .004 .252 .415 .666 a - - - .048 .048 b - - - .299 .299 y sc cv v f car a .117 .005 .155 .117 .272 b .134 .014 .374 .384 .758 y cv v f/B Y y + a .101 .008 .092 .125 .217 b .152 .050 .302 .331 . .633 * not corrected for primary non-disjunction in females ** corrected for primary non-disjunction in females a at 29°C b at 21.5°C 24 the ratio of y,.ts.,/0 males to y..ts../dl-49 females at the two temperatures. These ratios will be referred to as relative viability ratios or RVR. RVR were adjusted to eliminate distortion caused by misclassification vC of non-disjunctional females as w_ /y..ts... Although extensive precautions were followed to eliminate such misclassification, i t could not be eliminated completely. Control tests showed that the ratio of primary exceptional to regular females was 0.008 at 29°C and 0.050 at 21.5°C (Table III). Further-more, at both 21.5°C and 29°C, half of the exceptional-3? females showed mosaicism, thus contributing 0.004 at 29°C and 0.025 at 21.5°C to each of the mosaic and non-mosaic class ratios. These values were used to correct the ratios of the y..ts.. results. In a l l autonomy experiments, females could be unambiguously classified as primary non-disjunctional offspring only i f they were mosaic for external tissue since only those displaying y patches were tested or dissected to determine the presence of the dl-49 chromosome. Therefore, the ratios of matroclinous females (Table III) represent only non-disjunctional mosaic females. The corrections were made in the following manner; i f the frequency of verified non-disjunctional females in the crosses was greater than 0.004 25 Table IV Ratios of the Relative V i a b i l i t y Ratios of vr^-bearing fe males (21.5°C/29°C) chromosome tested mosaic non-mosaic total •« y sc cv E5 3.3 4.7 4.1 y sc E7 4.8 7.2 6.1 y sc cv E9 v car 3.1 4.0 y E25 5.2 5.0 5.1 \y sc E2? f car 13.*+ 4.4 6.1 y sc E34 11.4 9.1 10.0 y sc cv E45 10.3 5.1 6.4 y sc cv v E46 45.3 5.0 7.7 y sc E7& 5.7 6.7 6.3 y E82 v 8.3 7.2 7.5 y sc cv v E88 37.6 5.5 8.9 y sc E94 4.0 4.1 4.1 v X8 50.4 7.5 average 15.9 ,5.6 6.4 we bb1/BSY - 6.2 6.2 y sc cv v f car 2.4 3.3 2.8 y cv v f/B SY y + 3.3 2.7 2.9 26 or 0.025 at 29°C and 21,5°C respectively, no change was made. However, if it was less than this figure, the difference was subtracted from the y..ts.. mosaic RVR. For non-mosaic females, the established control value was sub-tracted from the RVR of non-mosaics at both temperatures. An analysis of variance on the corrected and uncorrected ratios showed that the statistical distortion of mosaic and non-mosaic values by misclassification of non-dis-junctional offspring was negligible. These-.corrected mosaic and non-mosaic values at 21.5°C (Table III) were taken as a ratio of the respective values at 29°C (Table IIj). Thus, a value greater than 1.0 was an indication of decreased viability at 29°C (Table IV). Similar ratios were calculated from the results of non-lethal control vC experiments. It can be seen that both classes of w_-bearing females of the controls decreased in frequency at Z9?0, and that this decrease was of the same order to magnitude in both phenotypic classes for both controls (Table IV, average ratio «? 2 .9) . This decrease never exceeded the values observed in tests of y..ts.. chromosomes, where it varied from 3 - 9 in non-mosaic females and 3 - SI in mosaic females. These control values may simply re-flect a decreased viability of females which carry the unstable ring at.29°C. 27 Results using the bb"*" mutant to obtain mosaic and non-mosaic frequencies for an autonomous non-temperature-sensitive lethal gave a ratio of wvC/we bb1 to we bbVdl-49 of 0.299 at 21.5°C. This was decreased by 6.3 times to 0.048 at 29°C These frequencies of surviving wvC/we bb1 females may indicate the proportion of zygotes in which no ring loss had occurred, loss occurred in tissue in which activity of the locus was not required, or loss occurred in-vC / ternally very late. Decreases in viabilities of w_ /y... ts.. females of sim-ilar magnitude were found for some ts mutants at 29°0 (Table IV). Before a closer analysis of the ratios is made, results indicating the penetrance of the ts genes will be investigated. Penetrance of a gene, that is, the actual phenotypic manifestation of an allele, has been shown to vary with differing environmental and genetic factors so that "Durchbrenner" or lethal-bearing "escapees" may either survive to the adult stage or die only at a later effective lethal phase (Hadorn 1951)• Upon outcrossing, some of the ts mutants survived as hemizygous males at 29°C, indicating that the lethal phenotype had been affected by genetic modifiers. Therefore, although all 13 y..ts.. mutant stocks were initially confirmed as complete lethals at 29°C, viability could be affected by altering the "environmental milieu" 28 upon outcrossing to the ring-X stock. Indeed, only two of the thirteen mu-tants tested (y sc E27 f car and y_X8) remained completely lethal upon out-crossing (Table II). Both y E82 v and y sc cv v E88 gave only one y. .'ts.. male (In the case of y E82 v, the male was dead.) in all crosses at 29°C and so can be classed with those that maintained their temperature-sensitivity. Another group of mutants showed a very slight leakinessj y_E2_5, y sc E34, y_ sc cv E45, y sc cv v E46, and y sc E94 with ratios of XO males to y..ts../ dl-49 females varying from' .002 to 0.008 at 29°C, and RVR of 0.142 to 0.526 at 21.5°C (Table III). A third class appeared to be more strongly affected by the change in genetic background for the autonomy tests showed, that X0 male survival had increased to give RVR of 0.013 for y sc E7, 0.085 for y sc  cv E9 v car, and 0.016 for y sc E?6 at 29°C However, the higher RVR fbr:' .: .• these mutants at 21.5°C indicated that their relative viabilities at the high temperature were s t i l l very low. In all these tests, the X0<males that did hatch at 29°G either died at eclosion or were stuck to the medium and therefore were adult lethals. Even in the case of y sc cv E5 where emergence of X0 males was high, all.adults died soon after eclosion. Since all surviv-ing males lacked a Y chromosome, the presence of the ts gene could not be verified genetically. 29 In order to determine whether the frequency of surviving XO males at 29°C, which could be a measure of the penetrance of the ts gene in a new genotype, was correlated with the frequency of females with mosaic patches at 29°C, a correlation test was carried out. The correlation coefficient, r, measures whether mutually dependent variables x ( the number of XO males) and y (the number of mosaic females) are related, and ranges in value from +1 (which shows perfect positive correlation) to -1 (which shows perfect nega-tive correlation); a value of 0 indicates no correlation. The correlation coefficient, r, may also be an index of the closeness of f i t of the observed points (n) to the estimated line of regression. The larger the absolute value of r, the closer the points will f i t the line; i f r « t l , every point will be exactly on the line. Also, r = ^(*-x) (y-,7) .—•• • where x and y are the means )T(x-x)E(y-y) of the x's and y's To test a hypothesis of correlation, t-tables can be used since t = r J l-r/n-2 The correlation coefficient was calculated on the raw data (that i s , with the number of mosaics as one variable and the number of XO males as the other). 30 Table Vi: Data collected in the scoring of "shift" cultures of y E25 time of # of shift, eggs iownfhr.) laid results 0 50 40 live adults 20 44 68 92 50 50 50 50 42 live adults 43 live adults 45 live adults 46 live adults 111 45 7 live adults, few dead third instar larvae 116 50 30 live adults, few dead third, instar larvae 120 30 a l l dead third instar larvae 140 86" Ifew. live adults, dead third instar larvae,' 152 160 59 3 t dead late pupae, partly eclosed dead.adults, _and dead adults 170 30 1 live adult, dead third instar larvae 175 541 fdead third instar larvae, dead early and late 185 188 50 50 Lpupae /mostly dead third instar larvae, some dead 190 ;;212 75j 100 pupae, dead partly eclosed. adults, dead adults mostly dead late pupae Death occurred in the culture that had been shifted "down after 111 hours at 29°C, therefore, TSP must begin sometime between 92 and 111 hours. LP extended from third larval instar to adult stage. time of # of shift eggs results up(hr.) laid 0 50 dead late pupae 20 40 dead late pupae 44 40 dead late pupae 68 4b1 [dead adults, dead partly eclosed adults, dead 92 32J (late pupa 111 40 0 live adults, dead adults and pupae 116 30 6 live adults, dead adults and pupae 120 76 19 live adults, dead adults and pupae 140 35 7 live adults, dead adults and pupae 152 38 6 live adults, dead adults and pupae 160 38 24 live adults, dead adults arid pupae 170 32 22 live adults, dead adults and pupae 175 43 25 live adults, dead adults and pupae I85 39 29~live adults, dead adults and pupae 188 30 11 live adults, dead adults 190 39 31 live adults, dead adults 212 30 16 live adults, dead adults. Live adults emerged in the culture that had been shifted up after 111 hours at 21,5°C, therefore, the TSP must have ended Sometime between 92 and 111 hours. 31 Table V XSP and. LP of the different mutants tested mutant tested TSP .LP y sc. cv E5 99 s indispensible dtf'i 150 hours (pupa) to adult late third instar to adult y sc E7 25 to 70 hours (first instar larva to third instar larva) first instar larva to adult y sc cv E9 v car 70 to 180 hour (third instar larva to late pupa) prepupa to adult y E25 92 tolll hours (end of third instar larva stage) late third instar to adult y sc E27 f car 60 to I65 hours (third instar larva to mid-pupa) third, instar larva to late pupa y sc E34 50 to 80 hours, (second instar larva to third instar larva) third instar to adult y sc cv E45 0 to 140 hours (egg to mid-pupa) second-third larval intermolt to adult y sc cv v E46 0 to 120-:.hours (egg to early pupa) first instar to eclosion y sc E76 0 to 240 hours (indispensible) first instar to adult y E82 v 60 to 140:-hours (third instar larva to mid-pupa) second-third larval intermolt to eclosion y sc cv v E88 80 to 150 hours (third instar larva to mid-pupa) third instar to late pupa y sc E94 28 to 125 hours (first instar larva to early pupa) first and second instar larva, pupa, adult y X8 82 to 97 hours (during late third instar larva) pupa to adult 32 The value of r was 0.33P and of t (11 degrees of freedom) was 1.158. With 11 degrees of freedom, the value of t at the 5% level of significance is 1.796; thus, no statistically significant correlation was indicated in this analysis. In comparing the RVR of XO males and mosaic females, r was found to be 0.593* The corresponding value of t ( l l degrees of freedom) was 2.444, while t at the 2.5% level of significance is 2.201. Therefore, at the Z . % level of significance, the null hypothesis that there is no correlation can be rejected, meaning that XO survival is related to viability of mosaics. Besides the penetrance of the ts gene, its TSP and LP might be expected to.affect the frequency of mosaicism at 29°C. The temperature-sensitive periods and lethal phases of a l l the mutants studied are shown in Table V. A sample of the method of scoring for one y..ts.. mutant is given in Table VI. It must be pointed out that the determinations of the TSP were very crude owing to considerable asynchrony in larval development. Because of this, organisms in shift cultures were of different ages and could, therefore, react to the temperature change in a number of ways. Although control shift experiments using the non-ts, y sc cv v f car/Y stock, were set up in an attempt to standardize developmental time and rates 33 at the two temperatures, these could not be used as stringent controls for the ts experiments since the rates of development may vary with each genotype. However, these controls did provide a measure of the amount of lethality in a culture at different developmental stages that might have resulted from the temperature change alone. This level of lethality was taken into account when the y..ts,, shift experiments were being recorded. i A statistical test was used to determine whether there was any correla-tion between the length of the TSP and the magnitude of the decrease encoun-tered by the mosaic class due to the temperature difference as measured by RVR21'5°C/RTO29°C# ^ t e s t g s h o w e d t h a t t h e r e w a g n o c o r r exation a t the 5% level of significance. In addition to the above results, information on three ts mutants was obtained from the autonomy and TSP experiments. Red pigment granules were found in the Malpighian tubules of larvae, pupae, and adults, both alive and dead, of the mutant y sc E27 f car at 29 °C. y.^ sc cv EV? males developing up to the late pupal stages at 29°C had dark pigment deposits on their dorsal abdominal surfaces. These abnormalities were definitely associated with the ts gene since they did not occur at 21.5°C. Also, they did not occur at 29°C in non-ts individuals which had the other recessive markers. The sexual 34 dimorphic nature of y sc cv E5 (Tarasoff 1968) was confirmed (Table 35 DISCUSSION The study of cellular autonomy is of biological interest as a method of gaining an insight into the nature of cell to cell-interactions. The influ-ence of one functioning cell on the activity of another is an integral part of the differentiation and regulation of a multicellular organism. From the definition of autonomy used in this study, genetic cell to cell interactions can be studied by determining whether cells of one genotype can alter the phenotype of a genotypically different cell. Although such a condition does not normally arise in a developing individual, such genetically contrived mosaics may, in fact, mimic states of differential genetic activity in d i f -ferentiated cells. Thus, studies of autonomy could parallel the. process, of.: differentiation, and whatever information about genetic interactions between cells that is gained from these studies might reveal interactions taking . place during development. These studies of autonomy involved the analysis of mosaics in which celis or tissues of one genotype are juxtaposed to genetically different cells. Thus, i f a mutant genotype being studied is autonomous and viable, mosaic patches of mutant tissue will be detectable phenotypically, adjacent to wild type tissue. The size and location of mosaic tissue may suggest the devel-36 opmental time at which genetic activity is initiated and repressed and its tissue specificity. In these studies, however, the phenotype of each mutant was lethality and therefore chromosomes carrying the lethal gene were marked with autonomous visible mutants in order to detect the presence of non-auton-omous mutant tissue. In such a case the size and location of mosaic tissue may suggest the time after which autonomous genetic activity is repressed and/or tissue in which the lethal gene does not function. A demonstration of non-autonomous genetic behaviour of a mutant suggests the presence of a diffusible substance in the wild type tissue which can modify the phenotype of mutant cells. Thus, the recognition of non-auton-omous mutants may provide a bio-assay which would permit the isolation of diffusible factor(s) produced by the wild type tissues. Characterization of the. requirements of such "supplementable" mutants could ultimately permit cell culture selection techniques comparable to those used in microorganisms. These studies of autonomy were facilitated by the use of temperature-sensitive lethal mutations. Previous studies, of autonomy in Drosophila mela-nogaster involved the use of non-conditional lethal mutations (Demerec 1934ji Ephrussi 1934; Stern 1934; Poulson 1948; and Oster and Sobels 1956).where the frequencies of mosaic patches in flies heterozygous for the lethal 3? mutant were compared with the frequencies of mosaics in non-lethal-bearing individuals. The ts mutant provides a more rigid control since mosaic fre-quencies in flies of the same genotype can be compared at 29°C and 21.5°C. Genotypic control and the ease of imposition of the selective condition greatly enhance the study of lethal autonomy. In addition, in studies of non-ts lethals, it cannot be said definitely that the mosaic patch of mutant tissue was produced during lethal activity of the mutant. The mutant patch could have been produced..after lethal activ-ity had ceased. However, the TSP of a ts mutant is the time during which genetic activity can be altered to result in lethality of the mutation. Thus, mosaic patch production after exposure of the developing fly to lethal tem-peratures during its TSP may be the result of non-autonomy. Yet, it must be remembered that mutant activity will be expressed only in tissue requiring genetic functioning of that particular locus. So, although by using a ts mutant, i t can be said that lethal genetic activity was being expressed dur-ing a specif ic;. time interval, this activity results in lethality of certain tissue causing death of the whole individual. It does not necessarily mean a small patch of other tissue will be affected by the ts lethal mutant. In order to say specifically that .the mosaic patch was expressing mutant activity, 38 knowledge of both TSP and tissue specificity is required. In this respect, the use of a ts mutant is no more advantageous. However, once autonomy has been confirmed, the advantages of the ts mutant in further investigations are numerous. Turning now to the experiments performed, the technical difficulties in evaluating the results will be outlined initially. A major problem arises because the mere presence of mosaic patches at 29°C does not necessarily in-dicate non-autonomy of a mutant since other factors may contribute to the production of mosaic tissue. In the following discussion a number of these will be considered and i t will be determined whether they affect the frequency of mosaics scored in these tests. Mosaic patches can be produced by abnormal genetic events not involving actual loss of the t s + gene? for example, loss of fragments of the ring-X chromosome unmasking only certain recessive markers while maintaining the wild type allele of the locus has been suggested (Singer 1969, personal com-munication). In the present experiments, i t was found that in most cases where y tissue was detected, other recessive visible markers linked to y (sc, cy, v, f, and car) were also expressed. Thus, i f loss of only small fragments of the X-chromosome. occurs, i t is infrequent and the loss generally involved the 39 entire ring-X (including the locus of the t s + gene). Moreover, since the final analysis of the data was made by comparing the relative frequencies of mosaics at the restrictive and permissive temperatures, this factor should cancel out i f the rate of loss of y + was independent of temperature.' Thus, generation of mosaic patches through loss of small regions of the ring-X, I feel, is probably unimportant. The production of mutant tissue through somatic exchange also should not distort the- estimate of autonomy based on the frequency of recovery of mosaics. Somatic crossing over between the mutant marker and the ts lethal results in the formation of twin, spots, tissue homozygous for the lethal and the mutant marker. If death of cells homozygous for the ts lethal does not k i l l the fly, non-autonomy of the lethal, could be suggested by surviving mosaic tissue. Also, i f somatic recombination is independent of temperature, the contribu-tion to mosaic frequencies by somatic crossing over should be similar at both temperatures. Moreover, since most mitotic crossing over occurs in proximal heterochromatin (Stern 1936) the marker and the ts lethal should remain linked. The possible distorting effects of a number of other factors have also been ruled out. The presence of females resulting from primary non-disjunc-tion and therefore having mosaic patches which do not carry the ts lethal 40 was found to be too small to affect the estimated frequencies of lethal mo-saics. The markers (j, sc, cv, f, and car) used to identify the mosaic patches of ts tissue were shown to be autonomous (Sturtevant 1932) at both temperatures. The number of recessive markers linked to the ts gene would not affect the relative frequencies at the two temperatures unless there was a drastic and unexpected temperature sensitivity of the markers. In spite of the elimination of these possibilities, the presence of ;;. mosaics at 29°C does not confirm the non-autonomy of a ts lethal. Mosaics could survive i f hemizygosis of the y..ts.. chromosome at 29°C took place after the time that ts. gene lethal activity had taken place. Although the tissue specificity of the mutants was not investigated, the TSP was deter-mined. It was expected that the longer the TSP, the greater would be the i vC chances of w loss during that interval. Thus, it was anticipated that there would be a slightly decreased frequency of mosaic survival with non-autonomous mutants and a markedly decreased frequency of mosaic survival with autonomous mutants of long TSP's at 29°C. Analysis of the results showed that the length of the TSP and the magnitude of the decrease in the mosaic class at 29°C were not statistically related. This could mean that both non-autonomous and autonomous mutants were present in the sample. On the other 41 Table VI'I Description of y..ts.. mosaics surviving at 29°C y sc cv v E46 1. half of sixth abdominal tergite yellow; half of f i f t h abdominal tergite. missing 2. eyes vermilion, half of f i f t h and sixth abdominal tergites yellow, genitalia mosaic 3. parts of f i f t h and sixth abdominal tergites yellow.,, half of fourth abdominal-tergite missing 4. half of f i f t h and sixth abdominal tergites yellow, genitalia mosaic eyes partly vermilion 5. f i f t h and sixth abdominal tergites yellow, genitalia mosaic, f l y s " u : : k stuck to medium 6. half of fi f t h and sixth abdominal tergite yellow, genitalia mosaic 7. scutellar bristles missing, genitalia mosaic 8. left half of abdominal tergites yellow, missing scutellar bristles y sc cv v E88 1. left legs yellow, genitalia mosaic* 2. left half of abdominal tergites yellow, genitalia mosaic 3. half of sixth abdominal tergite yellow 4. genitalia mosaic 5. a l l abdomen yellow except for half of the first, second and third abdominal tergites 6. left half of head yellow, left eye vermilion, f i f t h and sixth abdo-minal tergites yellow, genitalia mosaic 7. left half of abdomen yellow 8. right eye vermilion, right antenna yellow 9. right half of abdominal tergites yellow 10. half of f i f t h abdominal tergite yellow 11. half of f i f t h and sixth abdominal tergites yellow 12. half of f i f t h and sixth abdominal tergites yellow 13. half of f i r s t to f i f t h abdominal tergites yellow 14. bristles on head yellow 15. not described *only mosaic patch detected on legs; could be due to primary excep-tional female y X8 1. half of fourth abdominal tergite,.yellow 2. half of fourth and f i f t h abdominal tergites .yellow, genitalia yellow, dead "3. parts of abdominal tergites yellow, other parts missing, genitalia abnormal 4. male genital arch, stuck to medium 5. one antenna yellow 6. one wing and half thorax yellow, individual dead 7. parts of head and thorax yellow, individual dead 8. small patches of tissue all-over the body yellow, dead 9-11. mosaics stuck and dead, yellow patches a l l over the body 42 hand, i t could be argued that a l l of the mutants are autonomous but are re-quired only in certain tissues during the TSP; mutant tissue not requiring activity of the ts. gene at this time will survive and yield mosaics. There-fore, only when i t is known in which tissue the ts mutant is lethal during the TSP will the TSP be of great significance in understanding autonomy. Thus, in these studies, only when total absence of mosaics or. complete ab-sence of one type of tissue displaying mosaicism is demonstrated, can a mu-tant definitely be called autonomous. Mutants E46 and E88, which yielded no mosaic patches on the thorax, wings, and legs (Table VII) at 29°C, are exam-ples of the latter. The survival of y.,ts.. males at 29°C ln some of the tests probably re-sulted from altered gene expression under different genetic and environmental conditions. It must be asked whether such survival might result from a dif-ferent mechanism such as the loss of the ts mutant by somatic crossing over. Hinton (1955) established that XO males result primarily from early somatic loss, of the wvG chromosome from x/wvG zygotes. Therefore, the occurrence of a somatic double exchange in the earliest stages of cleavage before or at the time.of w_j elimination, could result in replacement of the ts. lethal by its wild type allele, permitting survival of the XO male. A test for 43 such an elimination could not be performed since these males were XO and therefore sterile. Somatic exchange, if i t occurs, can be used to account for the survival of these XO males at 29^C, but the possibility of the early occurrence of such an event spanning only the ts locus, followed by wv^ loss, is remote since the frequency of mitotic double crossovers is itself rare (Stern 1936). Thus, y..ts.,/0 m a l e H S u r v i v a l is undoubtedly primarily the consequence of "escapee" activity. Many of the y,.ts../0 males that did hatch at 29°C died immediately or stuck to the medium and died shortly thereafter. The weakness of these males probably resulted from a prolongation of the LP into late pupal and early adult stages. Five y..ts.. mutants (E46, E88. E27, E82, and X8) that gave virtually no XO males did not have LP's in the adult stage, whereas all of the others which yielded some XO male "breakthroughs" had LP's extending into the adult stage. The higher frequency of XO adult male of y E5 was expected since its lethal period is exclusively from pupal to adult stage. So, the survival of XO males to the adult stage due to "escapee" activity at 29°C appears to be enhanced by the presence of a lethal period in adults. Analysis of results showed that higher mosaic frequencies at 29°C were correlated with XO "breakthrough" frequencies at 29°C. XO male survival in 44 turn has been found to be greatest in those ts mutants with adult lethal per-iods. Therefore, mosaic frequencies at 29 °C are higher among those mutants having an adult lethal period. Such results may mean that in some cases the ts gene in the hemizygous mosaic tissue, as in XO males, is susceptible to similar genotypic and environmental modifications. Such altered activity must not be misinterpreted as non-autonomous behaviour. Besides mosaic tissue production at 29°C, which does not reflect the autonomous property of the ts gene, the manipulation of the data could affect estimates of mosaic frequencies. Since the evaluation of the results is based on the ratios of the frequency of a particular class to the frequency of sib-ling In(l) dl-49/(X-chromosome from the male) females, and since these rela-tive viability ratios (RVR) were used in further calculations, it is impor-tant to establish the validity of using the frequency of these heterozygotes as a common denominator. Are discrepancies caused by differential viability of the In(1) dl-49/(X-chromosome from the male) female at 29°C and 21.5°C which might then distort the relative viabilities of the mosaic class? If temperature did affect the development of this class, decreased viability at higher temperatures is expected since i t has generally been shown that the frequency of emergence of adults from cultures kept at higher temperatures 45 i s much lower than the frequency at optimal temperatures. Parsons (1959) demonstrated such decreased v i a b i l i t y by testing the effect of 31 °C as com-pared with 24°C on various wild type stocks and their F^ hybrids. Therefore, in the present experiment, the ratios at 29°C would yield an overestimate of mosaic v i a b i l i t y and tend towards classification of a mutant as non-autonomous. In fact, the ratios at 29°C of both mosaic and non-mosaic classes were greatly reduced over that at 21.5°C. Thus, even i f the ratio were overestimated at 29°C, decreased v i a b i l i t y was indicated. If temperature had no effect on the denominator, then this ratio would have been much smaller. Since i t i s of interest to look at the amount of decrease encountered at the higher tem-perature, i t should be kept in mind that the magnitude of the decrease meas-ured would be minimal by these c r i t e r i a . Therefore, even i f temperature did affect.the v i a b i l i t y of the In(l) dl-49/(X-chromosome from the male) female, the inferences drawn from the ratios would be conservative but reasonable. Comparison between RVR of different ts's are a different matter. In these cases, differences in heterozygote v i a b i l i t i e s in each cross would af-fect the ultimate ratios compared. Although adequate tests of dif f e r e n t i a l heterozygote v i a b i l i t y in different crosses were not carried out, there are no compelling reasons for expecting severe differences between the crosses. 46 Thus, in the discussion which follows, i t will he assumed that viability of the heterozygote remains relatively constant from experiment to experiment. Although a l l y..ts.. mosaic and non-mosaic frequencies were decreased at the restrictive temperatures, the total reduction cannot be wholly at-tributed to the lethal effects of the y..ts.. mosaic patches. It was found vC that non-ts control w -bearing mosaics and non-mosaics also underwent an approximate three fold reduction at 29°C. The decrease may result from a reduced viability of flies mosaic for male and female tissue at higher tem-peratures. This basic level of decrease must be considered when discussing ts mutant activity. In this experiment we are comparing survival of mosaic tissue at 21.5°C and 29°C. This includes external as well as internal mosaic tissue. A dif-ficulty arises in not knowing how representative external mosaicism is of internal mosaicism. The degree of internal mosaicism which is not detectable vC externally can be estimated by looking at the total decrease of a l l ,w -bearing females at 29°C. The decreased frequency of the mosaic class at 29°C represents the lethality incurred by flies mosaic both externally and internally at 29°C, whereas the decreased numbers in the non-mosaic class re-flect additional lethality of zygotes which are completely mosaic internally. The results of the bbJ: experiments can also he used as a base level to measure the total mosaic frequency of the %s mutants. The frequency of we bb^ /wv(^ females recovered is a measure of females which are non-mosaic in both internal and external tissue i f i t is assumed that loss of bb+ activity results in lethality at any time in any cell. Since Ritossa et. al. ( 1 9 6 6 ) have shown that the bb locus directs the synthesis of ribosomal RNA, i t is highly probable that this assumption is indeed valid. The ratio between the two classes is about 0 . 3 0 at 21.5°C, so i t can be said that about three times out of ten, wv^/bb^ females have few i f any cells in which the ring is lost in tissue vital for viability. In other words, seven out of ten developing w^ p/bb3 females suffered ring loss in vital tissue and therefore were lethal vC mosaics. The ratio of external mosaic to non-w -bearing females as shown by control experiments, is also about 0 . 3 0 (ie,, three out of ten ring-bearing vC \ females suffered loss of w in external somatic cells;. If these mosaics in the controls can be considered representative of the mosaic females re-sulting from loss of in any external tissue (although they are based on the numbers of females mosaic for the specific markers, y, sc, cv, v, _f, and car) and i f the numbers seven out of ten are taken to be an estimate of the frequency of females that undergo any loss of (although i t is specifically 46 the frequency pertaining to any loss in tissue affected by bb activity), then i t can be said that external mosaics represent less than half of a l l individ-uals in which the ring-X is lost at some stage. The results obtained from tests of the autonomous mutant, bb^ , also prd-vide values of the viability ratios against which the ts values may be com-pared. The decreases of the RVR of the wvC/we bb1 females at 29 °C compared to the ratio at 21.5°C (6.3 times) gives a measure of the lethality incurred vC by individuals carrying an autonomous lethal and the w chromosome at 29°C. This decrease would result from reduced viability of the wvC-bearing females at 29°C plus death due to mosaicism for an autonomous non-ts lethal. An av-erage decrease of 6.4 times was found for a l l y..ts.,/wvC females at 29 °C, a value very similar to the bbj- decrease. If bb^ is truly autonomous, i t sets an upper limit on viability ratios of autonomous lethal mutants. The ts mutants that have decreases greater then 6.3 times therefore must be auton-omous? the greater values may result from an interaction between.the ts and vC w chrdmbsomes at 29°C or from a greater lethal effect of the ts mutant in mosaic tissues. This figure will be used as a basis for the classification of the viability ratios of the. ts mutants. The fo lowing mutants yielded relative ratios of wv -bearing females 49 (21.5°C/29°C) greater than the ratio observed in wvC/bb1 females ( 6 . 3 ) and therefore were considered to be autonomous: EJ4 (10.0), E4_5_ ( 6.4), E46 ( 7 . 7 ) , E82 ( 7 . 5 ) . E88 ( 8 . 9 ) , and X8_ ( 7 . 5 ) . The greatest decrease of the w^-bearing female class was shown by E_3_4, The reduced viability in the mosaic and non-mosaic classes are 11.4 and 9 . 1 times respectively; so both classes are reduced equally. It should be pointed out that the decrease suffered by the non-mosaic class in E34 is the greatest of all the ts mutants tested (average decrease = 5 * 6 ) while the decrease in the mosaic class is below average ( 1 5 . 9 ) . Since decreases in the non-mosaic class reflect lethality of internal mosaics, it can be said that E34 is more effective as a lethal in internal tissue than in external tissue. The very low frequency of mosaic tissue is correlated with a very low frequency of escapees. XO survival frequency is 0 . 0 0 7 at 29°C as compared with 0.199 at 21.5°C E82, which can also be classed as autonomous, is like E34 in that i t shows the same amount of decrease in both classes (mosaic reduction =8 .3 times, non-mosaic reduction = 7.2 times). But unlike E3_4, escapee activity cannot account for the survival of mosaics since X0 flies are inviable at 29°C and no adult lethal period was observed. Since mosaic patches are found 50 on a l l external parts of the body, i t could be speculated that this mutant is lethal exclusively in internal tissue. Such speculation seems reasonable when i t is noted that the lethality of the non-mosaic class is greater than average. If this is true, then ts lethality of E82 is specific for internal tissue. vC E46 and E88. which have reductions of the w -bearing female class of 7.7 and 8.9 times, respectively, have very similar properties. Besides the over-vC a l l reduction of the w -bearing female class, the mosaic and non-mosaic classes show a similar pattern of decrease: in E46 the mosaic class was re-duced by a factor of 45.3 times and the non-mosaic class by 5»0 times and in E88 the reduction factor for the mosaic class was 37.6 and for the non-mosaic class, 5«5» This means that both are more effective as lethals in external tissue. In both, XO male survival is negligible (0.001 at 29°C). While the TSP for E46 is prolonged from egg to early pupal stage, the TSP for E88 was confined to the third instar to mid-pupal interval. Another striking property common to both is the complete absence of external mosaic patches on the thorax, wings and legs;, tissues which develop from the wing and leg imaginal discs. Also, morphological abnormalities in the same tissues and the absence of parts of these tissues were frequent in non-mosaic and mosaic females. 51 The occurrence of mosaic patches on the scutellum, as indicated by the absence of scutellar bristles in E46. may be a result of mutant tissue lethality at 29°C and not necessarily of scute phenotype manifestation due to the survival of mutant tissue. Therefore, these genes appear to function autonomously in cells of the wing and leg discs that will eventually be located on the exter-nal surface. Whether the similarity between these mutants is fortuitous or whether they are genetically related, cannot be said at this time. However, they are genetically distinct with respect to map position, E46 being located to the right of car and E88 mapping at wy, X8 is another autonomous mutant which is more active in external tissue (the mosaic class was reduced by 50^ as compared with the non-mosaic class reduction of 4.9), Cf the 11 mosaic adults recovered out of a total of 2865 progeny scored at 29°C, only 3 appeared to be fully viable, the rest were poorly viable.(stuck to the medium) or dead, and a l l had mosaic patches a l l over the body. Also, many abnormalities in external morphology were noted vC in the w -bearing females. Since i t cannot be disputed that this gene is autonomous and functional in external tissue, how can the survival of the few mosaics be explained when there was complete absence of XO males at 29°C? wyC loss after the very specific TSP at the end of the third"larval instar 52 is a reasonable explanation. This assumption is strengthened when i t is noticed that most mosaic patches involved very small, areas (Table V), the largest covering half a thorax. By the standards set with the b_b3 experiments, E&5 is also autonomous. The decrease in mosaic and non-mosaic classes at 29°C are 10.3 and 5.1 respect-ively, so this lethal appears to be more active in external tissue. The very low XO survival (0.008 at 29°C) and the presence of an adult lethal period can account for the viability of the mosaics which involve a l l body parts. Nothing more can be said about the genetic activity of this ts ex-cept that the dead y..ts.. males that developed up to the late pupal stage at 29°C had dark pigment deposits on their dorsal abdominal surfaces. This phenotype is a temperature-sensitive phenomenon, but how i t is related to the ts lethal is not known. vC Reduction of w -bearing females at 29°C similar in magnitude to that found with bb} occur in E76 (6.3 times) and E_7 (6,1 times). Nothing excep-tional was noted from the results of E?6. Mosaic and non-mosaic classes were equally susceptible to 29°C, being reduced by 5.7 and 6.7 times respectively. The observed escapee activity and the presence of an adult lethal period ex-plainsthe survival of mosaics. This mutant is temperature-sensitive at a l l 53 times so the XO males that do pupate are all dead, at hatching time. The mutant, E7, is exceptional. It has an unexpected pattern of decrease: the non-mosaic class undergoes a much greater reduction (7.2 times) than does the mosaic class (4.8 times). Such results can only mean that E_7 is most active in internal tissue. The data are made more interesting in light of the fact that the TSP occurs early in development, between the first and third instar. More can be said about the genetic activity of E7 but i t would all be conjecture. However, i t can definitely be stated that E? functions auton-omously in internal tissue. vC Although the survival of the w -bearing female with the rest of the mutants is reduced to a lesser degree than with the bb3, it cannot be said that these mutants are non-autonomous. E27/wv(^ females are 5.9 times less viable at 29°C but its mosaic classois reduced by 15.5 while its non-mosaic class is reduced by only 3.7 (N.B., control decrease is about 3.0). Such data suggest that E2? is autonomous in external tissue but non-functional or non-autonomous in internal tissue. Although some individuals recovered had mosaic patches all over the body at 29°C, these mosaics were either dead or nearly dead (stuck to the medium) at the time of eclosion. It is interesting to note that like E4_5, this mutant has a temperature-sensitive phenotype -'54 red pigment granules in the Malpighian tubules i n individuals developing at 29 °C The mosaic and non-mosaic classes with E2_5 are reduced to a similar ex-tent at 29°C (5.3 and 5,0 respectively) to give a total.reduction of the vC vrj-bearing female of 5»1« This pattern of decrease i s reminiscent of those shown by EJ4 and E82 except the degree of v i a b i l i t y i s greater at 29°C in this case. Mosaic survival does not reflect XO breakthrough activity which i s negligible in this case (0.004 at 29°C as compared with O.526 at 21,5°C). The. very limited TSP of about twenty hours at the end of the third larval instar may account for survival of females with mosaic patches i f w^ loss occurs after this period. If this were so, relatively small patches of tissue should be mosaic. However, viable mosaics involving more than half the whole organ-ism are recovered. Therefore, i t can be argued that like E3_4, E 2 5 i s auton-omous and more active in internal tissue, but unlike EJ34 and E82, i t s period of activity i s short-lived so that i t s lethal-inflicting, a b i l i t y i s curtailed. The sexually dimorphic ts mutant, E5, has a TSP from I50 hours to the adult stage in males and continuously in females. Decreases in v i a b i l i t y of E5 were not much greater than those found in the controls, as might be ex-pected since the male TSP occurs very late in development with a LP exclu-55 sively in the late pupal and adult stage. It is interesting to note that the pattern of genetic activity, of EJj in heraizygous tissues reflected the male pattern even when i t was surrounded by female tissue. Therefore, with respect to sexually dimorphic temperature-sensitive activity, E5_ is autonomous. Of all mutants studied, E94 appears to be the most non-autonomous. This ts mutant has equal decreases of 4,0 in the mosaic class and 4,1 in the non-mosaic class. External mosaicism was detected throughout the body. The de-creased magnitude of lethality cannot be explained by a short TSP as in E25 since its TSP lasts from first instar to early pupal stage. Nor can it be accounted for by "Durchhrenner" activity since XO male survival frequency at 29°C is only 0.002 compared with 0.216 at 21.5°C. The temperature-sensitive lethality was least noticeable in the case of E9, which had decreases in the mosaic class of 4,2 and in the non-mosaic class of 3*8 at 29°C. Survival of both external and internal mosaics can be wholly explained by the X0 survival frequency, which is only slightly changed by temperature (0,088 at 29°C and 0.131 at 21.5°C), an indication of the exces-sive leakiness of the mutant upon outcrossing. The above discussion demonstrates that a study of somatic mosaicism re-sulting from unstable ring loss can indicate the relative autonomy of the ts 56 mutants but is not precise enough to identify a non-autonomous mutant une-quivocally. In order to prove non-autonomy within this scheme, methods must be devised to exclude the production of mosaics in tissues where mutant ac-tivity is not lethal at 29 °C, A more difficult task is the elimination of survival of mosaics made possible by "Durchbrenner"effects, By choosing ts lethals that are not influenced by changes in genetic background and whose tissue specificity is known, non-autonomous behaviour can be detected i f mo-saic patches are found in tissue requiring functioning of that locus. However, this study has proven fruitful in other respects. It has shed light into the tissue specificity of some ts mutants. With a more detailed investigation of the occurrence of mosaic patches in mutants such as E46 and E88, the tissue affected by..a given lethal may be pin-pointed more exactly. 57 SUMMARY The survival of mosaic patches of tissue at 29 °C cannot be used as a valid criterion for the non-autonomous behaviour of sex-linked recessive temperature-sensitive lethal mutants. However, the relative degrees of autonomy of the mutants were determined after considering the relative viability ratios of mosaics and non-mosaic fe-males, the XO survival frequencies, the lethal periods, and the temperature-sensitive periods. Those thought to be autonomous are ET34, E45, E46, E82 , E88, and X8, E46 and X8 are definitely.autonomous in cells of the thorax, wings and legs, since no mosaic patches appear in these tissues at 29°C. E76 is not as strict an autonomous lethal as the preceding ones while E7 is thought to function autonomously in internal tissue only. E2/\ on the other hand, is thought to act autonomously in external tissue. E25 is another lethal acting only in internal tissues but not as effectively as E7_. The sexual dimorphic ts mutant, E5_, functions autonomously according to its sexual dimorphic nature. Of all the ts mutants studied, E94 appears to be the least autonomous. The apparent non-autonomous character of E£ can be .explained by a high XO survival frequency. Therefore, although it cannot be said definitely that any of the ts mutants act non-autonomously, their potency as lethals in mosaic patches varies. 58 BIBLIOGRAPHY Baillie, D., D. T. Suzuki and H. Tarasoff, 1968. Temperature-sensitive mu-tations in Drosophila melanogaster. II. Frequency among second chromosome recessive lethals induced by ethyl methane-sulfonate. Can. J. Genet, Cytol. 10: 412-420. Bodenstein, D., 1950. The postembryonic development of Drosophila. In "Biology of Drosophila", ed. M. Demerec, pp. 284-286, Hafner Publishing Co., New York. Bonner, J. T., 1947. Evidence for the formation of cell aggregates by chemo-taxis in the development of Dictyostelium discoideum. J. Exptl. Zool. 106: 1-26. Demerec, M., 1934. Biological action of small deficiencies of X-chromosome of Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. 20: 354-359. Ephrussi, B., 1934. The absence of autonomy in the development of the effects of certain deficiencies in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. 20: 420-422. Epstein, R. H.A Bqlle, CM. Steinberg, E. Kellenberger, E. Boy De La Tour, R, Chevalley, R. S, Edgar, S. Susman, G. H. Denhardt, and A. Lielausis, 1963. Physiological studies of conditional lethal mutations of bacteriophage T 4 D . Cold Spring Harbour Symp. Quant. Biol. 28: 375-392. Hadorn, E., 1951* Developmental action of lethal factors in Drosophila. Advan. Gen. 4: 53-85, Hannah, A., 1953. Non-autonomy of yellow. J. Exptl. Zool. 12J3: 523-560. Hinton, C. W., 1955. The behaviour of an unstable ring chromosome of Droso- phila melanogaster. Genetics 40: 9 5 I - 9 6 I . Jockusch, H., 1966. Relations between temperature sensitivity, amino acid replacements, and quaternary structure of mutant proteins. Biochem. Biophys. Res. Commun. 24: 577-583. Landsley, D. L. and E. H. Grell, 1968. Genetic variations of Drosophila  melanogaster. Carnegie Inst. Wash. Publ. 627. 59 Morgan, T. H., and C, B. Bridges, 1919. Contributions to the genetics of Drosophila melanogaster. I.. The origin of gynandromorphs, Carnegie Inst. Wash. Publ. 2?8: 1-22. Naona, S. and F. Gros, 196?. On the mechanism of transcription of the lambda genome during the induction of lysogenic bacteria, J. Mol. Bio. 25J 517-536. Oster, I. and F. H. Sobels, 1956. "Natural implantation" of a lethal muta-tion in Drosophila melanogaster. Amer. Nat, 9_0_! 55-60. Parsons, P. A,, 1959. Genotypic environmental interactions for various tem-peratures in Drosophila melanogaster. Genetics 44: 1325-1333. Poulson, D. F,, 1945. Somatic mosaics and the differentiation of imaginal discs in Drosophila. (Abstract) Genetics 30*-17. Ritossa, F. M,, K. C. Atwood, and S. Speigelman, 1966. A molecular explan-ation of the bobbed mutants of Drosophila as partial defi-ciencies of "ribosomal" DNA. Genetics $±i 819-834. Stern, C, 1934. The effects of yellow-scute gene deficiency on somatic cells of Drosophila. Proc. Natl. Acad. Sci, U. S. 21:374-379. Stern, C., 1936. Somatic crossing over and segregation in Drosophila mela-nogaster. Genetics 21: 625-730. Sturtevant, A. H., 1932. Use of mosaics in the study of developmental effects of genes. Proc. 6th Intern. Congr. Genet. Ithaca, N. Y, 1: 304-307. Sundaram, T. K. and J, R. S. Fincham, 1967. Hybridization, between wild-type and mutant Neurospora glutamate dehydrogenase in vivo and in vitro. J. Mol. Biol. 2£: 433-439. Sussman, M. and F, Lu, 1955- Interactions among variant and wild-type strains of cellular slime moulds across thin agar membranes. Proc, Natl. Acad. Sci. U. S. 4 1 : 7O-78. Suzuki, D, T., 1969. Temperature-sensitive mutations in Drosophila melano-gaster. V. Genetic positions, and viability indices of sex-linked recessive lethals. Manuscript to be submitted to Genetics. 60 Suzuki, D. T., L. K. Piternick, S. Hayashi, M. Tarasoff, D. Baillie, and U. Erasmus, 1967, Temperature-sensitive lethal mutations in Drosophila melanogaster. I. Frequency among sex-linked reces-sive lethals induced by different mutagens. Proc. Natl. Acad. Sci. U. S. 2£i 907-912. Suzuki, D, T. and D. Procunier, 1969. Temperature-sensitive mutations in Drosophila melanogaster. III. Dominant lethals and semi-lethals on chromosome 2. Proc. Natl. Acad. Sci. U. S. 62: 369-376. Tarasoff, M., 1968. The use of conditional lethals in the analysis of devel-opment of Drosophila melanogaster. A thesis submitted in par-t i a l fulfilment of the requirements for the degree of Masters of Science in the Department of Zoology, the University of British Columbia, 

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