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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 TEMPERATURESENSITIVE LETHAL MUTANTS IN DROSOPHILA»MELANOGASTER by B.Sc,  SHIZU HAYASHI 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 an a d v a n c e d the  this  degree  Library shall  I further for  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  that  it  permission  purposes  may be  representatives. thes,is  for  freely  It  financial  for  by  the  understood  gain  of  /^yuX^  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  shall  LJuMiWtt"  requirements  reference copying  of  I agree and this  be  copying  or  allowed  without  for that  Study. thesis  Head of. my D e p a r t m e n t  that  not  l^d^tf Columbia  for  extensive  permission.  Department  Date  available  granted  is  British  the  or  publication my  ii ABSTRACT The autonomous behaviour of sex-linked recessive  temperature-sensitive  lethal mutants in Drosophila melanogaster could be demonstrated by the presence 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-autonomous.  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 affected 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 frequencies, lethal periods, and temperature-sensitive periods,have been placed on  iii 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 TABLE OF CONTENTS Page INTRODUCTION  1  METHODS AND MATERIALS  7  RESULTS  22  DISCUSSION  35  SUMMARY  57  BIBLIOGRAPHY  58  LIST OF TABLES Table  Page  Ia  General genetic properties of stocks tested for temperaturesensitivity  9  Ib  Description of stocks tested for temperature-sensitivity  8  II  Number i n each class of offspring of the cross J X wvC/dl-49 <M  v.. ts. . A d d  1  21  III  Ratio of the number of w ^-bearing females and exceptional males to the number of y. .ts. ./dl-4-9 females  23  IV  Ratio of the Relative Viability Ratios of w -bearing females (21.5°C/29°C)  25  V  TSP and LP of the different mutants tested  VI  Data collected i n the scoring of "shift" cultures of y. E25  VII  Description of y..ts.. mosaics surviving at 29°C  v  vC  31 30 41  y,i.  LIST OF FIGURES i  Figure  Page  1  Crosses made for different aspects of the study  2  Crosses carried out for a determination of the autonomy of  16  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  vii 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 o f a gene i s d e f i n e d  as autonomous i f the  oped In a g i v e n t i s s u e r e f l e c t s the g e n e t i c  i s not  phenotype d e v e l -  c o n s t i t u t i o n of that t i s s u e  i n f l u e n c e d by the genotypes o f n e i g h b o u r i n g c e l l s .  and  Non-autonomous g e n e t i c  b e h a v i o u r , on the o t h e r hand, i s i n d i c a t e d when a c e l l u l a r phenotype does  c o r r e s p o n d t o the g e n e t i c  not  c o n s t i t u t i o n o f the c e l l s which produce i t owing t o  an i n f l u e n c e o f 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  gene f u n c t i o n a r e g e n e r a l l y s t u d i e d  (Hadom  1951).  These a s p e c t s of  i n h i g h e r organisms by s u r r o u n d i n g c e l l s  or t i s s u e o f the genotype under i n v e s t i g a t i o n w i t h c e l l s o f a d i f f e r e n t geno-  type.  The  d e f i n i t i o n o f autonomy i m p l i e s t h a t phenotypes can be i n f l u e n c e d  the a c t i o n o f s u b s t a n c e s produced by genes o f a d i f f e r e n t genotype.  ence o f such a substance has  been demonstrated by Sussman and  the mechanism o f i t s a c t i o n remains s p e c u l a t i v e .  substance d e s c r i b e d  e a r l i e r by Bonner  (19^?) a s  type myxamoeba o f the s l i m e mould D i c t y o s t e l i u m  an a g a r membrane and  wild type centres  cause a g g r e g a t i o n l e s s  of aggregation.  Lu  The  (1955)  They showed t h a t a  by  exist-  although  diffusible  a c r a s i n , produced by the  wild  d i s c o i d e u m , c o u l d pass through  mutants t o c l u s t e r d i r e c t l y  opposite  However, t h e y found t h a t d i f f e r e n t , p a i r w i s e  combinations o f m o r p h o g e n e t i c a l l y d e f i c i e n t v a r i a n t s , each o f which cannot  com-  2 plete the normal developmental sequence i n 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, i n general, the sex and phenotype of thirty different sexlinked characters i n gynandromorphs produced by the occasional elimination of one of the X chromosomes from a female cleavage nucleus, were completely autonomous in development.  Later, Sturtevant (1932), studying mosaics produced by  chromosome elimination caused by M(l) -n (see Lindsley and Grell 1968) found 2  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 segments, they arrived at different conclusions.  Demerec claimed that the lethal  expression of deletions was autonomous in development, whereas, Ephrussi recovered 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 questioned 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 X ^ chromoc  some which i s 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 ( 1 9 5 3 ) . 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 nonautonomous 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 transplanted" into wild type hosts, Hadorn (1951) bas concluded that the phenotypic behaviour of. the mutants i s influenced by the followings l ) phase specificity of the mutant, referring to the different times in the development of the organism 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 genotypic milieu, sex, temperature, nutritional conditions, and other environmental influences,  A l l these studies point to the complex nature of c e l l to c e l l  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 i n which the locus i s 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 ° , in somatic cells (Hinton 1955) i s used to generate 2  patches of tissue in which recessive mutants carried on the homologous X chromosome are expressed due to the loss of the corresponding wild type alleles when the ring X i s 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 i s non-autonomous or i s 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 i s 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 a l l e l e influenced 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 sexlinked 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 genetic 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 w i l l 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  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  E88  y sc cv v  E94  yy sc  c?  <?••  d"  homozygous  X8  y  homozygous  M10  y  d>  6IV ES  EIII E52,  y cv  homozygous  9 Table l a General genetic properties of stocks, tested for temperature-sensitivity  mutant  mutagen  . v i a b i l i t y index* i21.5°C 29°e  viability regional , at 29°C 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  0.08  at sn  E45  EMS  homo=60.3  homo»0.0  v-f  B46  EMS  homo=35»6  homo=0.0  0.98  MS  0.17  0.0  car-1  E82  EMS  0.77  0.0  at cn,  E88.  EMS  (0.54  0.0  at wy  E94  EMS  0.92  0.0  cv-sn  E76  3  •  •S'  ... - hot-de.terirlined .... ... .i.  X8  <^-rays  M10  Mitomycin p 6IV BS EMS EIII E52 EMS 4  homo=47.0  homo=«0.0  right of y  0.42  0.0  f-car,  0.13  0.0  g-f  0.75  0.0  V.-1JLY.  j .  •/  not ts  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 manner except at different temperatures.  2  Example:  3  Males not f e r t i l e at 29°C  v-sn means that the mutant is located betwen.y. and .sn  4.. Homozygous females not f e r t i l e 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 i n males by mating to females carryint 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 i n Tables Ia and Ib (mode of origin, v i a b i l i t y indices, > genetic positions, markers on the ts chromosomes).  II. Determination of autonomy The unstable ring X chromosome, In (l) X , w , c2  vC  (referred to as w^p),  was generated by irradiation, with X-rays, of the ring chromosome, R(l)2, containing the inversion, In (l) w  vC  (Hinton 1955). The resulting chromo-  vC some, _w_ t i s characterized by variable degrees of instability which produces gynandromorphs, XO males, and dominant lethals i n progeny of w^-rbearing females,  w_ -bearing males are sterile and the ring i s maintained i n females  balanced over the In (l) dl-49. y w l z chromosome (referred to as dl-49) s  Q  contributed by f e r t i l e males i n the stock.  The males also carry the sc • Y  11  chromosome which includes the entire Y chromosome and the t i p of the X i n cluding l ( l ) J l ' , j , and s c . f  +  +  Virgin w ^/dl-49 females raised at room temperature and less than 3 v  days old were mated to y..ts... males also raised at room temperature. 20 pairs were allowed to mate and lay eggs f o r 5 days at 29°C i n quarter pint bottles containing standard Drosophila medium. The same procedure was car-: ried out at 21.5°C.  The  f l i e s were then discarded and the F  to develop at these temperatures.  1  f l i e s allowed  A l l viable progeny including those that  adhered to the medium or remained i n the pupal cases were counted and classified.  Dead unhatched pupae were classified i n the 29°C cultures by dis-  secting them to determine the phenotype of the developing imago. Any morvG / phologlcal abnormalitiesiin the w_ /y..ts., females, whether they were stuck to the medium, dead, or unhatched were noted. In crosses of w /dl-49 $ x y..ts../Y vC  primary non-disjunction i n  females generated w /dl-49/Y females which are phenotypically similar to vC  w /y..ts.. females. vC  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 i n 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 d l - 4 9 chromosome was in fact carried. If this chromosome was detected, the females were classified as primary exceptional females.  However, most of these females were sterile.  They were i n -  cluded i n 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. likewise and classed as products of primary mented Malpighian tubules or testes. y..ts.. mosaics.  These were dissected  non-disjunction i f they had unpig-  Otherwise, these were considered to be  At 21,5°C mosaic females were classed as y..ts..-bearing  females unless patches of w l z tissue occurred. A l l other non-y females s  which did not show mosaic patches under a dissection microscope at 25X magnification were, at both temperatures, classed as u regards external characters.  -bearing non-mosaics as  Henceforth, females manifesting external mosa-  icism w i l l be referred to as mosaics while those females bearing the w  vC  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,  w Vdl-49 females were mated to y sc cv v f car/Y v<  males and to y cv v f/B Y y* males in the same manner as described above to S  measure the survival of non-lethal mosaic and non-mosaic females.  They were f  bfrVB^ Y males to measure these frequencies with a non-ts  also mated to w  e  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 i n 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  w Vdl-49/Y females resulting from primary non-disjunction v{  ,>  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/B Y y eft? x w /dl-49 S  +  vC  21.5°C and 29 °C  score w /dl-49/B Y y+ $| ,w/y_ cv v f 2 ; y cv v f/dl-49 $; y cv v f / 0 <? n-ritnaV-us ' ' primary primary exceptions exceptions wild type.§$ with mosaic patches (nonr-mosaic)' (mosaic)' vC  S  V c  B  B. Controls to determine regular mosaic and non-mosaic frequencies y sc cv v f car/Y dtf  w /dl-49 vC  x  .  .  21.5°C and 29°C  score  ^w^/y sc cv v f car^ £ v  mosaic  y sc cv v f car/dl-49 $  non-mosaic  C. Test of a non-temperature-sensitive autonomous, lethal r  w b-b/BY dB..::v . w /dl-49 9$ e  1  S  ,  vC  _21.5°C and 29°C score  /we , wvC / bb. 1 $,, 7jion-mosaic)  w bbVdi-49 9 e  15 Figure 2 Cross carried out for a determination of the autonomy of each temperature-sensitive mutant  y..ts../Y dS  1  x  w /dl-49 9? vC  21.5°C and 29°C  score  y. .ts../dl-49 $j  ^w  v  $$ with ^ t i s s u e (mosaic)  /y..ts.. ^;  y..ts  '..completely wild type (non-mosaic)  primary exceptional 9?  others  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  21.5°C  x FM-6/y $ 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 $ 29 °C no y..ts../Y ^  select y.,ts../Y ^ x select y..ts../y..ts 29 °C no survivors  17 mimic wX /y.»ts.. females, their frequencies of occurrence had to be estiG  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 detected. The frequencies of y..ts../0 exceptional males does not give an indication of the rate of primary non-disjunctional female production at permissive temperatures since XO males are produced at higher frequencies than XXY females in crosses involving w  vG  (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 estimate the number resulting from paternal non-disjunction. Summaries of a l l 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 sensitive period or TSP) and also of the time at which exposure to the restrictive temperature during the TSP manifests i t s e l f phenotypieally by death (the lethal period or LP), these periods were determined by the following procedure.  Groups of 50 - 100 pairs of f l i e s 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 periods.  hour  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 cultures of homozygous ts lethals were examined for the number of adults, for the presence of pupa cases containing dead f u l l y 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 f i 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 f i r s t 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 progeny. 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 I I Number iri each class of o f f s p r i n g of the cross: y. .ts../Y dtj x w ./dl-49 99 8  vC  dl-49 .non-mosaic • mosaic xo 1 Excepy..ts, others •total d tional y . t s . y. .ts,,. 9 9 a 206 1284 79 65stuck 94stuck . 835 5 8 y sc cv E5 b 9 10 7 3 c 10 377 171 67 99 816 3 a' 1760 22 386 2402 127 103 3 38 y sc E7 b 22 14 17 3 9 c 211 726 371 267 1674 3 86 a 62 431 30 1 47 510 29 6 y sc cv E9 v car b 11 2 21 12 44 41 c 146 336 706 13** 5 4 4 1221 a 92 956 109 : 52 12 10 y:.E25 b 16 152 63 546 166 4 c 78 270. 287 1355 80 a 18 6 1 893 163 1161 6 y sc E2? f car. b 14 6 7 c 364 84 1754 149 895 253 9 a 886 6 285 1276 29 51 9 y sc E34 b 104 31 9 48 9 ... . c 372 110 1680 750 149 293 7 2524 222 19 71 , 13 383 3233 2 6 6 11 8 y sc cv E45 b 494 222 70 150 5 73 1014 c 1241 8 a 110 302 1671 3 5 y sc cv v E46 >b .10 20 16 11 10 c 852 384 238 148 132 1709 15 a 874 61 14 44 126 1120 1 y sc E76 , b - 10 8 6 9 9 656 c 70 307 49 1276 187 5 a 76 1465 37 •I'dead 1153 3 195 2 14 49 y E82 v , b 25 25 290. 620 12 117 71 c 33 1203 a 1542 234 1922 1 125 15 5 y sc cv v E88 b 14 18 17 15 c 494 222 6 116.. 1134 179 117 a 100 154 2 1423 229 1913 3 y~sc E 9 4 b 17: 6 7 13 3 c 216 40 1399 731 254 158 a 194 2209 11 4-33 2865 15 y X8 b 23 30 87 19 28 1110 c 212 326 2444 4 303 487 2 187 9. 219 w bb /B Y b 6 -• 3 4 c 24 219 98 1073 732 a 86 556 921 136 65 65 3 6 y sc cv v f car b 18 9 6 13 c 210 562 216 8 75 147 1214 1045 a 106 .8 - 96 131 897 2284 y cv v f/B Y y b 18 12 6 13 1 77 c 206 188 560 1708 98 623 31 a a t 29°C b' unhatched pupa a t 29°C c- a t 21.5°C chromosome tested  1  4  a  e  1  S  +  T  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 w ^/dl-49 females at both 21.5°C and v  29°G (at both temperatures, a number of white opaque eggs which usually represent 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 investigation. 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 i n 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 w ^/y..ts.. mosaic and non-mosaic females to v  the number of y..ts../dl-49 females at 21.5°C and 29°C, respectively. degree of leakiness of the t§_ mutants when outcrossed was estimated by  The  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 non9* nosaic X0 r  y" sc cv E5  a .?0?8 , ;P95 t .276 .454 y sc E? a .058 .072 t .291 .511 y sc cv E9 v car a .087 .138 t .399 .435 a .054 .099 y E25 t .304 .495 y sc E27 f car a .020 .090 b .283 .407 y sc E34 a .033 .056 b .391 .496 y sc cv E45 a .028 .088 b .304 .449 y sc cv v E46 a .006 .089 b .279 .451 y sc E76 a .050 .070 b .285 .468 y E82 v a .032 .066 b .264 .468 y sc cv v E88 a .010 .081 b .362 ...449 y sc E94 a .070 .108 b .295 .347 a .005 .088 y b .273 .439 we bbVB Y a x 8  s  b  y sc cv v f car y cv v f/B Y y  * ** a b  +  a b a b  matro-mosaic non0** mosaic clin0** ous 9 . 7006 .078 .091 .169 .178 ,00£ .259 .429 .688 .01j .002 .056 .068 .124 .118 .004 .270 .486 .756 .085 .00; .086 .134 . .209 .01f .389 .410 .829 .131 .004 .004 .054 .095 .149 .526 .00? .286 .470 .756 .001 .00? .020 .086 .106 .166 .010 .268 .382 ;650 .007 .010 .033 .052 .085 .199 .009 .375 .471 .846 .008 .005 .028 .084 .112 .142 .010 .289 .424 .713 .002 .004 .006 .085 .091 .174 .018 .272 .426 .698 .016 .001 .047 .066 .113 .107 .008 .268 .443 .711 .001 .003 .031 .062 .093 .115 .019 .258 .443 .701 .001 . 0 0 3 .009 .077 .086 .237 .001 .338 .424 .762 .002 .001 .O67 .104 .171 - .270 .422 .692 .216 - .007 . 0 0 5 .084 .089 .191 .004 .252 .415 .666 - .048 .048 - .299 .299 .117 .005 .155 .117 .272 .134 .014 .374 .384 .758 .101 .008 .092 .125 .217 .152 .050 .302 .331 . . 6 3 3  not corrected for primary non-disjunction in females corrected for primary non-disjunction in females at 29°C at 21.5°C  24 the ratio of y,.ts.,/0 males to y..ts../dl-49 females at the two temperatures. These ratios w i l l 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). Furthermore, 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 i n the following manner; i f the frequency  of verified non-disjunctional females i n 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 f e males  (21.5°C/29°C)  chromosome tested  mosaic  nonmosaic  total •«  y sc cv E5  3.3  4.7  4.1  y sc E7  4.8  7.2  6.1  3.1  4.0  5.2  5.0  5.1  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  37.6  5.5  8.9  4.0  4.1  4.1  y sc cv E9 v car y E25 \y sc E2? f car  y sc cv v E88 y sc E94 v X8  50.4  average  15.9  w  e  -  bb /B Y 1  S  y sc cv v f car  2.4  y cv v f/B Y y  3.3  S  +  7.5 ,5.6  6.4  6.2  6.2  3.3  2.8  2.7  2.9  26  or 0.025 at 29°C and 21,5°C respectively, no change was made. However, i f i t was less than this figure, the difference was subtracted from the y..ts.. mosaic RVR. For non-mosaic females, the established control value was subtracted 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-disjunctional 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 i t varied from 3 - 9 in non-mosaic females and 3 - SI in mosaic females. These control values may simply reflect 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 to w  e  lethal gave a ratio of w /w bb vC  e  1  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 w /w bb females may indicate the vC  e  1  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 invC / 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 a l l 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 outcrossing (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 a l l 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 a l l 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 a l l surviving 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 i n value from +1 (which shows perfect positive correlation) to -1 (which shows perfect negative 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. r = ^(*-x) (y-,7) .—•• • )T(x-x)E(y-y)  Also,  where x and y are the means 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 0 20 44 68  results  50 50 50 50 50 45 50 30 86" 59 3t 30  40 live adults 42 live adults 43 live adults 45 live adults 92 46 live adults 111 7 live adults, few dead third instar larvae 116 30 live adults, few dead third, instar larvae 120 a l l dead third instar larvae 140 Ifew. live adults, dead third instar larvae,' dead late pupae, partly eclosed dead.adults, 152 _and dead adults 160 170 1 live adult, dead third instar larvae 541 fdead third instar larvae, dead early and late 175 50 Lpupae 185 188 50 /mostly dead third instar larvae, some dead 190 pupae, dead partly eclosed. adults, dead adults 75j 100 mostly dead late pupae ;;212 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 up(hr.) laid 0 50 20 40 44 40 68 4b1  results  dead late pupae dead late pupae dead late pupae [dead adults, dead partly eclosed adults, dead 92 (late pupa 32J 111 40 0 live adults, dead adults and pupae 116 6 live adults, dead adults and pupae 30 120 76 19 live adults, dead adults and pupae 140 7 live adults, dead adults and pupae 35 152 6 live adults, dead adults and pupae 38 160 24 live adults, dead adults arid pupae 38 170 22 live adults, dead adults and pupae 32 43 25 live adults, dead adults and pupae 175 I85 29~live adults, dead adults and pupae 39 188 30 11 live adults, dead adults 190 31 live adults, dead adults 39 212 16 live adults, dead adults. 30 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  .LP  TSP  99 s indispensible dtf'i 150 hours (pupa) to adult 25 to 70 hours sc E7 (first instar larva to third instar larva) 70 to 180 hour sc cv E9 v car (third instar larva to late pupa) 92 t o l l l hours (end of third instar E25 larva stage) 60 to I65 hours sc E27 f car (third instar larva to mid-pupa) 50 to 80 hours, sc E34 (second instar larva to third instar larva) 0 to 140 hours (egg to mid-pupa) sc cv E45  y sc. cv E5  late third instar to adult  y  first instar larva to adult  y y y y y  y sc cv v E46  0 to 120-:.hours (egg to early pupa)  y sc E76  0 to 240 hours (indispensible)  y E82 v y sc cv v E88 y sc E94 y X8  60 to 140:-hours (third instar larva to mid-pupa) 80 to 150 hours (third instar larva to mid-pupa) 28 to 125 hours (first instar larva to early pupa) 82 to 97 hours (during late third instar larva)  prepupa to adult late third instar to adult third, instar larva to late pupa third instar to adult second-third larval intermolt to adult first instar to eclosion first instar to adult second-third larval intermolt to eclosion third instar to late pupa first and second instar larva, pupa, adult 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 i s 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 i s 2.201.  Therefore, at the  Z . %  level of significance, the null hypothesis that there i s no correlation can be rejected, meaning that XO survival i s related to viability of mosaics. Besides the penetrance of the ts gene, i t s 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 i s 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 i n a number of ways. Although control shift experiments using the non-ts, y sc cv v f car/Y stock, were set up i n 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 correlation between the length of the TSP and the magnitude of the decrease encountered by the mosaic class due to the temperature difference as measured by RVR '5°C/ 29°C 21  RTO  #  ^  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  corre  xation  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 influence 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-autonomous 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 temperaturesensitive lethal mutations. Previous studies, of autonomy in Drosophila melanogaster 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 frequencies 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, i t 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 activity 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 temperatures during its TSP may be the result of non-autonomy. Yet, i t 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 during 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 i s required. the use of a ts mutant i s no more advantageous.  In this respect,  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 d i f f i c u l t i e s in evaluating the results w i l l be outlined i n i t i a l l y .  A major problem arises  because the mere presence of mosaic patches at 29°C does not necessarily i n 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  w i l l be considered and i t w i l l 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, munication).  personal com-  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 i s infrequent and the loss generally involved the  39  entire ring-X (including the locus of the t s gene). +  Moreover, since the  f i n a l 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, i s 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  f l y , non-autonomy of the lethal, could be suggested by surviving mosaic tissue. Also, i f somatic recombination i s independent of temperature, the contribution 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 mosaics.  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 determined. It was expected that the longer the TSP, the greater would be the i  vC chances of w  loss during that interval.  Thus, i t was anticipated that  there would be a slightly decreased frequency of mosaic survival with nonautonomous 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 f i f t h and sixth abdominal tergite yellow, genitalia mosaic 7. scutellar bristles missing, genitalia mosaic 8. l e f t half of abdominal tergites yellow, missing scutellar bristles y sc cv v E88 1. l e f t legs yellow, genitalia mosaic* 2. l e f t 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 f i r s t , second and third abdominal tergites 6. l e f t half of head yellow, l e f t eye vermilion, f i f t h and sixth abdominal tergites yellow, genitalia mosaic 7. l e f t 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 exceptional 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 required only in certain tissues during the TSP; mutant tissue not requiring activity of the ts. gene at this time w i l l survive and yield mosaics.  There-  fore, only when i t i s known i n which tissue the ts mutant i s lethal during the TSP w i l l the TSP be of great significance in understanding autonomy. Thus, i n these studies, only when total absence of mosaics or. complete absence of one type of tissue displaying mosaicism i s demonstrated, can a mutant 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 examples 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 d i f -  ferent mechanism such as the loss of the ts mutant by somatic crossing over. Hinton  (1955)  loss, of the w  vG  established that XO males result primarily from early somatic chromosome from x/w  vG  zygotes. Therefore, the occurrence of  a somatic double exchange i n the earliest stages of cleavage before or at the time.of w_j elimination, could result in replacement of the ts. lethal by i t s 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, i f 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 w^ loss, v  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 a l l 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 periods. 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 sibling In(l) dl-49/(X-chromosome from the male) females, and since these relative viability ratios (RVR) were used in further calculations, i t is important 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 a t optimal temperatures. demonstrated  Parsons (1959)  such decreased v i a b i l i t y by t e s t i n g the e f f e c t of 31 °C as com-  pared with 24°C on various wild type stocks and t h e i r F^ hybrids.  Therefore,  i n the present experiment, the r a t i o s at 29°C would y i e l d an overestimate of mosaic v i a b i l i t y and tend towards c l a s s i f i c a t i o n of a mutant as non-autonomous. In f a c t , the r a t i o s a t 29°C of both mosaic and non-mosaic classes were greatly reduced over that a t 21.5°C.  Thus, even i f the r a t i o were overestimated at  29°C, decreased v i a b i l i t y was indicated.  I f temperature had no e f f e c t on  the denominator, then t h i s r a t i o would have been much smaller.  Since i t i s  of interest to look a t the amount of decrease encountered at the higher temperature, i t should be kept i n mind that the magnitude of the decrease measured would be minimal by  these c r i t e r i a .  Therefore, even i f temperature d i d  affect.the v i a b i l i t y of the I n ( l ) dl-49/(X-chromosome from the male) female, the inferences drawn from the r a t i o s would be conservative but reasonable. Comparison between RVR of d i f f e r e n t ts's are a d i f f e r e n t matter.  In  these cases, differences i n heterozygote v i a b i l i t i e s i n each cross would a f f e c t the ultimate r a t i o s compared.  Although adequate tests of d i f f e r e n t i a l  heterozygote v i a b i l i t y i n d i f f e r e n t crosses were not carried out, there are no compelling reasons f o r expecting severe differences between the crosses.  46 Thus, i n the discussion which follows, i t will he assumed that v i a b i l i t y 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 attributed 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. reduced v i a b i l i t y of f l i e s peratures.  The decrease may result from a  mosaic for male and female tissue at higher tem-  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 i s of internal mosaicism.  The degree of internal mosaicism which i s 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 f l i e s mosaic both externally and internally at 29°C, whereas the decreased numbers in the non-mosaic class reflect 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 w  e  bb^/w ^ females recovered i s a measure of females which are non-mosaic i n v(  both internal and external tissue i f i t i s assumed that loss of bb activity +  results in lethality at any time in any c e l l .  Since Ritossa et. a l .  (1966)  have shown that the bb locus directs the synthesis of ribosomal RNA, i t i s highly probable that this assumption i s indeed valid.  The ratio between the  two classes i s about 0 . 3 0 at 21.5°C, so i t can be said that about three times out of ten, w ^/bb^ females have few i f any cells in which the ring i s lost v  in tissue v i t a l for v i a b i l i t y .  In other words, seven out of ten developing  w^p/bb3 females suffered ring loss in v i t a l tissue and therefore were lethal mosaics.  vC The ratio of external mosaic to non-w -bearing females as shown  by control experiments, i s 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 resulting 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 i s 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 individuals in which the ring-X i s lost at some stage. The results obtained from tests of the autonomous mutant, bb^, also prdvide values of the viability ratios against which the ts values may be compared. The decreases of the RVR of the w /w bb females at 29 °C compared vC  e  1  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 w -bearing females vC  at 29°C plus death due to mosaicism for an autonomous non-ts lethal. erage decrease of 6.4 times was found for a l l y..ts.,/w a value very similar to the bbj- decrease.  vC  An av-  females at 29 °C,  If bb^ i s 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 autonomous? 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 theThe viability ratios of the. ts mutants. following mutants yielded relative ratios of w  v  -bearing females  49 (21.5°C/29°C) greater than the ratio observed in w /bb females ( 6 . 3 ) and vC  1  therefore were considered to be autonomous: EJ4 (10.0), E82  ( 7 . 5 ) .  E88  ( 8 . 9 ) ,  and  E4_5_ ( 6 . 4 ) ,  E46  ( 7 . 7 ) ,  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 a l l 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, i t 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 i n internal tissue.  Such speculation seems reasonable  when i t i s noted that the lethality of the non-mosaic class i s greater than average.  If this i s true, then ts lethality of E82 i s 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 reduced 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 i s negligible (0.001 at 29°C).  While the  TSP for E46 i s 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 i s 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 w i l l eventually be located on the external surface.  Whether the similarity between these mutants i s 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 i s another autonomous mutant which i s more active in external tissue (the mosaic class was reduced by 5 0 ^ 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 f u l l y 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 i n external morphology were noted  vC in the w  -bearing females.  Since i t cannot be disputed that this gene i s  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? w C loss after the very specific TSP at the end of the third"larval instar y  52 is a reasonable explanation.  This assumption i s strengthened when i t i s  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 i s also autonomous. The decrease in mosaic and non-mosaic classes at 29°C are 10.3 and 5.1 ively, so this lethal appears to be more active in external tissue.  respectThe  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. phenotype i s a temperature-sensitive  This  phenomenon, but how i t i s related to  the ts lethal i s 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). tional was noted from the results of E?6.  Nothing excep-  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 explainsthe survival of mosaics.  This mutant is temperature-sensitive  at a l l  53 times so the XO males that do pupate are a l l 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 a l l be conjecture. However, i t can definitely be stated that E? functions autonomously 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,  i t cannot be said  that these mutants are non-autonomous. E27/w ^ females are 5.9 times less v(  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 a l l 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 i n the Malpighian tubules i n individuals developing a t  29 ° C The mosaic and non-mosaic classes with E2_5 are reduced to a s i m i l a r extent a t 29°C (5.3 and 5,0 respectively) to give a total.reduction of the vC v r j - b e a r i n g 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 i n  t h i s case.  Mosaic s u r v i v a l does not r e f l e c t XO breakthrough a c t i v i t y which  i s n e g l i g i b l e i n t h i s case (0.004 a t 29°C as compared with O.526 a t 21,5°C).  The. very l i m i t e d TSP of about twenty hours at the end of the t h i r d l a r v a l i n s t a r  may account f o r s u r v i v a l of females with mosaic patches i f w^  a f t e r t h i s period.  be mosaic.  l o s s occurs  I f t h i s were so, r e l a t i v e l y small patches of tissue should  However, viable mosaics i n v o l v i n g more than half the whole organ-  ism are recovered.  Therefore, i t can be argued that l i k e E3_4, E 2 5 i s auton-  omous and more a c t i v e i n i n t e r n a l t i s s u e , but unlike EJ34 and E82, i t s period  of a c t i v i t y i s short-lived so that i t s l e t h a l - i n f l i c t i n g , a b i l i t y i s c u r t a i l e d .  The sexually dimorphic ts mutant, E5, has a TSP from I50 hours to the  adult stage i n males and continuously i n females.  Decreases i n v i a b i l i t y of  E5 were not much greater than those found i n the controls, as might be ex-  pected since the male TSP occurs very l a t e i n 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 a l l 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 nonmosaic class. External mosaicism was detected throughout the body. The decreased 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 i t 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 excessive leakiness of the mutant upon outcrossing. The above discussion demonstrates that a study of somatic mosaicism resulting from unstable ring loss can indicate the relative autonomy of the ts  56 mutants but i s not precise enough to identify a non-autonomous mutant unequivocally.  In order to prove non-autonomy within this scheme, methods must  be devised to exclude the production of mosaics in tissues where mutant act i v i t y i s not lethal at 29 °C,  A more d i f f i c u l t task i s 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 i s known, non-autonomous behaviour can be detected i f mosaic patches are found in tissue requiring functioning of that locus. However, this study has proven f r u i t f u l in other respects. light into the tissue specificity of some ts mutants.  It has shed  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 females, the XO survival frequencies, the lethal periods, and the temperaturesensitive 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 a l l 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 i t 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 mutations in Drosophila melanogaster. II. Frequency among second chromosome recessive lethals induced by ethyl methanesulfonate. 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 chemotaxis 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, C M . 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 T4D. 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 Drosophila 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 mutation in Drosophila melanogaster. Amer. Nat, 9_0_! 55-60. Parsons, P. A,, 1959. Genotypic environmental interactions for various temperatures 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 explanation of the bobbed mutants of Drosophila as partial deficiencies 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. 21:374379.  Stern, C., 1936. Somatic crossing over and segregation in Drosophila melanogaster. 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 melanogaster. V. Genetic positions, and viability indices of sexlinked recessive lethals. Manuscript to be submitted to Genetics.  60 Suzuki, D. T., L. K. Piternick, S. Hayashi, M. Tarasoff, D. Erasmus, 1967, Temperature-sensitive lethal Drosophila melanogaster. I. Frequency among sive lethals induced by different mutagens.  Sci. U. S. 2£i 907-912.  Baillie, and U. mutations in sex-linked recesProc. Natl. Acad.  Suzuki, D, T. and D. Procunier, 1969. Temperature-sensitive mutations in Drosophila melanogaster. III. Dominant lethals and semilethals on chromosome 2. Proc. Natl. Acad. Sci. U. S. 62:  369-376.  Tarasoff, M., 1968. The use of conditional lethals in the analysis of development of Drosophila melanogaster. A thesis submitted in part 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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