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A study of a dominant suppressor of the purple eye-color mutant in Drosophila melanogaster Firth, James Dawrant 1985

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A STUDY OF A DOMINANT SUPPRESSOR OF THE PURPLE EYE-COLOR MUTANT IN DROSOPHILA MELANOGASTER by JAMES DAWRANT FIRTH B . S c , The University of Bri t i sh Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to tke^required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1985 © James Dawrant Firth In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Department of Date &£'S~. /fU~. DE-6(3/81) ABSTRACT The subject of this study is a new dominant suppressor mutation Su(pr) which acts on the purple eye-colour mutant (pr) of Drosoph11 a  mela.noga.ster. The Induction of Su (pr) was original ly associated with the synthesis of a compound-2_: chromosome In SD72/cn b_ females. The suppression of p_ was f i r s t observed in combination with a homologous p_-bearing compound-2_ chromosome. Suppressed-px f l i es appeared to have a ful ly wild eye phenotype. The intention of this study was to determine the chromosomal constitution necessary for Su(pr) induction, and to map the suppressor s i t e . To do th i s , many compound-2_ chromosomes were synthesized from several combinations of standard seconds. It was found that SD72 must be present to produce a suppressing compound-2_. The SD72 second carries a pericentric inversion that results in a duplication of 2_ heterochromatIn, and an associated deficiency of 2_ heterochromatin in the compound-2T3 S u (p r) chromosome. Suppression, therefore, Is associated with the pericentric inversion found only on SD72. The role of this segmental aneuploidy was studied by detaching several C(2L)pr;  C(2R)SD72/,cn bw suppressed strains such that both arms of the Su(pr) compound autosome were recovered independently and established in standard s t r a i n s . Suppressing and non-suppressing detachment products were recovered with a frequency that varied according to the compound-2R Su(pr) strain from which they were derived. The chromosome mechanics involved in the process of C(2R)SD72/cn bw formation and subsequent detachment implicates a l t e r a t i o n s to a segment of proxlmial 2R heterochromatin from SD72 in Su(pr) Induction. Loss of Su(pr) in the i ! i detachment process correlates predominantly with deletions generated In 2R heterochromatIn. Recombination mapping relative to the two v i s ib le heterochromatic markers, I ight and rol led , revealed that Su(pr) I ies to the le f t of rolIed. SpectrophotometrIc measurements of eye pigments revealed that suppressed-px and suppressed-pru W f l i e s had pigment levels that exceeded the wild type. The lethal a l l e l e p r c 4 . was not found to be suppressible. iv TABLE OF CONTENTS Page Abstract it Table of Contents iv L i s t of Tables vi L i s t of Figures v i i i Acknowledgement ix Chapter 1. General Introduction 1 Chapter 2. Analysis of Compound Second Autosomes Introduction 11 Materials and Methods 19 Mutations and Chromosomal Rearrangements 19 Synthesis of Compound Autosomes 19 Cytological Analysis 22 Visual Examination for Su(pr) 22 Spectrophotometric Measurement of Eye Pigments . . . 23 Results and Discussion 24 Chapter 3. Analysis of C(2R)Su(pr) Detachment Products Introduction 48 Materials and Methods 59 Mutations and Chromosomal Rearrangements 59 Recovery of Detachment Products 59 Lethality Tests 59 Complementation Tests 63 Results and Discussion 64 V Chapter 4. Recombination Mapping of Su(pr) Introduction 94 Materials and Methods 98 Mutations and Chromosomes Used 98 Synthesis of Recombinant Chromosomes 98 Testing Recombinants for Su(pr) 98 Results and Discussion 101 Chapter 5. Summary 112 Literature Cited U8~ ' Appendix 123 v i L I S T OF TABLES T a b l e P a g e C h a p t e r 2 1. Description of second chromosome mutations used 21 2. Description of compound second chromosomes used 21 3. Drosopterin levels in unsuppressed C(2L) strains 35 4. Drosopterin levels In C(2R)VK43.SD72/cn bw strains 37 5. Drosopterin levels in C(2R)VF5.SD72/cn bw strains 38 6. Drosopterin levels in C(2R)VF10.SD72/cn bw strains 39 7. Drosopterin levels in C(2R)VF30.SD72/cn.bw strains 40 8. Drosopterin levels In C(2R)VF12.SD72/cn bw strains 42 9. Drosopterin levels in C(2R)VF3.SD72/+ strains 43 C h a p t e r 3 1. Description of second chromosome mutations and chromosomal rearrangements 60 2. Recovery of detachment products from C(2R)Su(pr) 66 3. Recovery of suppressing and non-suppressing detachments . . . 67 4. The establishment of detachment-bearing stocks 69 5. Lethality tests of detachment products 72 6. Heterochromatic deletions carried on detachment products . . . 74 7. The retention of Su(pr) on detachments not carrying heterochromatIc deletions 76 8. The retention of Su(pr) in the presence of heterochromatic deletions 78 9. The retention of Su(pr) on detachment products bearing deletions of 2J_ 79 v i i Table Page 10. The retention of Su(pr) on detachment products bearing deletions of 2RJ] 81 11. A G-statisticaI test for the retention of Su(pr) in the VD3 deletion classes 82 Chapter 4 1. Description of second chromosome mutations used 99 2. Recombinants recovered from detachment products 102 3. Test of b_ r_L recombinants for the presence of Su (pr) 103 4. Test of H± ii recombinants for the presence of Su(pr) . . . . 105 5. Test of t 11 recombinants for the presence of Su(pr) 107 6. Disparate recovery of suppressing and non-suppressing recombinant classes 108 7. Test of l i t recombinants for the presence of Su(pr) 110 8. Test of b_ H rJ recombinants for the presence of Su (pr) . . . 111 v i i i LIST OF FIGURES Figure Page Chapter 2 1. Diagram of the adult compound eye and of a single ommat i d i urn 15 2. (A) Synthetic pathway for the drosopterins and sepiapterin (B) Chemical structures of the common drosopterins of Drosophila 18 3. Photomicrograph of the distal region of a polytene chromosome preparation of C(2R)VF5.SD72/cn bw 26 4. Photomicrograph of the proximal region of a polytene chromosome preparation of C(2R)VF5TsD72/cn bw 28 5. Absorbance of drosopterln extracts from 450 - 550 nm 34 Chapter 3 1. Alternate configurations of chromatid breakage and reunion in C(2R)SD72/cn bw synthesis 51 2. Detachment product classes possible from the C(2R)SD72/cn bw configuration shown in Figure 1(A) 54 3. Detachment product classes expected from the C(2R)SD72/cn.bw configuration shown in Figure KB) 58 4. Genetic map of the centric region of chromosome-2 62 5. Deletion classes recovered In detachment products 85 Chapter 4 1. Recombination products expected from suppressing detachments In combination with Tft p x c 4 i ± L i 97 ix ACKNOWLEDGEMENT I wish to sincerely thank Dr. David G. Holm for his valuable advice, support and patience throughout this project. 1 CHAPTER 1 GENERAL INTRODUCTION 2 Suppression occurs when the effects of one mutation are compensated for by a second mutation, such that the wild phenotype Is part ia l ly or f u l l y r e s t o r e d . 1 The study of suppression has provided valuable information on the mechanisms of mutagenesis and the function of the s t r u c t u r e s a f f e c t e d . S tudy ing the r e l a t i o n s h i p between two counterbalancing mutations can also identify functional re la t ionships between d i s t i n c t components of a genetic system. This has revealed several aspects regarding the normal control of gene expression. Examples of suppression In many different organisms have been reviewed by Gorini and Beckworth^ and Hartman and Roth.-^ Mutations may be suppressed intragenicaI Iy by additional changes to the base pair sequence. Missense and nonsense mutations can be suppressed by a second change within the mutated codon that creates an alternative t r i p l e t , coding for the correct amino ac id .^» 5 Frameshlft mutations can be suppressed by a second frameshlft that restores the proper reading frame. Mutations which occur outside the structural sequence of a gene, in the upstream control sequence, may prevent transcription by disrupting the promoter. In such cases, suppression may act at a transcriptional level by giving rise to a new promoter.^ Mutations may also impede normal translation if they occur in the polypeptide Initiator codon . Suppression at the translationaI level can occur when a second mutation creates a novel init iator codon.^ Some mutations which cause the loss of a protein's functional conformation, and therefore i t s a c t i v i t y , can be suppressed at the level of the polypeptide. Such a mutation occurs at the site of polypeptide chain interaction, preventing the protein from assuming its proper tert iary or 3 quaternary structure. The mutation at the residue that precludes normal Interaction may be counteracted by a change at a second residue that reinstates the normal folding.8, 9 jhe same mechanism has been shown to reinstate the functional conformation of doubly mutant tRNAs.10 Altered tRNAs are also involved in informational suppression. This Is a type of Intergenlc suppression in which the mutant gene s t i l l provides an altered mRNA, but the altered region Is misread by a mutant tRNA. The result is a functional protein.^ Mutant tRNAs can suppress nonsense^ and missense^ mutations through base substitutions in the anticodon loop. If a frameshift mutation is caused by the insertion of an additional base pair, then it can be corrected by a complementary tRNA containing a 4 base pair anticodon l o o p j 4 Few examples of Informational suppression have been found in eukaryotes. Suppressor tRNAs have been found for a l l three nonsense mutations in two species of Saccharomyces. S. cerevisiae and S. pombe. A possible nonsense suppressor that acts on s p e c i f i c a l l e l e s of many genes has been described in CaenorhabdItis  elegans (reviewed by K u b l P 5 ) . Suppressor tRNAs have not yet been confirmed in Drosoph iI a  melanogaster. Several suppressor stocks have been tested for deviation from wild type tRNA patterns by two-dimensional gel electrophoresis. The only aberration from wild type was seen in the dominant suppressor of  deltex (Su(dx)) r which showed one additional spot In the region of a larger tRNA species.^ This suggests that Su(dx) may contain an additional isoacceptor, but it has not been characterized, nor has its role in suppression been directly demonstrated. The translation of viral message 4 by the tRNAs of several suppressor stocks has been tested in v i tro . The deviant tRNA TYR jsoacceptor found in one a Ilele of the suppressor of  sable (Su(s) 2) differed from that found in a l l other stocks tested by being able to suppress a TMV-RNA stop codon.17 As well as the direct interaction of d i f f erent gene products, intergenic suppression can occur through more general changes in the intracel lular mil lieu (reviewed by Hartman and Roth^). In the case of conditional mutants, some suppressor mutations alter certain intracellular conditions, such as pH, ionic concentrations, or the concentrations of effector molecules. Changes In each of these conditions are capable of res tor ing the functional conformation of some mutant macromolecules. Intergenic suppression often involves changes in the flow of metabolites down a biosynthetic pathway. The suppressing mutation may increase the flow down an alternate pathway, or alter a protein such that it acts on the substrate of the mutated enzyme, or greatly Increases the level of mutated enzyme, such that adequate act iv i ty levels are maintained.^ An example in Drosophila melanogaster i l lustrates how modulation of a biosynthetic pathway can be suppressive. The black body mutant (bj is caused by a deficiency In beta-aIanine, which is necessary for the normal tanning and melanization of the c u t i c l e . ^ » ^ » 2 0 There are two sources of beta-alanine in the f ly ; a small amount Is produced by the pyrimidine pathway, while most of the beta-alanine Is derived through the alpha-decarboxylation of aspartic acid. I n b. mutants a lesion in this second pathway greatly reduces the levels of beta-alanine produced from aspartate. The amount of this compound normally provided through the pyrimidine pathway is Insufficient to prevent the b. phenotype. 2 1* 2 2 The 5 b mutation is suppressed by the dominant suppressor of black (Su(b)). The Su(b) does not, however, act on the damaged aspartate pathway. Instead, Su(b) elevates the flow down the pyrlmidlne pathway, such that this normally minor source of beta-alanine compensates for the deficiency in the mutated pathway.23 Of the more than 30 suppressor mutations known in Drosophila melanogaster ( l i s t e d in LIndsley and GreW^), the most extensively studied is the suppressor of sable (su(s)). This suppressor acts on mutations at four loc i : sable (s.) and speck (§42), both of which affect body colour, and purple (px) and vermlllion (v.), which both affect eye colour. Most of these studies have used the spontaneous a l l e l e su(s) 2 in combination with y_. The y_ gene is the structural locus for tryptophan pyrrolase (TP) which catalyses the f i r s t step in the production of the ommochrome eye pigments.25 The action of su(s)2 was long thought to be recessive, but recent studies have revealed a small but appreciable level of suppression in su(s)2 heteroyygotes. The level of suppression In the heteroyygote varied according to the a l l e l e used.26 A search for the suppressive mechanism concentrated on differences In tRNA species between wild-type and mutant su(s) strains. The only difference found was an altered distribution of the two major isoacceptors of tyrosyl-tRNA, tRNA TYR a n c j tRNA TYR. In su(s) mutants, tRNA TYR which Is normally the predominant species, is significantly reduced and the level of tRNA TYR increases proport ionately.27, 28 This shift In Isoacceptor pattern was taken to indicate Informational suppression.27 This hypothesis was eventually refuted by the findings of several studies. The most significant of these showed that the mutant, wild-type and 6 suppressed-v tryptophan pyrrolase were a l l of the same molecular weight. The hypothesis of informational suppression predicts a lower molecular weight for the mutant enzyme caused by a fai lure to complete trans I at ion. 29 It was found, however, that the mutant enzyme had an altered Km and pH optimum relative to the wild type and su(s):v 2 . It was suggested that su(s) 2 might induce changes in the ce l lu lar environment which reinstated the functional conformation of the enzyme.30, 31 Jacobson 3 2 alternatively hypothesized that tRNA TYR a c-f-s a s a n inhibltor of mutant TP, but not wiId type TP. In su(s)?;v r the el imination of most of the second isoacceptor, or its conversion to an inert form, would abolish Inhibition and release enzyme function. In this hypothesis, the su(s) 2 gene product functions to produce the mature form of tRNA TYRf or to Interconvert it with the other isoacceptor spec i e s . 3 3 Consistent with th is , when the level of tRNA TYR j n s u ( s ) 2 . v w a s r a j s e d by dietary conditions, there was no change to the suppressed y_ phenotype. But, in the s.u(s)+;v. in which the wild type tRNA TYR w a s presumably inhibiting mutant TP, reductions In this isoacceptor by dietary modification resulted in a y_ phenocopy.3^ The mechanism of su(s) action has s t i l l not been determined, but recent work has focused on the possible role of inserted transposable elements discovered at both the suppressor and target l o c i . Seven spontaneous a l le les of su(s) have been cloned, and a l l have been found to contain an insertion of the gypsy element. Also, six more su(s) a l le les have been induced by P element insertion, and subsequently cloned. DNA sequencing has revealed that in a l l 13 a l le les the elements were inserted within a 2.2 kb region adjacent to the 5' end of the transcribed region 7 of the gene. As well , Insertions of the 412 element have been found at each of the four suppressible target loci.^5 It Is beginning to appear that the role of these inserted elements in suppression may be to modulate transcription at the target locus. Evidence for this comes from two different suppressors in Drosoph iI a  melanogaster. both of which affect the wh Ite eye-colour gene (w_). The mutant a I lele wh ite-apricot (w_a) is caused by an Insertion of the copia element into an Intervening sequence at the w locus.36, 37 -rne w_a phenotype is part ia l ly suppressed by the suppressor of whIte-aprIcot (su(w a)). which results in a darker eye colour. Conversely, w_a can be enhanced to produce a paler eye colour by the suppressor of forked (su(f)). Also, there is a partial revertant of w_a, containing a single long terminal repeat insertion In place of the whole copia, that is not affected by either su(wa) or su(f).58 Enhancement of w_a coincides with the very low level of mutant w. t r a n s c r i p t i o n being s t i l l further reduced in the presence of su(f). Suppression of w_a is coincident with a several fold Increase in the w_ transcript in the presence of su(wa).58 It has been proposed that transcription of the copia element Inserted at the w_ locus causes the w_a mutation. An Increase in copia transcription by su(f).P to the further detriment of w_ transcription, would enhance the mutation. Conversely, a reduction in copia transcription by su(wa) with a concomitant increase in w_ transcription would suppress w_a. F inal ly , removal of copia, save one of i t s long terminal repeats, would eliminate the ab i l i ty of either su(wa) or su(f) to modulate transcription of the partial rever tant . 4 0 8 It is not yet known what significance there Is to the interaction between transposable elements and the suppressor a l l e les . Nor is it known what function the wild-type a l l e l e of the suppressor serves. It has been suggested that suppressor genes may normally produce developmental signals that control the timing and level of transcription at target genes. 3 8 The normal target genes may be different from those involved In suppression. Also, It may be that the elements discussed here are not Indigenous components of a developmental control system. It may only be that they are susceptible to the same types of transcriptional control that governs gene expression during development. Either way, the discovery of several cases of suppressor modulated transcription of target loci suggests that this may be a common suppressive mechanism. 3 8 The subject of this study is the suppression of the purpIe eye mutant (r_) by a new dominant suppressor of purple (Su(pr). The suppressed-px phenotype was original ly associated with the synthesis of a specific type of radiation induced chromosomal rearrangement, a compound-2R chromosome (C(2R)). In a C(2R). the two right arms of the normally metacentric second chromosome are attached to the same centromere. Such a rearrangement can be maintained In a stock bearing the complementary compound-2_ chromosome (C(2L) ) , 3 9 The suppressing C(2R) (C(2R)Su(pr)) was synthesized In females that c a r r i e d the second chromosome combination of I n(2LR)SD72/cn b_. The Segregation Distorter components (S_) are carried on SD72. A pericentric inversion on SD72 has its break-points near the heterochromatic junction on both sides of the centromere. 4 0 This determines that C(2R)SD72/cn bw will be deleted for one copy of 2R-heterochromation (2J_t) and duplicated 9 for one copy of 2L heterochromation (2L£i). When the C(2R)SD72/cn bw was tested in combination with a homzygous b. px-bearing C(2L). the black body phenotype was present but the eyes looked f u l l y wild type. This suppression was not seen In combination with any other CJ2RJ.41 The purpose of this study is to characterize and map Su(pr). In Chapter 2, the constitution of C(2R) necessary for Su(pr) induction Is analysed. To do th is , many C(2R) were synthesized from SD72 In combination with different homologues. Another SD-bearing second, S_PJ>, was also used In C(2R) synthesis to test the Implication of S_Q in Su(pr) induction. Al so , the e f fects of S u (p r) on eye pigmentation were quant i f ied spectrophotometrical ly. In Chapter 3, C(2L)pr?C(2R)Su(pr) were detached to reconstitute standard seconds. This tested whether Su(pr) could persist through further chromosomal rearrangement. Deletions generated during the detachment procedure were also analysed in order to localize a region necessary for S u (p r) ac t iv i ty . In Chapter 4, Su(pr) was mapped by recombination to the v i s ib le heterochromatic markers, IIght and rolIed. 10 CHAPTER 2 ANALYSIS OF COMPOUND SECOND AUTOSOMES 11 Introduction A compound second autosome Is a s p e c i f i c type of chromosomal rearrangement In which two Identical autosomal arms are attached to the same centromere. The formation and meiotic behaviour of compound autosomes has been reviewed by Holm. 3 9 Compound autosome formation Involves a translocation-1 ike event which occurs at the four-strand stage of melosis. For a viable product to be formed the two homologous chromatids involved must each be broken, and the two break-points must I ie on opposite sides of the centromere. Rejoining at these break-points between the centric fragment of one chromatid and the acentric fragment of the other chromatid results in the formation of a new compound autosome. Fl ies carrying one type of compound autosome (eg C(2L)) may maintain diploidy by also carrying the complementary compound autosome ( C ( 2 R ) ) , 3 9 Both compound autosome formation, and the detachment of compound autosomes to reconstitute standard chromosomes occurs spontaneously, but only at low frequency. Both types of chromosomal rearrangement can, however, be induced by gamma Irradiation. In Drosophila melanogaster females most chromosomal rearrangements have t h e i r break-points in h e t e r o c h r o m a t i n . 4 2 On chromosome-2, heterochromatin const i tutes approximately 20% of the prometaphase length of the le f t arm and approximately 25% of the prometaphase length of the right a r m . 4 3 Heterochromatic break-points often occur at a distance from the centromere. In come cases, the break-points fa I I distal to the vital genes that have been identified within this region. A compound autosome synthesized from fragments thus broken wil l not be isogenic, Insofar as 12 it is not diploid for alI functional genetic loc i . In an analagous manner, duplications and deletions of proximal genes may also be generated in the detachment process . 4 4 The detailed mechanics of these processes, and the ut i l izat ion of deletions generated this way to map gene function is the topic of Chapter 3. The break-points that generate the two chromatid fragments probably occur at random between any two chromatids in the tetrad. It has been shown that subsequent compound autosome formation can result from both sister and non-sister attachment.39 However, In the case of C(2R)SD72 formation, the poss ib i l i t ies for strand attachment are limited by the presence of a pericentric inversion on SD72. The break-points of this Inversion have been determined on the polytene chromosome. The break-point on the left arm is very near the euchromatic-heterochromatic border at 39D3-4. The break-point on the right arm is a short distance Into the euchromatin at 4 2 A . 4 2 The juxtaposition of 2Lh to the right arm of chromosome-2 determines that a C(2R) formed by s ister strand attachment wil l be deleted for essential ly al I of 2RJ] and a smal I segment of euchromatin. The euchromatic deletion would encompass sjtw. Such a deletion is lethal. Viable C(2R)SD72 chromosomes must, therefore , be formed from non-s i s ter chromatid attachments to ensure the presence of at least one copy of every v i t a l , proximal gene. In addition to being deleted for one copy of 2Bh, a C(2R)SD72 will be duplicated for one copy of 2Lfc). Compound-2R formation by sister strand attachment of the unrearranged homologue is possible. In this study the formation of several types of C(2R) were tested for their ab i l i t y to modulate expression at the purple locus. Changes in 13 purple gene expression were measured through differences in eye pigmentation that exist between mutant and wild-type f l i e s . Here, px will denote any purple mutant whereas the specific mutant a l le les used are designated as DTJ, p x 2 » BJ:c4> and p x D W . The normal eye phenotype of DrosophI la melanogaster Is due to the accumulation of two classes of pigment, the brown ommochromes and the red pteridines. The biochemistry and genetics of eye pigmentation have been reviewed by P h i l l i p s and F o r r e s t . 4 5 The ommochrome present Is xanthomattin. The pteridines present are a subset of this group called p ter ins , or more commonly, drosopterins. The term drosopterin also designates a specific member of this group. Both types of eye pigment are synthesized In the eye. Protein granules with a diameter of 0.4 -0.8 m are synthesized simultaneously In the eye and association of the pigments with the protein results in a mature pigment granule. The ommochrome and pteridlne pigments are separately associated with the protein granules. 4 ^ The two types of pigment granules have a dist inct ive deposition within the eye. The f ly ' s eye Is composed of about 700 c y l i n d r i c a l units known as ommotldia, that radiate from the optic lobe of the brain to the surface. The ommatidia are organized into a hexagonal array of lenses, or facets, as shown in figure 1. In the wild-type eye, each ommatidia derives its pigmented appearance from the deposition of the two types of pigments in two groups of c e l l s . These are referred to as primary and secondary pigment ce l l s (Figure 1). Brown xanthomattin granules are deposited in the primary pigment ce l l s and the proximal end 14 F l o u r e 1 Diagram of the adult compound eye and of a single ommatidium. (From P h i l l i p s and Forrest, 1980.) b, br i s t l e ; bm, basement membrane; BNG, bris t ler nerve group; C, cornea, CC, cone c e l l ; ps, pseudocone; PPC, primary pigment c e l l ; RC, retlnular c e l l ; 7 RCN, 8 RCN, nuclei of seventh and eight retinular ce l l s ; rh, rhabdomere; SPC, secondary pigment c e l l . 1 5 16 of the secondary pigment eel Is. Granules carrying the red drosopterlns are mostly found in the secondary pigment ce l l s where they are concentrated toward the distal end. A small amount of drosopterin granules are found directly under the basal membrane of the ommatIdlum.46 The pigment granules function to absorb and diffract incident light in a controlled manner. Drosopterins absorb In the blue and near UV region of the spectrum. Xanthomattin absorbs v i s ib le l ight. The presence of eye pigment Is essential for visual acuity. White eye mutants have v ir tua l ly no eye pigment and consequently have no visual acuity. The visual acuity of other eye mutants varies roughly In accordance to the pigment levels of each.46 The p u r p l e gene p a r t i c i p a t e s In the production of the red drosopterins. The purple locus is the site of the structural gene for sepiapterin synthase. 4? As shown In Figure 2, this enzyme catalyses the conversion of dihydroneopterin triphosphate to sepiapterin in the second step of the pteridlne pathway. At least five major compounds, a l l of which are derivatives of a Ipha-amino-4-hydroxypteridIne, result from this pathway (figure 2). Drosopterin and isodrosopterin are enantiomers. The exact pathway from sepiapterin to the end products has not been confirmed. There are other related compounds for which a chemical structure has not been worked out (reviewed in Ph i l l ips and Forrest 4 ^). Purple mutants are characterized by lower levels of drosopterin, i sodrosopter I n and an u n i d e n t i f i e d compound ( fract ion e) . Even greater decreases of aurodrosopterIn and sepiapterin are found in the px f ly.^8, 49 17 Figure 2 (A) S y n t h e t i c pathway for the s y n t h e s i s of the d r o s o p t e r i n s and seplapterln (after P h i l l i p s and Forrest , 1980). The pathway begins with GTP which is converted by GTP eye Iohydrolase to dIhydroneopterin tr iphosphate. Sepiapterin synthase c a t a l y s e s the next step t o produce sep iapter in . The broken arrow indicates that the remainder of the pathway is uncharacterIzed. (B) Chemical structures of the common drosopterins of Drosophila. 18 (A) GTP- D I H Y D R O N E O P T E R I N T R I P H O S P H A T E H 0 . C O C H O H C H , S E P I A P T E R I N D R O S O P T E R I N S (B) H N 2 - A M I N 0 - 3 , 4 - D I H Y D R O -4 - 0 X 0 P T E R I D I N E ( " P T E R I N " ) H . , N ^ H N I S O X A N T H O P T E R I N X A N T H O P T E R I N .N M \ ^ V - N H . 0^ " " C H 2 O H W / " C H . C H C H , I I 3 O H O H H N ^ N \ , ^ N ^ | I  I O H O H HH K D R O S O P T E R I N B I O P T E R I N N E O P T E R I N 19 Materials and Methods Mutations and chromosomal rearrangements: A brief description of the genetic markers used in this study Is given in Table 1. Further detail on these mutations can be found In Lindsley and G r e l l . 2 4 A brief description of the chromosomal rearrangements used In this study is given in Table 2. Synthesis of C(2L) chromosomes: Three types of px-bearing C(2L) chromosomes were synthesized for use in this study. The new C(2L) chromosomes were homoyzgous for either px1, b_ px1* or px b w . Repeated attempts to synthesize C(2L)pr 2 were unsuccessful. Each type of C(2L) was synthesized separately by treating about 1,000 homoyygous females with approximately 2,500 rads of gamma radiation from a ^°Co source. Groups of 25 treated females were then mated to C(2L)P.b;C(2R)P.px males in half pint bottles at 25 degrees cels ius. The f l i e s were transferred to new bottles every five days for a total of three broods. New C(2L)pH and C(2L)pr b w were recovered as px p_x. Three new CC2L)pr? were recovered at a frequency of approximately .3 per 100 females treated. Two new C(2L)b prl were recovered as b_ px p_x progeny at a frequency of about .3 per 100 females treated. Each new C(2L) was established as a separate line with C(2R)P.px. Each new C(2L) was assigned an alphanumeric code according to the system described by Holm. 3 9 Synthesis of C(2R) chromosomes: Four standard seconds were used for C(2R) synthesis; SD72 carries SD and also carries a pericentric Inversion as described In the Introduction to this chapter and a smaller para-centric Inversion on distal 2R. The SD5 chromosome also bears SD and 2 20 Table t Description of second chromosome mutations used. The chromosome-2 centromere is at 55.1 Symbol Name Map P o r t i o n Description V vermiI I ion 1 - 33.0 bright red eye b black 2 - 48.5 black body pr1 purpIe 2 - 54.5 purple eyes p r 2 purpIe 2 - 54.5 darker purple eyes prbw purp le 2 - 54.5 brownish purple eyes SD . SegregatIon Distorter 2 - 55 SD/+ males exhibit meiotlc drive It I Ight 2 - 55.1 yelIowIsh-p ink eyes rl ro I I ed 2 - 55.1 rolled w ing edges cn cinnabar 2 - 57.5 br Ight red eyes , colourless ocelIi px pIexus 2 - 100.5 extra wing veins bw brown 2 - 104.5 brown eyes 21 Table 2 Description of compound second chromosomes used. Symbol Description C(2L)SH3,+ Left arm, no genetic markers C(2L)VY1,b pH Left arm, homozygous for h_ px] C(2L)VH2,It Left arm, homozygous for JJ; C(2L)P,b Left arm, homozygous for b_ C(2R)VK43,SD72/cn bw Right arm, SD72/cn bw C(2R)SH3,+ Right arm, no genetic markers C(2R)P,px Right arm, homozygous for p_x 22 non-overlapping inversions on the right arm.50 An unrearranged second bearing cn b_ was used as was a wild-type second from an OR-R stock. New C(2R) chromosomes were synthesized In females from three different SJ2 heterozygotes: SD72/cn b_, SD72/+ and SJ_/c_n b_. Females in each experiment were treated as discussed above. Groups of 25 females were mated to compound-2 males and cultured as above. New C(2R)SD72/cn. bw were recovered with C(2L)P.b as i> progeny, while s ister strand attachments from SD72/cn b_ treated females were recovered as J_ on bjtf individuals. New C(2R)SD72/+ were recovered with C(2L)VH2.It as suppressed-li progeny, while the s ister strand attachments were recovered as _ individuals. New C(2R)SD5/cn bw were recovered in combination with C(2L)P.b as b_ progeny and sister strand attachments were recovered as above. Each new C(2R) was established in a separate line with C(2L)P.b and assigned an alpha-numeric code. The number and frequency of each type of new C(2R) Is presented in the Results and Discussion. Cytological Analysis: Polytene chromosomes from salivary glands of late third instar Iarvae were examined using the method described by Hil l iker.51 The salivary glands were dissected in 45% acetic acid on a depression s l i d e . The i s o l a t e d glands were transferred to a drop of 2% aceto-1acto-orcein on a s i l iconized sl ide and covered with a covers I ip. The covers I ip was gently tapped, then more firmly pressed to spread the preparation. The preparation was observed and photographed using a Zeiss photomicroscope equipped with phase contrast o p t i c s . ^ Visual Examination for Su(pr): The newly synthesized C ( 2 R ) , and C(2R) VK43. SD72/c,n... bw were tested for the ab i l i ty to suppress px in combination with several C(2L). All the C(2R) were tested In combination 23 with C(2L)VY1.b prK In addition, each C(2R) formed by non-sister strand attachment was tested in combination with at least one other C(2I )h pr-1. two C(2L )pr1 and both C(2L)prhw. New C(2R) formed by s ister strand attachment were tested in combination with one C(2L)prDW. Each C(2L) was used in combination with several C(2R). When a C(2R) demonstrated Su(pr) a c t i v i t y , the C(2L) bearing the suppressed px was outcrossed to C(2L )P.b:,G(2R)P.px and the p_x progeny were visually examined for the re-emergence of the px phenotype. Several C(2R)Su(pr) were also tested for their abil ity to suppress y_ by visual examination of v/v;C(2L) PC(2R)- $M(pr? females and v/Y:C(2L):C(2R)Su(pr) males. SpectroDhotometr ic measurement of . eve p laments: Several C(2R) were selected for measurement. Each was tested In combination with C(2L)VY1.b pxi, G(2LJVF1.b pr 1, C(2UVF1 pr 1 and C(2L)VF1. prbw. Measurements were also taken for p r 1 and OR-R controls. Stocks of f l i e s cultured as described above were co l l ec t ed at 0 to 24 hours post e c lo s ion . Measurements were taken for one genotype at a time plus a simultaneous measurement of the wild type control. Five determinations were done for each genotype tested. Typically 20 f l i e s , 10 males and 10 females were decapitated for each determination. The heads were placed In 1.0 ml of \$ NH4OH/O.25 M beta-mercaptoethanol in a microcentrifuge tube on ice and sonicated for 20 seconds. The homogenate was centrifuged at high speed for one minute. The absorbance of 0.5 ml of the clear supernatant was immediately recorded at 495 nm on a Unicam SP1750 u l t r a v i o l e t spectrophotometer. 24 Results and Discussion Thir ty -one putative C(2R)SD72 were recovered from approximately 1,600 treated females. The frequency of recovery was about 2 per 100 females treated . Examination of polytene preparations confirms the cons t i tu t ion of these C(2R). As shown in Figure 3, the paracentric inversion on the right arm of SD72 forms an Inversion loop in pairing with its unrearranged homologue. A difference in the length of the heterochromatic region caused by the pericentric inversion on SD72 is also evident. As shown in Figure 4, the difference in arm length of the homologues caused by the pericentric inversion prevents the proximal pairing. Five CD b_ sister strand attachments were also recovered at a frequency of approximately .3 per 100 females treated. The number of C(2R) of both types was undoubtedly higher than the number recovered, but only one quarter of the eggs bearing newly formed C(2R) are expected to be f er t i l i zed by a complementary sperm to produce a diploid viable zygote . 3 9 Twenty-eight of the thirty-one new C(2R)SD72/cn bw were found to suppress r_1. All these C(2R)Su(pr) were seen to ful ly suppress a l l C(2L)pr^ and C(2L)b pr^ against which they were tested. This shows that Su (pr) action is not peculiar to a specific C(2L). In addition, a l l C(2R)Su (pr) a lso looked f u l l y wi ld-type in combination with both C(2L)pr^w. This shows that Su(pr) is not a l l e l e specif ic . Each of the suppressed C(2L) was outcrossed to C(2R)P.px. Visual Inspection of these f l i e s revealed that full pr_l and pr D W had fully re-emerged. This demonstrates that the px locus had not undergone a 25 Figure 5 Photomicrograph of the proximal region of a polytene chromosome prepara-tion of C(2R)VF5.SD72/cn bw. The arrow points to the inversion loop formed by pairing of the right arm of SD72 with its unrearranged homologue. 2b 27 Figure 4 Photomicrograph of the proximal region of a polytene chromosome preparation of C(2R)VF5. SD72/cn bw. The arrows Indicate an unpaired region adjacent to the chromocenter. 29 permanent a l t e r a t i o n , and that the presence of Su (pr) is necessary for continued suppression. The three non-suppressing C(2R)SD72/cn bw had a ful ly mutant phenotype. In no case was an Intermediate eye colour observed. It was also found that in no case did a C(2R)Su(pr) suppress y_. This is interesting because px and y_ are both target mutations of su(s) 2 . This difference in target specif ic i ty plus the fact that Su(pr) action Is dominant while su(s ) 2 Is a recessive mutation, suggests that the mode of action of these two suppressor genes is different. The speci f ic i ty of C(2R) constitution necessary for Su(pr) induction was tested two ways. F i r s t , C(2R) were synthesized in SD72/+ females. Both C(2R)SD72/+ formed by non-sister strand attachment, and C(2R)+ formed by sister strand attachment were expected. Testing the C(2R)SD72/t for suppression in combination with C(2L)pr chromosomes, was intended to determine whether a wild-type second could substitute for the QQ _ -bear!ng homologue original ly used. Testing the C(2R)+ for the ab i l i ty to suppress was intended to determine whether the presence of a SD72-donated fragment was essential for the formation of a C(2R)Su(pr). The two types of C(2R) expected were c lass i f ied according to their ab i l i ty to suppress _ on C(2L)VH2.It. A C(2R)SD72/+ can carry a _ duplication on the inverted chromatid fragment donated by SD72. Only if SD72 is broken to the right of the centromere, distal to I t , will the resulting C(2R)SD72/+ not carry a l i t dupl ication. A JJ_ dupl ication may also be donated to a new C(2R)SD72/+ by the other homologue. This can occur when the chromatid fragment donated by the wild-type second carries a break-point to the left of the centromere, distal to _ . Similarly, a C(2R)+ can carry a 1 _ duplication if one of the wild-type progenitor 30 52 standards was broken distal to i t . Gibson found that approximately 90$ of a l l C(2R) chromosomes formed from unrearranged standards will carry their break-points proximal to i t . Consequently, the suppression of i t wil l accurately classify most of the compound seconds synthesized here as C(2R)SD72/+. However, absolute confidence in this c lass i f icat ion could only be gained through the cytological analysis of each of these compounds. Five C(2R)SD72/+ were recovered out of about 1,500 females treated, a frequency of approximately .33 per 100 females treated. It is Interesting to note that the frequency of recovery of C(2R)SD72/+ was lower by an order of magnitude than that of C(2R)SD72/cn bw. Three of the five C(2R)SD72/+ showed Su(pr) act ivi ty with al I the C(2L) that they tested with: two C(2L)b prK two C(2L)pr1 and both C(2L)prbw. This demon-strates that the presence of the CD bjj-bearlng homologue Is not necessary for Su(pr) induction. Nine C(2R)+ chromosomes were also recovered in this experiment, a recovery frequency of approximately .6 per 100 females treated. None of them was able to suppress prl or p r b w on any C(2L). This suggests that the presence of a chromatid fragments from SD72 is essential for C(2R)Su(pr) formation. The recovery of s i s ter strand attachments was greater than the recovery of non-sister strand attachments In this experiment. This is the opposite of what is expected. One sixth of recovered attachments induced are expected to be of the sister strand type and four sixths of the non-sister strand type.^9 Another one sixth are lethal as discussed above. The deviation from the expected r a t i o and the low recovery frequency relative to C(2R)SD72/cn bw. suggests that C(2R)SD72/+ may have poor v iabiI i ty . 31 The constitution necessary for Su(pr) Induction was tested in a second way. Compound-2R chromosomes were synthesized from the c_ _ bearing homologue and another S_ bearing second, SD_. Unlike SD72. SD5 does not carry a pericentric inversion.50 Twenty-three C(2R)SD5/cn bw were recovered from approximately 1,000 females treated at a frequency of about 2.3 per 100 females treated. This agrees with the recovery rate for C(2R)SD72/cn bw. This agreement substantiates the suggestion that the much lower recovery rate of C(2R)SD72/+ is Indicative of poor v i a -b i l i t y . None of the 23 C(2R)SD5/cn bw could suppress either pxl or r_r_ . The cn b_ s ister strand attachments recovered In this experiment cannot be used to study suppression since ex b_ is epistatic to px. These findings suggest that the presence of SJ_ is not responsible for Su(pr) induction. It substantiates the suggestion that SD72 makes an essential contribution to Su(pr) Induction. It also raises a question regarding the presence of the pericentric inversion, present only on SD72. This inversion confers upon the C(2R)SD72 a proximal constitution unlikely in C(2R)SD5. or any other C(2R) not synthesized from an inverted standard second. The C(2R)SD72 carry a 2 _ duplication and a 2P_ deletion that is probably unique to this type of C(2R). Also, the C(2R)SD72 can carry 2Lh and 2Rh in a juxtaposition not possible in any other type of C(2R). A deta i led study of the possible constitution necessary for Su (pr) Induction Is covered in Chapter 3. Suppression of px was quant i f ied using the spectrophotometric measurement of eye pigment. This technique is based on the fact that aqueous ammonia extracts of wild type Drosoph iI a heads absorb strongly in the blue region of the spectrum. Analysis of such extracts has revealed 32 that most of the absorbance is due to the presence of drosopterins. 4^ As shown In Figure 5, extracts made from the wild type have an absorption maximum at 495 nm. In the px! extract, however, there is a very large reduction in absorbance throughout this region, and the peak at 495 nm is undetectable. This large difference in absorbance also exists between suppressed and unsuppressed px! f l i e s . A comparison between the px! absorbance curve for a standard second and a C(2R) reveals that they are identical . Likewise, the absorbance curve of suppressed-px! and the wild type are indistinguishable (Figure 5). Hence, the mean absorbance of head extracts measured at 495 nm (A495) is an effective technique for quantifying drosopterin levels. Drosopterin measurements were consistent for a l l C(2R)Su(pr) recovered. Consequently, the results of tests on C(2R)VK43.SD72/cn bw. and three newly synthesized C(2R)SD72 are presented as representative of a l l C(2R)Su(pr) tested. The results include a representative of each type of C(2L) used. Drosopterin levels are expressed as a percentage of the wild-type levels. Table 3 shows the control values for the wild type and unsuppressed px. The C(2L)SH5.+;C(2R)SH3.+ drosopterin levels agrees with the wild-type level. This shows that C(2R) formation does not alter eye pigmentation per £ £ . This fact is supported by the synthesis of many difference C(2R) in other studies without any alteration of eye pigmen-t a t i o n . 4 1 Although the black gene does not participate in drosopterin synthesis, 33 Figure 5 Absorbance of drosopterin extracts from 450-550 nm. 34 450 T—rTTTn— i 1 i 500 550 W A V E L E N G T H ( n m ) z: <C CO CO C£ O 0 0 ™ CQ <: C ( 2 L ) V F l , p r 1 C ( 2 R ) P , p x C ( 2 L ) V F l , p r J C ( 2 R ) V F 5 , S D 7 2 / c n bw 450 B — m m m • n i — a a — • — — — —B B O B B M — 500 550 W A V E L E N G T H ( n m ) 35 Table 3 Drosopterine levels is unsuppressed C(2L) strains Extracts Mean Standard Drosopterin levels Genotype Tested A495 Deviation ( % of OR-R ) wild type (OR-R) 5 .780 .073 100 C(2L)SH3,+; 5 .760 .073 97 C(2R)SH3,+ C(2L)P,b; 5 .885 .078 113 C(2R)P,px C(2L)VY1,b pr 1; 5 .395 .069 51 C(2R)P,px C(2L)VF1,b pr 1; 5 .391 .059 50 C(2R)P,px C(2L)VF1,pr1; 5 .252 .068 32 C(2R)P,px C(2L)VF1,pr b w; 5 .142 .059 25 C(2R)P,px 36 the presence of b on some C(2L) was found to s l ight ly increase the A495. This increase was consistently about 0.1 absorbance units above bj\ This results in the A495 of C(2L)P.b being approximately 13$ greater than C(2L)SH3.+ and the C(2L)b prl about 15$ above C(2L)pr1 (Table 3). The elevation of A495 in the presence of b_ must be considered In the discussion of suppressed-px drosopterin levels. The C(2L)VF1.pr1:C(2R)P.px strain was found to have 32$ of the wild type drosopterin levels. This Is consistent with the levels found for homozygous px! on standard seconds. 2 6 The C(2L)VF1 fprD W:C(2R)P fpx was found to have drosopterin levels that were 25$ of those in the wild type (pr+) strains. Again, this agrees measurement for this a l l e l e on standard seconds.47 The drosopterin measurements for the four C(2R)Su(pr) presented are contained In Tables 4 - 7 . All C(2R)Su(pr) showed drosopterin levels in excess of the wild type. In a l l tests, the C(2L)pr D W which had the lowest unsuppressed drosopterin levels, also had the lowest suppressed leve l s . The Su (pr);pr D W drosopterin levels consistently exceeded the wild-type levels by only 1 - 3$. This Increase, of 75 - 80$ over the mutant levels, was found for both C(2L)pr b w in combination with every C(2R)Su(pr) tested. The drosopterin levels in Su(pr);vpr 1 /pr 1 also exceeded those in the wild-type, but by a greater amount. The suppressed pr 1 levels were in the range of 106 - 110$ of wild type for every C(2R)Su(pr) tested. The C(2L)bpr1 was found to have the highest suppressed pigment levels. All 37 Table 4 Drosopterin levels in C(2R)VK43.SD72/cn bw strains Extracts Mean Standard Drosopterin levels Genotype Tested A495 Dev iat ion ( % of OR-R ) wild type (OR-R) 5 .792 .045 100 C(2L)VY1,b p r l ; 5 1.012 .052 128 C(2R)VK43,SD72/cn bw C(2L)VF1,b p r 1 ; 5 1.009 .080 127 C(2R)VK43,SD72/cn bw C(2L)VF1,pr 1; 5 .872 .076 110 C(2R)VK43,SD72/cn bw C(2L)VF1,pr b w ; 5 .802 .071 101 C(2R)VK43,SD72/cn bw 38 Table 5 Drosopterin levels in C(2R)VF5.SD72/cn bw strains Extracts Mean Standard Drosopterin levels Genotype Tested A495 Deviation ( % of OR-R ) wild type (OR-R) 5 .788 .055 100 C(2L)VY1,b p r 1 ; 5 .985 .070 125 C(2R)VF5,SD72/cn bw C(2L)VF1,b p r 1 ; 5 .961 .043 122 C(2R)VF5,SD72/cn bw C(2L)VF1,pr 1; 5 .839 .061 106 C(2R)VF5,SD72/cn bw C(2L)VF1,pr b w ; 5 .831 .083 105 C(2R)VF5,SD72/cn bw 39 Table 6 Drosopterin levels in C(2R)VF10.SD72/cn bw strains Extracts Mean Standard Drosopterin levels Genotype Tested A495 Deviation ( % of OR-R ) wild type (OR-R) 5 .785 .059 100 C(2L)VY1,b p r l ; 5 .966 .046 123 C(2R)VF10,SD72/cn bw C(2L)VF1,b p r l ; 5 .950 .048 121 C(2R)VF10,SD72/cn bw C(2L)VF1,pr1; 5 .865 .059 110 C(2R)VF10,SD72/cn bw C (a )VF1,pr D W ; 5 .809 .062 103 C(2R)VF10,SD72/cn bw o 40 Table 7 Drosopterin levels in C(2R)VF50.SD72/cn bw strains Extracts Mean Standard Drosopterin levels Genotype Tested A495 Deviation ( j of OR-R ) wild t y p e (OR-R) 5 .795 .060 100 C(2L)VY1,b p r ' ; 5 .969 .071 122 C(2R)VF30,SD72/cn bw C(2L)VF1,b p H ; 5 .962 .045 121 C(2R)VF30,SD72/cn bw C(2L)VF1,pr 1; 5 .859 .062 108 C(2R)VF30,SD72/cn bw C(2L)VF1,prbW; 5 .818 .078 103 C(2R)VF30,SD72/cn bw 41 Su(pr);b prVb pr 1 drosopterin measurements exceeded 120$ of the wild type and the range Induced by the four C(2R)Su(pr) presented here is 121 - 128$. When the contribution to absorbance by b_ is subtracted, there is good agreement between suppressed drosopterin levels in C(2L)pH and C(2L)b prl. Suppressed drosopterin measurements were in agreement in C(2L)VY1.b prl and C(2L)VF1.b prl although the former had s l ightly higher levels throughout. This variation is partly due to pecul iarit ies of the C(2L). since it was also seen in the absence of C(2R)Su(pr) (Table 3). Overall , the suppression of pr 1 was associated with drosopterin levels approximately 10$ greater than wild type, once the contribution to absorbance by ii (if present) was accounted for. These results were consistent for each combination of pxl-bearlng C(2L) and C(2R)Su(pr) tested. The drosopterin measurements in each of the three nonsuppressing C(2R)SD72/cn bw strains confirmed the visual inspection by exhibiting f u l l y mutant pigment l eve l s . Table 8 shows the drosopterin levels produced by one of them, C(2R)VF12.SD72/cn bw. A comparison between these values and those of unsuppressed C(2L)pr (Table 3) reveals no significant difference. Table 9 shows the drosopterin measurements in C(2R)VF3.+. These measurements agree with those in the C(2R)SD72/cn bw. Drosopterin measurements were found to be consistent in a l l suppressing C(2R)SD72P irrespective of whether they carried a wild-type chromatid fragment, or one from the cn bw second. These findings agree with ear l ier studies in which suppressed-pr1 and suppressed p r h w drosopterin levels induced by various su(s) a l le les 42 Table 8 Drosopterin levels in C(2R)VF12.SD72/cn bw strains Extracts Mean Genotype Tested A495 wild type (OR-R) 5 .778 C(2L)VY1,b p r 1 ; 5 .403 C(2R)VF12,SD72/cn bw C(2L)VF1,b p r 1 ; 5 .365 C(2R)VF12,SD72/cn bw C(2L)VF1,pr 1; 5 .259 C(2R)VF12,SD72/cn bw C(2L)VF1,pr D W ; 5 .205 C(2R)VF12,SD72/cn bw Standard Drosopterin levels Deviation ( j of OR-R ) .017 100 .080 52 .037 47 .049 33 .052 26 43 Table 9 Drosopterin levels in C(2R)VF3.SD72/+ Genotype Extracts Tested Mean A.495 Standard Deviation Drosopterin levels _ _ Of OR-R ? wild type (OR-R) C(2L)VY1,b pr 1; C(2R)VF3,+ C(2L)VF1,b pr 1; C(2R)VF3,+ C(2L)VF1,pr1; C(2R)VF3,+ C(2L)VF1,prb«; C(2R)VF3,+ 5 5 .795 1.088 1.050 .836 .802 .045 .066 .0458 .059 .057 100 137 132 105 101 44 were measured. As in this study, Yim et a_47 found that suppressed px levels exceeded the wild type, and suppressed-pxi levels exceed those of suppressed-prbw. Yim e_ aj. 4^ and Jacobson e_ aj. 2^ also measured the sepiapterin synthase activity levels and compared them to drosopterin levels in suppressed and unsuppressed mutants. Jacobson e± aj. 2^ found that a su(s) mutant al l e l e need only raise enzyme activity from the 15$ activity level found in su(s) +:pr 1. to 20$ of wild-type activity in order to produce wild-type levels of pigment. This shows that above 20% of wild-type activity, the sepiapterin synthase activated step is not rate limiting on drosopterin production. Jacobsen e_ aj. 2^ also found that suppressed-pxi enzyme activity d i f f e r e d when in combination with different su(s) alleles. Enzyme activity and drosopterin levels were found, In most cases, to be roughly proportional. The weakest a l l e l e used, su(s)e5«6 raised r__ enzyme activity to approximately 34% wild type, and accumulated 105$ wild-type pigment levels. The most effective a l l e l e , su(s) 2 raised sepiapterin synthase activity to 75$ and pigment levels to 125$ of the wild type. The discrepancy between enzyme and drosopterin levels in su(s):pr 1 suggests that the drosopterin measurements for Su(pr);pr^ may, likewise, not accurately reflect suppressed enzyme activity. If Su(pr) is similar to su(s) in this respect, then different C(2R)Su(pr) chromosomes may dIffer w idely in their abiI ity to el evate enzyme activ ity, wh iIe exh ib it ing pigment levels that agree quite closely. Also, Su(pr) may not have to raise enzyme levels by much to produce the suppressed-px phenotype. The su(s) x 4;pr 1 produced drosopterin levels in the range of 110$ of wild 45 type, (l ike Su(pr) ; p H ) . by raising enzyme activity to only 50$ of the wild type. It is also interesting to note that the difference between enzyme and pigment levels induced by su(s) 2 varies between suppressed px! and suppressed p r D W . Yim ej a j 4 7 found that, unlike the large difference found in su(s):pH . in su(s):pr b w there was a close correlation between drosopterin pools and sepiapterin synthase act iv i ty . Suppressed-r^!^ f l i e s had enzyme and pigment levels that were both very close to the wild type. Hence, in contrast to the suppression of px!, the suppression of p r b w can not be produced with less than wild type levels of enzyme act iv ity. The findings of the studies on su(s) pose two questions that are pertinent to the study of Su(pr). F i r s t , why does su (s ) 2 raise the act iv i ty of one target a l l e l e (px!) to 75$ of the wild type, yet raise the act iv i ty of another target a l l e l e (pr D W) to approximately 100$ of the wild type? This is e spec ia l l y puzzl ing because p r b w has a lower unsuppressed act iv i ty level than px!. Second, how can the lower enzyme act ivity in su(s ) 2 :pr 1 result in greater drosopterin accumulation (125$) than is found in su(s ) 2 ?pr b w (approximately 100$ of the wild type). Perhaps, answers to both these questions Involve su(s) induced changes In the development prof i le of target a l l e les . Tobler ej; a j . 5 3 have found that there are two main peaks of sepiapterin synthase act iv i ty . The f i r s t peak occurs very early in larval development, and the second peak begins late in the pupal stage then recedes at about three days post e c l o s i o n . It is th i s second peak that Is responsible for eye pigmentation. 5 3 Drosopterin levels in excess of the wild type could 46 accumulate in f l i es with less than wild-type enzyme act iv i ty , if the second a c t i v i t y peak began ear l i er than normal. Alteration in the developmental profi le could also explain the different suppressed-pr1 pigment levels found In combination with different su(s) a l le les . Such variation could be accomplished if peak expression of p_l was induced at different times by different su(s) a l l e l e s . This explanation posits that the greater the di f ference is between enzyme a c t i v i t y levels and drosopterin levels, the earl ier the increase in suppressed enzyme act iv i ty must begin. The difference in the effect of su(s ) 2 on pj_ and p r p w can also be explained by differences in the developmental profi le between the two suppressed target a l l e les . The close agreement of su(s) 2 :pr D W enzyme act iv i ty levels and drosopterin pools to those of the wild type, suggests that the developmental p r o f i l e of both is v ir tual ly identical . In su ( s ) 2 :pr 1 . however, it may be that enzyme act ivi ty does in fact reach wild type levels, but much earl ier than normal. If so, It might already be receding at the point when it is measured. The suppressed-pr1 act iv i ty levels might be lower than either the wild type or suppressed p r b w at this time. At the same time, the earl ier onset of suppressed-r__ peak act iv i ty would result in higher drosopterin accumulation than found in normal or suppressed development. Considering the enzyme assay studies in su(s) 2 :pr. it is possible to envision how Su(pr) might modulate px. The poss ib i l i t i es Include changes in the onset of peak sepiapterin synthase act iv i ty , the profi le of the act ivi ty peak, the maximum enzyme act iv i ty , or the duration of suppressed act iv i ty levels. Time course enzyme act ivi ty studies in Su(pr):pr would make it possible to discern between these poss ib i l i t i es and, as such, would provide a better understanding of Su(pr). CHAPTER 3 ANALYSIS OF C(2R)Su(pr) DETACHMENT PRODUCTS 48 Introduction In the detachment process, the steps giving rise to compound autosome formation are reversed, and reconst i tuted standard chromosomes are r e c o v e r e d . 4 4 There are two main reasons for undertaking detachment studies on C(2R)Su(pr). F i r s t , since Su(pr) was induced during C(2R) formation, it is pertinent to ask whether its function is dependent on that specific chromosomal rearrangement. If Su(pr) act ivi ty Is dependent on C(2R)Su(pr) c o n s t i t u t i o n , then detachment of the component arms should eliminate Its expression. If, however, Su(pr) act ivity is not dependent on a specific chromosomal rearrangement, but arose through the process of rearrangement, then Su(pr) expression might persist In other types of rearrangement. If so, suppressing detachment products might be recovered. The second reason for detaching C(2R)Su(pr) is that It allows each component arm to be studied separately over other homologues. These detachment products can be tested with the known vi ta l markers In chromosome-2 heterochromatin. Lethality over these v i ta l genes can reveal deletions of heterochromatin. From th is , the constitution of the detachment products may be part ia l ly deciphered. Correlations between specific types of deletions, and the loss of suppression, may help to I oca Ii ze Su(pr). To understand how differences in heterochromatic constitution may help to characterize Su(pr). it is necessary to examine what types of rearrangement may be recovered In the detachment products. The particular heterochromatic constItutIon of any detachment Is i n i t i a l l y determined, In 49 part, by the way In which the progenitor C(2R) is made. The way in which duplications and deletions can be generated during compound autosome formation has been thoroughly analyzed by Hi l l Iker and Holm.44 The two central points revealed by that analysis pertinent to this study are: (1) al l genes lying proximal to a break-point on the centric fragment will be carried as duplications on the newly formed compound autosome, and (2) conversely, the free arms generated by such a break will be deleted for those proximal genes, as will a compound autosome that receives that arm. Since the centric fragment is donated by one homologue and the free arm by the other, differences in the positions of the break-points on each determines that the new compound autosome may be Isogenic, or carry a duplication, or a deletion, or both a duplication and a deletion. The same mechanism occurs during the detachment procedure, with the result that a reconstituted standard chromosome may undergo further a l t e r a t i o n s . 4 4 The mechanics involved in C(2R)SD72 synthesis are further complicated by the presence of the p e r i c e n t r i c Inversion. This inversion is fundamental in determining the content and configuration of heterochromat in In C(2R)SD72. As shown in Figure 1, the pericentric inversion determines that there are two possible ways that the C(2R)SD72 can be composed, depending on whether SD72 or Its homologue donate the centric fragment. If SD72 donates the centric fragment, as Is part A of Figure 1, then the break-points on both homologues will have occurred in 2RJ]. The resulting C(2R) will carry a proximal segment of 2Rh from SD72. This segment may Include vital genes, if the break-points on SD72 fe l l distal to their loci. Vital 2RJ] genes may also be duplicated on the captured arm, If the 50 Figure 1 Alternate configurations of chromatid breakage and reunion in C(2R)SD72/cn _ synthesis . (A) Chromatid fragments are generated by break-points in 2P_ of both homologues. (B) Both chromatid fragments are generated by breaks in 2Lh. 52 break-point on the cn b_-bearing homologue fe l l proximal to them. This C(2R) wil l be duplicated for a l l of 2J_. The second possibi l i ty is shown in Figure 1B. In this case, SD72 donates the captured arm, and the centric fragment Is derived from Its homologue. The break-points on both standards wil l occur In 2|_. The resulting C(2R) will not carry any 2F_b from SD72 It will be duplicated for part, but not necessarily a l l of 2Lh. Either way, the C(2R) wil l be deleted for one copy of 2R to a point just distal to stw.. It is also known that no viable C(2R)SD72/cn bw wil l be deleted for any vital gene in the remaining 2P_ segment. The two possible ways of constructing a C(2R)SD72 determines that two different sets of detachment products are possible. Figure 2 shows the four detachment classes that could possibly be recovered from the C(2R) in Figure 1A. Among the possible variations considered here are (1) the donation of the centric fragment from the C(2L) and the acentric fragment from the C(2R). (2) the reciprocal association, (3) the donation of an acentric fragment derived from either arm of the C(2R) and, (4) variations in the position of the break-point on the acentric fragment. Since It is the constitution of the C(2R) that is under investigation, variation in the position of break-points on the C(2L) are not considered here. Such variations do occur, however, and in practice they w i l l complicate detachment analysis. Detachments 1 and 2 (Figure 2) both had their centric fragment donated by the C(2R)Su(pr). As well, both classes may be composed of chromosomal segments from three sources: the C(2L). SD72. and Its C_Q to-bearing homologue. As shown, class 1 detachment resulted from break-points occurring in the 2P_, on the cn b_ side of the C(2R)Su(pr). If 53 Figure 2 Detachment product classes possible from the C(2R)SD72/cn bw configuration shown in Figure 1(A). The junction between the acentric and centric chromatid fragments that formed the C(2R) is shown by dark arrowheads. Outlined arrowheads show the position of the break-point necessary to generate the four detachment classes. These are numbered on the C(2R) to designate the four classes. 55 the detachment-generating break-point f a l l s proximal to the CJ2RJ-generat ing breakpoint, then this class will not carry the segment derived from the cn b_w. side that is shown. In any case, the location of this breakpoint will determine which 2RJ] genes are donated from this segment. A break-point proximal to a gene in this region makes i t possible for the detachment product to carry a non-polar deletion, assuming that It was not duplicated elsewhere during C(2R)Su(pr) formation. This class will carry the S_D side of the C(2R)Su(pr) which means that i t will be deleted from some, or a l l , of 2Rh to a point just d i s t a l to stw. Presence of t h i s arm can be detected by stw pseudo-dominance. Class 2 detachments will carry the right arm from the on b_w. side of the C(2R)Su(pr). This class will maintain any dupl ications generated In ZBh during C(2R)Su(pr) synthesis. This class may also carry non-polar deletions for 2Lh. Class 3 and class 4 detachments are both composed of a captured free arm derived from the C(2R)Su(pr). Class 3 carries the free arm from the SD72 side. Al I 2Rb will be deleted In this class and some, or a I I, of 2Lh genes may be duplicated. If a deletion Is generated on the 2Lh segment donated by SD72. It will be covered by the corresponding C(2L)-donated segment. Class 4 detachments will carry the free arm from the c_Q bj/ side of the C(2R)Su(pr). It may be composed of chromosomal segments from three sources if the detachment-generating break-point is proximal to the C(2R)-generating break-point. Polar deletions In the 2gh segment donated by SD72 may be generated in the detachment procedure. However, such deletions may be masked by compensating dupl ications on the on b_w. frag-56 ment. Alternatively, if the break-point that generated the detachment is distal to the one that generated the C(2R). the SD72 2__ wil l be completely omitted. In that case, polar deletions may be generated In the distal 2J_ of the cj] _ fragment. Figure 3 shows the four possible detachment classes expected from a C(2R)Su(pr) synthesized as shown in Figure IB. As before, classes 1 and 2 contain a centric fragment derived from the C(2R)Su(pr). Detachments of these classes may contain segments from three donor chromosomes. Class 1 detachments contain the centric fragment bearing the SD72 side of the C(2R)Su(pr). The detachment-generating break-point may give r ise to non-polar deletions in the 2P_ from the C_Q b_ side. This class may also carry non-polar deletions in the SD72 2Lh that were generated during C(2R) formation. Class 2 detachments will bear the cj} _ arm of the C(2R)Su (pr). Polar and non-polar deletions of 2_ are possible. Classes 3 and 4 detachments both contain a free arm donated by C(2R)Su(pr). Class 3 detachments are composed of chromosome segments from three sources. They may also carry 2Lh duplications from the g_D _ side. They may also carry segments of SD72 2Lh. They should, however, carry no 2R_. Class 4 detachments contain the free arm from the c_o bj* side of the C(2R)Su(pr). On these detachments, polar deletions for QD _-donated 2Rh may be generated. 57 Figure 3 Detachment products expected from the C(2R)SD72/cn bw configuration shown in Figure 1(B). The junction between the acentric and centric chromatid fragments that formed the C(2R) is shown by dark arrowheads. Outlined arrowheads show the position of the break-point necessary to generate the four detachment classes. These are numbered on the C(2R) to designate the four classes. 58 59 Materials and Methods Mutations and chromosomal rearrangements: A brief description of some of the genetic markers and chromosomal rearrangements used is given in Table 1. The rest are described in Chapter 2, Materials and Methods. Further details can be found in Lindsley and G r e l l . 2 4 The lethal deletions and EMS-induced lethal mutations used are shown in Figure 4. These are discussed In detail in H i l l i k e r . 5 4 Recovery of detachment products: C(2R)VK43.SD72/cn bw was detached in separate experiments with C(2L)VY1.b pr 1 and C(2L)VD3.nub2 b 6 6 h p r 1 . The three newly synthesized C(2R)Su(pr). described in Chapter 2, were detached in separate experiments with C(2L)VD3.nub2 b^ 6 h p r 1 . Virgin compound-2 females were treated with 2,500 rads of gamma radiation and mated to Df (2L,H61,pr/|n(2L8)0,Cy d p 1 v l p r 1 c n 2 (ln(2LR)Cy0) males. The number of treated females varied between experiments. The procedures for mating and culturing are described in Chapter 2, Materials and Methods. Each detachment product was established in a separate lines with In(2LR)Cy0. Scoring of detachment products for Su(pr): All detachments were scored for suppression over In(2LR)Cy0 both by visual inspection as described in Chapter 2, Materials and Methods. LethaI ity t e s t s : Each detachment product was tested for recessive lethality by crossing males and females that carried a detachment over In (2LR)CyO.Cy pr c n 2 . and scoring for £y_" progeny. Each lethality test was done In duplicate. If the detachment carried a recessive lethal, no Cy + progeny were recovered. If the detachment product was homozygous 60 Table 1A D e s c r i p t i o n of second chromosome mutat ions used. The chromosome 2 centromere is a t 55.1 Symbol Cy Name Cur ly Map P o s i t i o n 2 - 6 .1 D e s c r i p t i o n w ings cur Ied upward homozygous l e t h a l nu b 2 b 66h nubb in b lack 4 7 . 0 2 - 48 .5 a l l e l e of nub; smaI I s p o o n - s h a p e d w i n g ; l e s s extreme than nub b lack body Table IB D e s c r i p t i o n of chromosomal rearrangements used. Symbol ln(2LR)0,Cy d p 1 v l p r 1 c n 2 D e s g r i p t i o n Chromosome-2 ba lancer w i th m u l t i p l e rearrangements Df (2L )161 , pr PX d e l e t i o n 61 Figure 4 Genetic map of the centric region of chromosome-2. Shown are the relative positions and lengths of proximal deficiencies, and EMS-induced lethal mutations used for complementation tests of detachment products. 45-10 31 pr r l 34-7 A" M2-S10 34-2 45-75 stw M2-S8 M2-S4 63 viable, the f l i e s heteroyygous of the balance (£y.) and f l i e s homozygous for the detachment product (£y_j") were recovered in approximately a 2:1 ratio. Complementation tests: All homozygous lethal detachment products were tested with second chromosome heterochromatIc deletions and EMS-induced lethal point mutations shown In Figure 4. Each lethal detachment was f i r s t tested with Df(2L)C and Df(2L)M2-S10. Every detachment that was lethal with either of the big deletions, was then tested for a l l lethal point mutations uncovered by that deletion. Duplicate crosses were done In every test. 64 R e s u l t s and D i s c u s s i o n The recovery of detachment products is summarized in Table 2. Each detachment class is designated by the letter D, followed by the same code assigned to i ts progenitor C(2R). The f i r s t four columns show the r e s u l t s of detaching the four C(2R)Su(pr)s in combination with C(2L)VD3.nub2 b 6 6 n p r 1 . These four classes, termed col lect ively as the VD3 class, are summarized in column 5. Column 6 contains the class comprised of detachments synthesized from the C(2L)VY1. b pr:C(2R)VK45.SD72/cn bw stra in . Detachments of this class are designated as DVK43A to distinguish them from the DVK43 detachments of the VD3 class . The combined totals of a l l the detachment classes are shown in column 7. Altogether, a total of 6,805 virgin females were Irradiated, Including 5,625 of the C(2L)VD3.nub2  b 6 6 " pr 1 bearing strains, and 1,180 of the C(2L)VY1.b prl bearing strains. The five compound strains used varied in their v i a b i l i t y , and this Is re f l ec ted In the number of females co l l e c t ed from each class for irradiation. Hence, the smallest number of virgins were collected from C(2L)VD3.nub 2 b ^ h pr:C(2R)VF30.SD72/cn bw and the largest from C(2L)VD3.nub2 b^h p r 1 :C(2R)VF10.SD72/cn bw. Also, C(2R)VK43.SD72/cn bw proved to be more v iab le with C(2L) VD3. nub 2 b 6 6 " p r 1 . than with Q(2UVY1, b pr1. Variation in the frequency of detachment recovery was seen, as shown in lines 2 and 3. The v i ab i l i t y of the progenitor C(2R) did not correlate with the frequency with which detachments were recovered from i t . The C(2L)VD3.nub 2 b66h p r 1 :C(2R)VF5.SD72/cn bw strain, which was not the weakest used, nevertheless produced the lowest frequency of detachments 65 at 3.30 per 100 treated females. In contrast, the DVF30 class members were recovered at more than twice that frequency (8.80 per 100 treated females), even though the progenitor compound stock was by far the weakest used. The other detachment classes were recovered at frequencies that f e l l between these two extremes. It is interesting that DVK43 detachments were recovered at a considerably higher frequency (6.76 per 100 treated females), and those of the DVK43A class (4.92 per 100 treated females). This indicates that differences between C(2L)VD3.nub2 b 6 6 h pr and C(2L)VY1.b pr 1 determine, in part, the altered v i a b i l i t y of detachment products. A total of 320 detachments of the VD3 class were recovered at an average frequency of 5.69 per 100 females treated. An additional 58 DVK43A chromosomes were recovered at a rate of 4.92 per 100 females treated. OveralI, a total of 378 detachments were generated at a frequency of 5.55 per 100 females treated. The most significant result of the detachment experiment is the recovery of both suppressing and non-suppressing chromosomes. This Is shown In Table 3, IInes 1 and 3. Of the 378 detachment products recovered, 178 (47.09$) carried Su(pr). Of these suppressing detachments, 123 were of the VD3 class and 55 were of the DVK43A class. The majority of detach-ments recovered were non-suppressing. A total of 200 (52.91$) had lost Su(pr). The retention of Su(pr) In the VD3 class (38.4$) was much less than in the DVK43A class (94.38$). There Is striking variation between detachment classes regarding the retention a loss of Su (pr) (Table 3, lines 2 and 4). At one extreme of this variation is DVF30, wherein 90.48$ of the chromosomes have lost the ab i l i t y to suppress. At the other extreme Is DVK43A, wherein 94.83$ of 66 Table 2 Recovery of detachment products from C(2R)Su(pr) . VD3 * VD5 COMB I NED DVF5 DVF10 DVF50 DVK43 TOTALS DVK45At TOTALS Number of females treated 1,000 2,150 775 1,700 5,625 1,180 6,805 Number of detach-ments recovered 33 109 63 115 320 58 378 Detachment recovery per 100 females treated 3.30 5.07 8.13 6.76 5.69 4.92 5.55 * VD3 denotes 4 classes of detachment products that were recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VD3.nub2 b 6 6 " p r 1 . t The DVK43A class of detachment products was recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VY1.b p r 1 . 67 Table 3 Recovery of suppressing and non-suppressing detachments VD5 * VD3 COMBINED DVF5 DVF10 DVF30 DVK43 TOTALS DVK43At TOTALS Number of Su(pr) detachments recovered 15 37 6 66 123 55 178 Frequency {%) of Su(pr) detachment recovery 45.45 33.94 9.52 57.39 38.40 94.83 47.09 Number of non-suppressing detachments recovered 18 72 57 49 197 3 200 Frequency {%) of non-suppressIng detachment recovery 54.58 66.06 90.48 42.61 61.56 5.17 52.91 * VD3 denotes 4 classes of detachment products that were recovered from females bearing a C(2R)Su(pr) in combination with C(2R)VD3.nub2 b 6 6 " p r 1 . t The DVK43A class of detachment products was recovered from females bearing a C(2R)Su(pr) in combinationwith C(2L)VYT.b p r 1 . 68 the detachments retained Su(pr). There Is a large difference In Su(pr) retention between DVK43 (57.39$) and DVK43A (94.83$), Indicating that the C(2L) used may be significant in the production of suppressing detach-ments. Even greater variation, however, Is seen within the VD3 class . This Indicates that different C(2R)Su(pr) have different propensities to retain Su(pr) during detachment. Many of the putative detachment-bearing individuals recovered were not viable, and 150 of them either died before mating, or failed to produce offspring. The number of surviving detachments, and the number of suppressing chromosomes and the number of non-suppressing chromosomes successfully established in stocks are shown in Table 4 on lines 1, 3 and 5, respectively. A total of 228 detachment-bearing stocks were establ ished for further ana IysIs. Overall , si ight I y more non-suppressing (63.50$) than suppressing detachments (56.74$) were viable. Great variation is seen in the rate of putative detachments which survived within each class, as shown on lines 2, 4 and 6. In the DVF5 class, for example, 100$ of the suppressing detachments survived, but only 42.11$ of the non-suppressing detachment-bearing individuals survived to establish stocks. The opposite Is seen in DVK43A. Here the great majority (90.91$) Of a l l non-suppressed f l i e s survived, while only 54.55$ of the suppressed Individuals survived. Therefore, the v iab i l i ty of these detachment products does not appear to correlate with the loss or persistence of Su(pr) act iv i ty . These findings show that suppression is not dependent on the overall cons t i tu t ion or integrity of the C(2R)Su(pr) chromosome. Rather, it 69 Table 4 The establishment of detachment-bearing stocks VD5 * y_P3 COMBINED DVF5 DVF1Q DVF30 DVK43 TOTALS DVK43At TOTALS Number of detachment stocks established 23 71 36 65 195 33 228 Percent v iabIe detachments viable 69.69 65.14 57.14 60.94 56.52 56.90 60.32 Number of Su(pr) stocks estabIished 15 16 6 34 71 30 101 Percent of viable Su(pr) detachments viable 100.0 43.24 100.0 52.31 57.72 54.55 56.74 Number of non-suppressing detachment stocks estabIished 8 55 30 31 124 3 127 Percent viable non-suppressing detachments 42.11 76.39 52.63 63.26 62.94 90.91 63.50 * VD3 denotes 4 classes of detachment products that were recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VD5.nub2 b 6 6 " p r 1 . t The DVK43A class of detachment products was recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VY1. b p r 1 . 70 appears that a stable alteration on one of the component arms induced Su(pr) ac t iv i ty , and this change may persist through subsequent chromosomal rearrangements. Since non-suppressing detachments were also recovered, it may be reasonably asked whether there are any detectable differences between suppressing and non-suppressing detachments. It may also be reasonable to expect that due to the v a r i a t i o n In the pos i t ion of break-points occurring during C(2R) attachment and detachment, the detachment stocks wil l vary signif icantly in heterochromatic content and arrangement. This v a r i a t i o n provides an opportunity to search for patterns of heterochromatic constitution that correlate with the loss or retention of Su(pr). Some types of alterations, such as heterochromatic inversions and duplications cannot be detected in this study. However, lethality with the vital heterochromat ic markers can be used to detect deletions on the detachment products. The position of radiation-induced break-points in heterochromatin is unpredictable, If not random. 3 9 Because of th is , generation of detachments from either arm of the C(2R)Su(pr) are expected In similar frequencies. However, when the detachment stocks were tested for sta pseudo-dominance, only two, DVF10-62 and DVK43A-147 were revealed to be carrying the SD72 side of the progenitor C(2R)Su(pr) Both were non-suppressors. Such a d i sproport ionate ly low recovery of detachments bearing the SD72 arm Indicates that these products had a very low v i a b i l i t y . The attempt to localize the suppressor s i te , therefore, employed detachments a l l bearing the sn Jb_a side of the C(2R)Su(pr). As noted above, lethal deletions wil l be generated In the detachment procedure if the break-points on the captured arm occur distal to a v i ta l 71 gene . 4 4 Deletions of a v i ta l s i te may also Include a considerable amount of flanking heterochromatin. Hence, If any such deletion encompassed a s i te or region responsible for Su(pr). It might be local ized by correlating the loss of suppression with specific lethal deletion classes. The detachment products were f i r s t tested for homozygous lethal ity. Lethality may indicate the presence of heterochromatic delet ions and these chromosomes were designated for further testing. The results are shown In Table 5. Line 3 shows that the percentage of homozygous lethal ity varied between detachment classes, from a low of 45.45$ for DVK43A, to a high of 100$ for DVF5. As shown on lines 4 and 5, there is no apparent correlation between homozygous lethality and the loss or retention of Sjjieri. Heterochromatic delet ions c a r r i e d on detachment products were Identified by using two large chromosome-2 deletions. Deletions for 2|_ were tested by using Df(2L)C'. which Is deficient for at least seven vital l oc i . Deletions for 2P_ were tested by using Df(2R)M2-S10 which lacks a l l 2P_ and uncovers at least six vi tal gens. When the homozygous lethals were tested over Df(2L)C' and Df(2R)M2-S10. it was found that the majority did not carry heterochromatic deletions. Whereas, 165 (72.37$) of the detachments were homozygous lethal (Table 5), only 79 (34.65$) detachments were deleted for chromosome-2 heterochromatin. This latter percentage agrees with recovery of detachment-generated deletions on the third chromosome by Hi l lker and Holm. 4 4 In that study, however, most homozygous lethal detachment products were found to carry heterochromatIc de le t ions . 4 4 The findings of this study dif fer , in that 86 lethals are not deleted for heterochromatin. Of these, 48 are 72 Table 5 Lethality tests of detachment products DVJ DVF10 DVF30 DVK43 DVK43A TOTALS Number of detachments tested 23 71 36 65 33 228 Number of lethal detachments 23 55 18 54 15 165 Percent lethal detachments 100.0 76.47 50.0 83.08 45.45 72.37 Number of lethal Su(pr) detachments 15 8 0 22 12 57 Number of lethal non-suppressing detachments 8 47 18 32 3 108 73 suppressors and 38 are not. Hence, there is not a strong correlation between this lethality and retention or loss of Su(pr). It Is known that compound chromosomes accumulate recessive lethals in the proximal region with t i m e . 5 5 The two C(2L) used In the detachment study had been in existence for some time and It is possible that they donated recessive lethal mutations to the detachment products. Table 6 shows the results of tests for heterochromatic deletions. The detachments are c lass i f ied as carrying either no vital heterochromatic de l e t i on , a 2JJ] de le t ion , a 2RJ] deletion, or a centromere spanning deletion (2LRh). Of the 228 chromosomes established In viable lines, 149 have no detectable heterochromatIc deletions. Another 79 have lethal heterochromatic deletions. As shown on the bottom row, there was variation in the percentage of lethal deletions found in each class . Three of the detachment classes (VF5, VF30, VK43) show close agreement with each other, and to the findings of H i l l i k e r and Holm. 4 4 In each of these cases, a l i t t l e more than 30$ of the detachments carried heterochromatic deletions. The greatest frequency of deletions (45.10$) was seen in DVF10. The DVK43A showed a much lower frequency of lethal deletion bearing detachments (15.15$). The C(2L)VY1.bpr1 used to generate detachments of this class Is known to carry a large 2RJ] duplication (Including the genetic marker r l + ) . This duplication could rescue many 2Rh deletions generated during detachment. 74 Table 6 HeterochromatIc deletions carried on detachment products DVF5 DVF10 DVF3Q DYK43 DVK43A TOTALS Number tested 23 71 36 65 33 228 2Lh deletion 0 7 4 5 0 16 2Rh deletion 8 24 9 16 5 62 2 LRh deletion 0 1 0 0 0 1 no deletion 15 39 23 44 28 149 Percentage deletions In class 34.78 45.10 36.11 32.31 15.15 65.35 75 The detachments were next analyzed for the relationship between deletions and the presence or absence of suppression. Table 7 shows the detachment strains that carry no detectable heterochromatIc deletions. Among the VD3 detachments, there is considerable variation with regards to the retention of Su(pr). All viable VF5 detachments were suppressors. In contrast, the majority of non-deleted DVF10 members had lost Su(pr). In the other two VD3 classes, DVF30 and DVK43, the majority of undeleted attachments were able to suppress. The combined VD3 results, shown in column 5, reveal that only a sl ight majority (52.89$) of detachments retained Su(pr). as opposed to those losing it (47.11$). Like DVF5, the non-deleted DVK43A detachments a l l maintained the ab i l i ty to suppress. The combined totals , shown in the last column of Table 7, Indicate a tendency to maintain Su(pr) on non-deleted detachments. Although suppressing detachments are in the majority in non-deleted classes, It is not a convincing correlation. Furthermore, while it might be true that non-deleted detachments are more likely to retain the ab i l i ty to suppress, there remains the problem of demonstrating what has occurred on the non-suppressing chromosomes. It may be that the s i te or region responsible for Su (pr) can be deleted, but does not contain v i t a l genes. If so, Su (pr) could be lost without generating lethal deletions. A second possibi l i ty is that the deleted area Is masked by a compensating duplication. If this were the case, loss of the dominant suppressor would be evident, but the lethal deletion would be reduced. A third poss ibi l i ty Is that some unknown mechanism which Induces Su(pr) during C(2R) formation, is reversed during the detachment presence of 76 Table 7 The retention of Su(pr) on detachments not carry ing heterochromatic delet ions I I ' i I ^ I I • I I I I I I I II I I  ' 1 . VD3* V.D3 COMBINED DVF5 DVF10 DVF30 DVK43 TOTALS DVK43At TOTALS Su(pr) 15 14 25 30 64 28 92 (52.89$) (100$) (61.74$) pr 0 25 18 14 57 0 57 (47.11$) (0$) (38.26) TOTALS 121 28 149 (100.0$) (100.0$) (100.0$) * VD3 denotes 4 c lasses of detachment products that were recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VD3.nub2 b 6 6 n p r 1 . t The DVK43A c lass of detachment products was recovered from females bearing a C(2R)Su(pr) In combination with C(2L)VY1.b p r 1 . 77 lethal procedure. The val idi ty of any of these explanations could only be demonstrated through an extremely detailed analysis of the progenitor compounds and each of their detachment products. Since this is not possible in this study, the undeleted detachments remain a class that cannot be sat isfactori ly explained. The relationship between the presence of lethality and the loss of Su(pr) Is more revealing, as shown In Table 8. Here a clear correlation emerges between lethal heterochromatic deletions and the loss of Su(pr). The great majority (90.54$) of deletion-bearing VD3 detachments had lost Su(pr) f compared to the small group that retained It (9.45$). The DVK43A detachments did not show such a strong correlation, but the class size was small. The overall findings indicate that loss of heterochromatin strongly predisposes suppressor chromosomes to the loss of Su(pr). The 70 non-suppressing detachments provide an opportunity to test whether their deletions fa l l in specific areas and hence define a region necessary for Su(pr) action. The nine suppressing detachments are also of use in this regard, especially If their deletions fa l l In regions dist inct from the non-suppressing detachments. In order to l o c a l i z e Su (pr) in this way, a l l detachments were c lass i f ied according to the presence of lethal deletions in 2Lh and/or 2Rh and the retention of Su(pr). Table 9 shows this comparison for 2Lh deletions uncovered by Df(2L)C'. AlI 16 detachments that were found to carry 2Lh deletions came from the VD3 class. Of these, s l ightly more had lost Su (pr) (10) than retained i t (6), but the difference is not s ignif icant. Since the majority of a l l VD3 detachments have lost Su(pr). with or without deletions, these few deletion-bearing non-suppressors 78 Table 8 The retention of Su(pr) In the presence of heterochromatic deletions VD3 * V_D3 COMBINED r_F_ DVHQ DYF30 DVK43 TOTALS DVK43At TOTALS Su(pr) 0 2 1 4 7 2 9 (9.45$) (40.0$) (11.39$) pr 8 30 12 17 67 3 70 (90.54$) (60.0$) (88.61$) TOTALS 74 5 79 (100.0$) (100.0$) (100.0$) * VD3 denotes 4 classes of detachment products that were recovered in females bearing a C(2R)Su(pr) In combination with C(2L) VD3. nub 2 b 6 6 h p r 1 . t The DVK43A class of detachment products was recovered from females bearing a C(2R)Su(pr) in combinationwith C(2L)VY1.b p r 1 . 79 Table 9 The presence of Su(pr) on detachment products bearing deletions of 2Lh VD3 * VD3 COMBINED DVF5 DVF10 DVF30 DVK43 TOTALS DVK43At TOTALS Su(pr) 0 1 1 4 6 0 6 (39.50$) (37.50$) pr 0 6 4 1 10 0 10 (62.50$) (62.50$) TOTALS 16 0 16 (100.0$) (100.0$) * VD3 denotes 4 classes of detachment products that were recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VD3. nub 2 b 6 6 " p x i . t The DVK43A class of detachment products was recovered from females bearing a C(2R)Su(pr) in combination with C(2L)VY1.b p r 1 . 80 may simply be part of the overall trend towards the loss of Su(pr). This is supported by the fact that the DVK43A class contained no non-suppressors with 2Lb deletions. However, since no 2|J] deletions were found in this class, the possible significance of 2L± deletions remains undefIned. A much clearer trend Is seen In the case of 2fih deletions uncovered by Df (2R)M2-S10. This is shown In Table 10. Of the 62 detachments carrying 2Rh deletions, 59 (95.16$) had lost Su(pr). while only 3 (4.84$) retained the ab i l i ty to suppress. Moreover, the trend seen for detachments not bearing deletions differed from the test involving 2Lh. Whereas, in the previous test suppressors lacking deletions had been in the minority, here they were in the majority. Of the 166 non-deleted detachments, 100 (60.24$) retained Su(pr). while 66 (39.76$) had lost It. Table 10 also shows that the trend towards the loss of Su(pr) being associated with 2Rh deletions is consistent for a l l VD3 classes. For three of these classes, DVF5, DVF30 and DVK43, a l l of the 2£h deletion-bearing detachments have lost Su(pr). In the DVF10 class, only 1 of 24 2RJ] deletion-bearing detachments maintained suppression. Only the DVK43A class varied from this trend In that two of the five deleted detachments which maintained suppress ion. The presence of Su(pr) in a l l deletion classes Is summarized In Table 11. A G-stat lst ical analysis was done on the distribution of detachments within the classes shown. 5 6 The analysis tests the hypothesis that the retention or loss of Su(pr) is not related to the absence or presence of a lethal heterochromatIc deficiency. The VD3 class alone was 81 Table 10 The retention of Su(pr) on detachment products bearing deletions of 2Rh VD5 * VD5 COMB I NED D _ _ DVF10 DV.F30 DVK43 TOTALS DVK43AT TOTALS Su(pr) 0 1 0 0 1 2 3 (1.75$) (40.0$) (4.84$) pr 8 23 9 16 56 3 59 (98.24$) (60.0$) (95.16$) TOTALS 57 5 62 (100.0$) (100.0$) (100.0$) * VD3 denotes 4 classes of detachment products that were recovered from females bearing a C(2R)Su(pr) in combinationwith C(2L)VD3. nub 2 b 6 6 h p r 2 . t The DVK43 c las s of detachment products was recovered fromfemales bearing a C(.2R)Su(pr) In combination with C(2LVY1. b p r 1 . Table 11 A G-statlst ical test for the retention of Su(pr) and a l l DVD3 deletion classes Deletion Type Su(pr) PX TOTAL,? None 64 57 121 2Lh 6 10 16 2Rh 1 56 57 TOTALS 71 124 194 p < .005 at 2 df 83 used because there were insufficient deleted DVK43A detachments for analysis. The G-statisticaI test confirms that these detachments do not represent a homogeneous population. The probability of this distribution being random is less than .005 (Table 11). Overall , these results Indicate that detachments not bearing a 2Rh deletion have an increased probability of retaining Su(pr). Conversely, loss of v i ta l 2Rjh almost always results in the loss of Su(pr). The size of the 2RJ] deletions on the 62 non-suppressing detachments was approximated by testing them over the previously generated deletions and EMS-Induced point mutations shown In Figure 5. The f ive polar deletions were generated by the detachment of C(2R) chromosomes, and uncover 5 groups of v i ta l genes. Subsequent EMS mutagenesis revealed at least 6 v i ta l loci in 2RJ]. 5 4 The position of these vital loci divides the 2Rh into 6 regions as shown, corresponding to the Intervals between them. This allows the size of the deletion on the non-suppressing detachments to be approximated by lethal pseudo-dominance over the EMS lethal mutations. A range of deletion sizes, like those in Figure 5, is expected if a l l detachment products are recovered. The Intent was to ascertain whether the loss of Su(pr) correlated with a specific type of deletion. It is also of Interest to test whether or not the three suppressing chromosomes carry a type of deletion that is dist inct from the non-suppressors. The results of these studies are summarized in Figure 5. Four c lasses of deletion were found corresponding to the interval in which their distal break-point f e l l . The distal break-point can fa l l anywhere within this Interval. Of the 62 detachments tested, 61 carried polar deletions. As in the study of 84 Figure 5 Deletion classes recovered in detachment products. 45-10 31 r l 34-7 A" 34-2 45-75 M2-S10 stw M2-S8 M2-S4 CLASS DVF5 DVF10 DVF30 DVK43 DVK43A TOTALS NUMBERS 6 2 21 1 1 7 2 15 1 55 4 I 1 86 H i l l i k e r and Holm 4 ^ the deletions vary considerably in size. These results di f fer , however, in that no deletion with a distal break-point proximal to EMS 34.7 was recovered. In the original study, two deletions of this type (A and B) were recovered out of 18 2_ deletions. It Is interesting that not one deletion of the type was recovered in t h i s study, even though almost four times as many deletion-bearing detachments were generated. The 62 deletions are grouped into four classes as shown in the summary of Figure 5. Two detachments comprised the type III c l a s s . These were both recovered from the SD72 side of the C(2R)Su(pr). as discussed above. One of these detachments came from the DVF10 class and one from the DVF30 class . These results confirmed that both detachments are deleted for a l l v i tal loci proximal to stw. that none of these loci had been duplicated from the C(2L). The constitution and source of the heterochromatin proximal to EMS 45.10 cannot be determined. If the detachment was generated from the centric fragment of the C(2R)Su(pr) with a captured 2L free arm, then these two detachments could carry a smalI undetected piece of 2R_ proximal to EMS 45.10 from the C(2R)Su(pr). Depending on the way in which the progenitor C(2R)Su(pr) was made, this 2Rh segment could have or ig inated from e i ther SD72 or i ts homologue. Alternately, If In the detachment process the captured arm came from the SD72 side of the C(2R)Su(pr). and if the C(2L) carried a 2P_ duplication, the detachments of class III might carry a segment of 2p_ proximal to F_$ 45.10 donated by the C(2L) centric fragment. This serves to i l lustrate that the role of 2 _ proximal to EMS 45.10 cannot be discerned In this study. 87 Four deletions of type I I were recovered, two from DVF1G and two from DVF30. This class was identified by the fact that its members were lethal over EMS 34.2 and Df(2DMS-24. but not over EMS 45.75. These four deletions are analagous to the A' deletion generated by H i l l i k e r and Holm. 4 4 Genetically, these deletions removed the great majority of 2gh. The position of EMS 34.2 Is quite distal In 2gh and EMS .45.75 is near, if not at, the euchromatic j u n c t i o n . 5 4 The small number of detachments in this class is due, at least in part, to two factors. F i r s t , the assumed random d i s t r i b u t i o n of break-points along the heterochromat In should result in the great majority of break-points occurring to the left of EMS  34.2. Secondly, the proximity of EMS 3.4.2 to EMS .45.75 presumably presents a relat ively small target, hence very few detachments will be generated from a break-point that fa l l s between them. These detachments have lost Su (pr) while retaining this small fragment of 2fih. Therefore class II rules out only the most distal region of 2£h for a Su(pr) s i te . One of the type IV detachments was picked up from the DVF30 class. This chromosome Is unusual in that Its deletion is apparently non-polar, being lethal over both 54.2 and Df(2R)M2-S4. From th is , we know that this deletion cannot extend as far as EMS 45.75. and its proximal limit must be distal to EMS .34.7 In the large region depicted by the dotted line. Hence the parameters that define this deletion are very wide. It Is hard to explain how a deletion was generated. Theoretically, if the C(2L) was duplicated past EMS 54.7. it could have donated the proximal markers, and a break-point on the captured arm between EMS 34.7 and EMS.45.75 would then have generated the observed deletion. But the C(2L) 88 used is known not to be duplicated as far as r_!» therefore some other explanation is required. A feasible explanation is suggested by the recovery, In a previous s tudy 4 4 , of an apparent deletion of a similar type in distal 2J_. Though genetically behaving as a non-polar def ic iency , examination of the polytene chromosomes revealed that it was In fact a quasi-rec Iproca I translocation. A three hit even during the detachment process had resulted in the translocation of a large block of 2_ to the right arm of chromosome 3. This Juxtaposition of hetochromatIn into a euchromatic region resulted in a position effect variegation for two of the three known loci in the transposed segment. The v i ta l gene closest to the break-point was total ly inactivated, and the eye marker _ showed the classical position effect variegation for light eyes. The third v i ta l gene was not affected, presumably because It lay far enough away from the break-point to evade the spreading effect. Examination of the polytenes was not done for the DVF30 detachment and so Its constitution is not known, but its apparent s imilarity suggests that It might likewise be the result of a multiple hit event. Alternatively, this detachment may have undergone a mutation at the EMS 34.2 loci coincident with the detachment procedure. Without a detailed characterization, this detachment does not help locate Su(pr). The type I deletions comprise the largest class with 55 members, 53 non-suppressing and 2 suppressing. This group is composed of detachments that are lethal over the four proximal v i ta l genes. As shown in Figure 5, the distance between the two loci that define the limits of this class, EMS 34.7 and EMS 34.2 define approximately one third of 2P_. The 89 extent of this region means that the size of deletions In this class may vary greatly. Even without knowing the exact size of each deficiency within this group, however, certain Inferences can be drawn regarding the placement of Su(pr). Such inferences are based on the assumption that the break-points that determine the distal boundary of deletions in this class occur with equal frequency throughout the region between EMS 34.7 and EMS 34.2. If this is true, and Su(pr) resides in this interval, then it could reasonably be expected that some detachments of this class would retain the ab i l i ty to suppress. Furthermore, the frequency with which suppressing and non-suppressing detachments of this class were recovered would Indicate the approximate position of Su(pr) within this interval. If Su(pr) was located equidistant from EMS 34.7 and EMS 34.2. then the occurrence of break-points on either side of the putative Su(pr) loci would result In the generation of approximately equal numbers of suppressing and non-suppressing members of this class. If the Su(pr) loci resided in the distal portion of the Interval, then a greater proportion of break-points would f a l l proximal to i t . Correspondingly, a greater number of detachments retaining Su(pr) would be recovered. Conversely, if Su(pr.). resided proximally In the interval, then more break-points would be expected to fa l l distal to i t . Hence it would be deleted from the majority of detachment products generated from the on free arm. In fact, however, only 2 out of 55 members of this class retained Su(pr). Because the proportion of suppressing detachments in this class is so smal l , i t argues that if Su(pr) resides in this region, it must be located very close to the proximal demarcation of this class, EMS 34.7. 90 There are, however, other considerations that would make the placement of Su(pr) d i s t a l to EMS 34.7 puzzling. The C(2R)Su(pr) studies in Chapter 2 implicated a segment of 2f_i from SD72 in the induction of Su(pr). If Su(pr)is a dist inct s i te lying distal to EMS 54.7. and is donated by SD72. then each C(2R)Su(p.r) would have to be duplicated for that segment of SD72 2Rh to a position past EMS 34.7. This would not be expected in more than 50$ of alI C(2R)SD72 synthesized. Yet over 90$ of C(2R)SD72/cn bw chromosomes recovered were suppressors. When this is taken into consideration, along with the fact that only 2 p r + detachments of this class were recovered, there is not a convincing case that Su(pr) resides distal to EMS 54.7. As an alternative explanation of the two exceptional detachments, perhaps they have had Su(pr) deleted, but coincidentally are p r + revertants. The bulk of the data from the detachment studies argues that Su(pr) resides in the proximal region of 2Rh. This is consistent with what is known of the chromosome mechanics involved in C(2R)SD72/cn bw formation and detachment. Unfortunately, could not be more accurately placed relative to the three proximal v i ta l loci because no deletions uncovering them were recovered. However, the large number of non-suppressing detachments in deletion class I argues that Su(pr) l ies either distal but very close to, or proximal to EMS 54.7. The c lass i f icat ion of the deletions recovered (Figure 6) can be used to part ia l ly deduce the constitution of the detachments on which they are carried. There are 8 possible detachment types, as described in the introduction to this chapter. The c l a s s i f i c a t i o n of the recovered detachments is restricted to the 4 classes bearing the CD _ side of the 91 C(2R)Su(pr) (Figures 2 and 3). Two of the possible detachment types are those of class 2 and class 4, shown in Figure 3. It would be possible to generate the observed polar deletions on the cu b_w. free arm in both these cI asses. These 2 classes are similar in that neither is expected to carry any segments of 2RJ] from SD.7.2. It cannot be determined In this study whether the v i ta l gene-conta In Ing segments of 2RJ} necessary for Su(pr) act ivi ty originated from S.D7.2 on the £n bjfl-bearing homologue. However, in Chapter 2 it was found that the cn. bjf second could be substituted with a wild-type second in a C(2R)Su(pr). It was also found that the presence of a chromatid fragment from SD72 was strongly Implicated In Su(pr) Induction during C(2R) formation. Hence, while It is possible that the two detachment classes under consideration might be found in the deletion classes, the findings presented in Chapter 2 suggest that class 2 and class 4 detachments (Figure 3) would be unIikely to carry Su(pr) even when they are deletion free. It is consistent with the findings of this study that these two rearrangement types at least part ia l ly comprise the non-deleted, non-suppressing detachment class (Table 7). The other 2 detachment candidates are found in class 2 and class 4 of Figure 2. Again, the recovery of both of these classes could generate polar deletions on the cu bjy free arm derived from a C(2R)Su(pr). In contrast to the 2 classes discussed above, both these classes could carry segments of 2RJ3 from SD72. It is most consistent with the finds of this study that one or both of these detachment types comprise the non-deleted suppressing class (Table 7). These findings also suggest that either or both of these detachment c lasses at least p a r t i a l l y comprise the deletion-bearing class that has lost Su(pr) (Table 10). CHAPTER 4 RECOMBINATION MAPPING OF Su(pr) 94 Introduction Recombination mapping was undertaken in order to locate Su(pr) more accurately within the region of 2_ defined by the deletion mapping. This study employed the two visible heterochromatic markers, _ , which resides near the border of 2L_, and r_L, which resides in 2P_. Rol I ed is thought to reside at a point which approximately bisects the smallest deletion class recovered. Mapping relative to rj., therefore, would reduce by half the region In which Su(pr) must reside. A chromosome was constructed for this purpose bearing the markers J_f_ p_c4 _ j _ . The recombination studies will also serve 2 other purposes. First, detachment products that have had p_r1 replaced by p_c4 w j | | be recovered. In the p _ c 4 homozygote, these recombinants will test whether Su(pr) can suppress this EMS-induced a l l e l e . Second, the recombinant class in which px 1 has been crossed off the Su (pr) detachment can be made homozygous, and tested for the re-emergence of the mutant phenotype. This will show that a fully mutant a l l e l e was present on the suppressing detachment. Figure 1 shows the JJ_ p x c 4 _ r j in combination with a suppressing detachment, and the five types of recombinants expected. It is the b_ _ (Interval 1) and h. Li (Interval 1 and 2) recombinants that will determine the position of Su(pr). The recovery of h. 11 Su(pr) recombinants would place Su(pr) to the right of i±, confirming the results of the deletion mapping in Chapter 3. The greater the proportion of suppressing b_ i t recombinants, the further it Is expected the Su(pr) will l i e to the right of i±. If no suppressing b_ r_L recombinants are recovered, then Su(pr) will be located to the right of r_. If» however, Su(pr) lies proximal to 95 r_[, then both suppressing and non-suppressing & cl recombinants are expected. The more proximal the Su(pr) locus, the greater the number of expected suppressing b_ r j recombinants. Single exchanges in Interval 1 or Interval 2 replace p_r! with px c 4 on the suppressing detachment (Figure 1). The Tft locus is t ightly linked to px so that it may safely be assumed that px c 4 is present, even if it is suppressed on a I f l pxc4 ^j- $ u (p r ) (interval 1) or U t pxc4 Su (pr) (Interval 2) recombinant. The & i t E l recombinant generated by a single exchange In interval 2 that wil l be used to test the re-emergence of px1 phenotype. Recombination events occur far less frequently In heterochromatin than euchromatin. T a t t e r s a l l 5 7 found that spontaneous exchanges between i t and rj. occurs at a frequency of 0.1$. However, radiation Induced recom-bination occurs at 2-6 times the spontaneous frequency in the heterochro-matic interval. For this reason, radiation Induced recombination was chosen to map Su(pr). Consequently, the recombination frequencies wil l not accurately reflect map distances. Also, the suppressor detachments used have undergone serial rearrangement, and t h e i r heterochromat ic region wil l be perturbed. This may also affect recombination. Hence, recombination products should be Interpreted as only indicating relative map positions, not actual map distances. 96 Figure 1 Recombination products expected from suppressing detachments in combination with T f t p r c 4 jj; r j . The recombination intervals of interest are the i±-r_L interval (1) and the px-J_t Interval (2). The recombination classes are categorized as products of single exchanges (Interval 1 or interval 2), or as double exchanges (interval 1 and interval 2). 97 b pr lt cn bw 98 M a t e r i a l s and M e t h o d s Mutations and chromosomes used; A brief description many of the genetic markers and chromosomes used in this study Is given In Table 1. The other mutations used are described in the Materials and Methods section of Chapters 2 and 3. Further details can be found in Lindsley and G r e l l . 2 4 The i ± c i chromosome used In this study Is described in Tattersal 1.57 The p _ c 4 cn and Tft r _ c 4 chromosomes used are described in Yim e_ aj.. 4? The Tft p _ c 4 _ r_ chromosome was constructed for this study in a Tft D_ c 4 / b . L i L i heterozygote female, by a spontaneous single exchange In the r _ - _ interval. Four suppressing DVK43A chromosomes designated as DVK43A-4, -16, -19 and -45 were used. None of the 4 possess known deletions and al l manifest good v i a b i l i t y . Synthesis of recombinant chromosomes: Recombinants were synthesized from each detachment in separate experiments. Recombinants were synthesized in females bearing a detached second in combination with the Tft r_r c 4 L i r_L chromosome. Virgin females were treated 24 hours post eclosion with 2500 rads of gamma Irradiation from a 6°Co source. Treated females were mated in batches of 25 to b, L i E l males and cultured as in Chapter 2, Materials and Methods. Recombinant individuals were recovered in five phenotypic classes; (1) l i t , (2) _ L i _ (or L i r_p, (3) _ _ (or _ ) , (4) Tft L i , and (5) b_ L i (or L i ) . Each recombinant was established in a separate line with as heterozygotes with In(2LR)CyO. Testing recombinants for Su(pr): Tft-bearing recombinants were tested in combination with the p _ c 4 cj] and ln(2LR)0. Cy dp1 v l pr 1 c n 2 chromosomes. Recombinants that did not carry Tft were tested with the I__ p x 0 4 _ _ 99 T a b l e 1 Description of second chromosome mutations used. The chromosome-2 centromere is at 55.1 Symbol Name Map Position Description Tft Tuft 2 - 55.2 dominant; extra brist les homozygous viabi I ity I ow p r c 4 purple 2 - 54.5 EMS-induced a l l e l e P_C c 4/px; purple eyes homozygous lethal 100 and In(2LR)CyO. Fl ies were scored for suppression by visual inspection as described in Materials and Methods section of Chapter 2 . 101 Results and PISCHSSIQH Results from the radiation-induced crossover experiement are presented in Table 2. The number of each type of female treated, the number and frequency of each recombinant type recovered, and the number of each recombinant type tested is shown. Exchanges In regions outside the 11-£l and the px-Lt Intervals were observed. Since these other exchanges do not affect the analysis, a l l recombinants were classified solely with regards to exchanges in the 2 intervals of interest. The h. recombinant class is indistinguishable from the parental type suppressing detachment, so b_ chromosomes were not tested. The analysis of the other 5 recombinant classes is presented below. The b . r l recombinants: This class is the result of an exchange in the H- r J interval (Figure 1). Twenty-eight chromosomes of this class were recovered as either rj. or Jb_ r l Individuals. Twenty-one chromosomes of this type were tested in a cross to the l i t p x c 4 It rl/ln(2LR)Cy0 tester stock. The results are shown in Table 3. Fifteen Jj r j recombinants showed wild-type eyes in combination with both tester chromosomes. Six b_ rj, recombinants expressed the fully px-eyed phenotype. The suppressing b_ E l chromosomes were the result of a cross-over between Su (pr) and rj.. This demonstrates that Su(pr) lies proximal to r_L in 2Eh. The recovery of such a large proportion of suppressing recombinants suggests that either there is a hot spot for chromosome breakage between Su(pr) and E l , or that the Su(pr)-rl interval is quite large. If the latter Is the case, then either Su(pr) fa I Is very close to the centromere, or the r j locus is more d i s t a l than previously thought. The 6 non-suppressing r_l recombinants were products of single exchanges 102 Table 2 Recombinants recovered from detachment products DVK45-.4 DVK45-16 DVK43-19 DVK45.-45 TOTALS Number of females irradiated 600 600 660 540 2,400 Number of progeny scored 6,050 4,825 6,870 3,670 21,415 Number of recombinants: (A) In the pr-lt Interval (1) Tft recovered 1(.02$) 5(.10$) 9(.13$) 6(.16$) 21(.10$) tested 1 4 9 5 19 (2) b It rl recovered 2(.03$) 2(.04$) 5(.07$) 0(0.0$) 9(.04$) tested 2 2 5 0 9 (B) In the It-rl interval (1) b rl recovered 7(.12$) 4(.08$) 13(.19$) 4(.11$) 23(.13$) tested 5 3 9 3 21 (2) Tft recovered 7(.12$) 6(.12$) 8(.12$) 3(.08$) 24(.11$) tested 7 6 5 2 20 (C) In the pr- l t & It-rl interval (1) b It recovered 3(.05$) 5(.10$) 2(.03$) 1(.03$) 11(.05$) tested 3 3 2 1 9 103 Tahle 3 Test of b r l recombinants for the presence of Su(pr) b pr1 r l x Tft . p r c 4 It r l b It r l Cy d p l v l p H c n 2 Progeny classes tested: (A) b p r M / T f t prc4|f r| Phenotyplc classes (1) Tft (2) Tft pr DVK45-.4 DVK43.-16 DVK43-19 DVK43-45 TOTALS 1 2 6 3 3 0 15 6 (B) b prV l/Cy d p 1 v l p r 1 c n 2 Phenotypic classes (1) Cy cn (2) Cy pr cn 4 1 1 2 6 3 3 0 15 6 104 between ] t and Su(pr). Considering the distance at which i ± is thought to reside from the centromere, fewer recombinant chromosomes of this type were recovered than expected. It may be that rearrangements In ZEh of the four suppressor detachments used might reduce crossing-over in this region. Alternatively, it may be that some exchange products were not recovered. The Tft It recombinants; The chromosomes are the reciprocal products of single exchanges in the l i - r J . interval. Twenty-four chromosomes of this class were recovered as either & Tft J_± or Tft i ± Fl individuals. These recombinants were tested in a cross to a px c 4 cn/1n(2LR)CyO tester stock. The results are presented in Table 4. Examination of the £y I f i F2 progeny tests for the presence of Su(pr) to the right of i ± . The abllIty of Su(pr) to suppress px c 4 can be tested in the Tft F2 progeny. F i r s t , considering the Qy Tft F2 class carrying the Tft px c 4 JL± and CyO £y px! on2 chromosomes; of the 20 chromosomes tested, 18 had wild type eyes and 2 expressed the px mutant. Hence, 18 chromosomes In this class picked up Su (pr) as the result of an exchange between J_i and Su.(pr). The two non-suppressing recombinants of this class resulted from an exchange in the Su(pr)-rl interval. Regarding the ab i l i ty of Su(pr) to suppress px c 4 , no Tft F2 progeny were recovered. This demonstrates that the Tft  p r c 4 11 p r c 4 gn class was lethal. Hence Su(pr) cannot suppress this EMS-induced a l l e l e of px, nor can it rescue its lethal effects. This in terpretat ion assumes that there are no other EMS-induced lethal s t ightly I inked to pxE4. The b It recombinants: Eleven recombinants of this type were recovered as h_ i ± or i l Individuals. They are the result of a double exchange, 105 Table 4 Test of Tft i ± recombinants for the presence of Su(pr) Tft prc4 i t cn bw x b It r l p r c 4 c n Cy d p ' v l p H c n 2 Progeny classes tested: (A) Tft prc4 | T r l cn bw Cy d y i v ' p H c n 2 Phenotyplc classes (1) Tft cn (2) Tft pr cn DVK43-4 DVK43-16 DVK43-19 DVK45-45 TOTALS 7 0 5 1 5 0 18 2 (B) Tft p r c 4 j t r l cn bw p r c 4 c n Phenotypic classes (1) Tft cn (lethal) (2) Tft pr cn (lethal) 106 with one in r _ - _ interval, and one in the _ - _ interval. Although fewer double exchange products were recovered than s ingle exchange products (Table 2) the number of chromosomes in this class was higher than expected. The analysis of this class with the r_c4 cn/ln (2LR) CyO tester stock Is shown in Table 5. Of the 9 t U recombinants tested, 8 were found to suppress px. These suppressing recombinants result from an exchange between _ and Su(pr). The one non-suppressing chromosome in this class resulted from and exchange between Su(pr) and j _ . As shown in Table 6, there is a disparate recovery of reciprocal products of exchanges in the i _ - r _ interval. The suppressing recombinant chromosomes In the Tft px c4 _ and _ recombinant class were expected in approximately the same frequency as the non-suppressing recombinant chromosomes in the _ recombinant class, since they were both generated by an exchange in the It-Su(pr) interval. Similarly, approximately equal numbers of suppressing and non-suppressing chromosomes were expected as the products of exchange in the Su(pr)-rl interval. In both cases, however, suppressor recombinant chromosomes were recovered predominantly. For the two classes produced from an exchange In the I t-Su (pr). 26 suppressed px and 3 did not. For the class resulting from an exchange in the Su(pr)-rl Interval, suppressing chromosomes were also predominant. There were 18 suppressing and only 2 non-suppressing r_ recombinants. As noted above, speculations based on the frequency of recombinant class recovery must be made cautiously in this case. However, these results 107 Table ;> Test of k i t recombinants for the presence of Su(pr) b pr1 It cn bw x p r c 4 c n . b It r l Cy dp1vI p r 1 c n 2 Progeny classes tested: DVK43-4 DVK45-16 DVK43-19 DVK45-45 TOTALS (A) b pr1 It cn bw Cy dyTvlpr'cn 2 Phenotypic classes (1) Cy cn 3 3 1 1 8 (2) Cy pr cn 0 0 1 0 1 (B) b pr1 It cn bw p r c 4 c n Phenotypic classes (1) cn 3 3 1 1 8 (2) pr cn 0 0 1 0 1 108 Table 6 Disparate recovery of suppressing and non-suppressing recombinant classes. Exchange interval Su(pr) px It-Su(pr) 26 3 Su(pr)-rl 15 6 TOTALS 41 9 109 raise the poss ibi l i ty that suppressing recombinant chromosomes were recovered preferentially. The Tft recombinants; Twenty-one recombinant chromosomes of this class were recovered as b_ Tft or Tft individuals (Table 2). These recombinants are the result of a single exchange In the px-ll interval (Figure 1). The results of testing these recombinants with the px c 4 cn/ln(2LR)CyO Is shown in Table 7. Of the 19 recombinants tested, a l l £y Tft progeny had wild-type eyes confirming the presence of Su(pr) to the right of px. No T f t progeny were recovered from any of the recombinants tested, confirming the Inability of Su (pr) to rescue the lethal effects of p r c 4 . These results agree with the testing of the Tft i± class (Table 4). The b It r l recombinants: The chromosomes that comprise this class are the reciprocal product generated by a single exchange in the px~i± Interval (Figure 1). Nine recombinants of this type were recovered as b_ ii r_l or Xt rj. individuals. No recombinants of this type were recovered from the DVK43A-45 suppressing detachment. All 9 recombinants were tested with the Tft pxc4 JJ- rl/ln(2LR)CyO tester stock as shown in Table 8. All progeny of both the Tf t and £y c lasses had ful ly px mutant eyes for each recombinant tested. This shows that the previous suppression of these px! a l le les is not due to reversion. It also confirms the findings In Chapter 2 that the suppression of px requires the continued presence of Sujpxi. 110 Table 7 Test of Tft recombinants for the presence of Su(pr) Tft prc4 c n bw x p r c 4 c n b It r l Cy dp1vI p r 1 c n 2 Progeny classes tested: DVK43-4 DVK43-16 DYK43-19 DVK43-45 TOTALS (A) Tft prc4 c n bw Cy dy 'v 'pr 'cn 2 Phenotypic classes (1) Cy Tft cn 1 4 9 5 19 (2) Cy Tft pr cn 0 0 0 0 0 (B) Tft p r c 4 c n bw p r c 4 c n Phenotypic classes (1) Tft cn (lethal) (2) Tft pr cn (lethal ) 111 T a b l e 8 Test of p. J_ Ci recombinants for the presence of Su (pr) b pr It r l x Tft prc4 | + r l b It r l Cy dplvl p H c n 2 Progeny.classes tested: DVK45-4 DV.K45-16 DVK43-19 DVK43-45 TOTAL.? (A) bpr It r l Tft p r c 4 l t r l Phenotypic classes (1) Tft It rI 0 0 0 0 0 (2) Tft pr It r l 2 2 5 0 9 (B) br Ii- r l , Cy d p 1 v l p r 1 c n 2 Phenotypic classes (1) Cy 0 0 0 0 0 (2) Cy pr 2 2 5 0 9 CHAPTER 5 SUMMARY 113 The preceding sections of this thes i s describe a new dominant suppressor of purple, Su(pr). which is located in the chromosome-2R heterochromat i n, to the left of the genetic marker rj.. The heterochromat in In Drosophi l a . and many other species, differs from euchromatin in cy to log i ca l appearance, genetic content, and types of DNA sequence. Cytologically, heterochromatin is characterized by staining properties that differ from euchromatin. Its denser stained appearance is due, In part, to its tendency to acquire a far more compacted state than euchro-matin. The distribution of heterochromatin in the genome Is non-random, and similar in many species. Heterochromatin is most often adjacent to the centromere, at the telomeres, and near the nucleolar organizer.58 Heterochromatin has long been thought to be genetically inert, but it Is now known that there are genetic loci are located in the hetero-chromat in of al I chromosomes of Drosoph 11 a melanogaster 4 3. In chromosome-2 heterochromat in, deletion mapping and EMS mutagenesis revealed the presence of 13 v i ta l loci.44, 54 None of the EMS-induced lethals behave as a de f i c i ency , and several loc i exh ib i t extensive and complex i n t e r a l l e l i c complementation. This strongly suggests that these loci ex i s t as interspersed unique sequence genes with v i ta l functions.54 It has also been demonstrated that two of the genetic elements associated with the Segregation Distortion (S_Q) phenomenon are also located within chromosome-2 heterochromatIn.59, 60 These loci exist at only about \% the gene density found in euchromatin.43 Heterochromatin may differ from euchromatin in that these unique sequences may be interspersed with the highly repeated sa te l l i t e DNA sequences. In Drosoph i la melanogaster. sa te l l i t e sequences are only 5 - 1 2 base pairs long, often A-T or G-C 114 r i ch , and can be present in a mil lion or more copies per genome (reviewed by Skinner^ 1). This study has identified an additional genetic function associated with chromosome-2R heterochromatin. There is not, however, sufficient evidence to indicate that Su(pr) is a unique sequence gene. Presently, loss of Su(p.r) Is associated with a specific class of deletion in 2_ heterochromatin. Consequently, it Is not possible to discern whether it is the deletion of a specific s i te , or of a larger region which causes the loss of suppression on these chromosomes. An EMS mutagenesis study of Su(pr) would be useful, in this regard, by establ Ish Ing whether suppression is the product of a mutable s i te . If an EMS-induced point mutation abol ished Su(pr) ac t iv i ty , there would be a strong suggestion that Su(pr) is a unique sequence gene. Failure to abolish suppression by point mutation would suggest that Su(pr) might be caused by repeated sequences, and possibly a position effect. An example of position effect in Drosoph iI a melanogaster Involving chromosomal rearrangement i l lustrates how this phenomenon might pertain to Su(pr). The recessive-visible eye mutant, facet-strawberry ( f a S W D ) . is defined as a very small deletion at the 5' end of the Notch locus. In preparation of the polytene X chromosome, the deletion is seen to be restricted to interband material. It appears that f a S W D arises because this deletion abuts the Notch locus against the nearest upstream band. This juxtaposition apparently induces a position effect, which allows genetic act iv i ty in the adjacent region to Interfere with the normal functions at Notch. When f a s w b is placed is c_L§ with upstream deletions in this region the wild phenotype returns. Apparently, t h i s occurs 115 because the interfering genetic functions that acted on Notch at close range are e l i m i n a t e d . Consequently, normal Notch function is re-established.62 It is possible to envision a similar occurence In the Induction and subsequent loss of Su(pr) ac t iv i ty . Deletions may be generated during the synthesis of new C(2R) chromosomes.44 It may be that during C(2R)SD72 synthesis, normally separated sequences are juxtaposed, and one of them establ i shes a position effect over the other, thus creating a novel genetic function. The entire region responsible for Su(pr) Induction could subsequently be deleted during the detachment process, thereby reinstating mutant target gene expression. As well, unique juxtapositions of heterochromatin are possible when C(2R) is synthesized from a chromo-some bearing a pericentric inversion. This Is true even if no deletions are generated in C(2R) synthesis. This may explain why suppressing C(2R) has been induced only from the inverted SD72. despite the fact that many C(2R) chromosomes have been synthesized from other types of second chromosome. Alternatively, it can be considered that by chromosome breakage, Su(pr) is induced through the activation of a previously s i lent . An example of this occurs in Zea mays in which the dominant suppressor, Dotted (D_) returns normal act iv i ty to a mutant a l l e l e of the anthocyanin gene (A). The A. gene produces pigment which is deposited in the pigment layer of the kernel. The D_ gene is a transposable element that may exist in an active or inactive state at several locations in the genome. Plants in which D± is introduced along with non-functional anthocyanin a l le les (a.) produce kernels with dots covering the aleurone layer of the 116 endosperm. These dots represent patches of the kernal in which D_ has suppressed § and reinstated normal A pigment production (reviewed by McCI in tock 6 3 ) . The f_ phenotype was f i r s t observed in Zea mays plants that were suspected of having undergone chromosome breakage. McCI Intock^4 performed an experiment to determine whether chromosome breakage could induce de novo Dt ac t iv i ty . This was tested In a plant that was homozygous for an a mutant on chromosome 3, and heterozygous for an Inverted duplication on one end of chromosome 9_. During meiosis in this plant, crossovers between the standard chromosome 9 and Its inverted homologue cause the formation of a dicentric bridge. When the fused homologues migrate during anaphase, the dicentric bridge breaks. The experiment had been designed so that only pollen grains carrying the inverted chromosome 9 would be viable. When the kernals were examined, suppression was confirmed by the appearance of a number of them in which patches of normal gene expression appeared as dots. Each dot was derived from a single cel l in which a s i lent D± element had been activated on chromosome 9 . 6 4 » 65, 66 The p_ element then suppressed the a mutation on chromosome 3. This gene expression persisted In a l l ce l l s of that lineage. The activation of D_ bears some s imi lar i t ies to Su(pr) Induction: (1) in both cases, suppression Is associated with chromosome breakage, (2) both Su (pr) and D± are dominant in their act iv i ty , and, (3) both suppressors are localized in heterochromatin. This study has not provided any evidence for the involvement of transposable element action In Su(pr) ac t iv i ty . However, the s imi lar i t ies between the suppressor studied here and D__ as well as the widespread implication of transposable elements In suppression,38 indicates an interesting avenue for future research on Su(pr). 117 LITERATURE CITEP 1. Reiger, R . , Michael i s , A. and Green, M.M., 1976. Glossary of Genetics and Cytogenetics. Springer-VerIag, Berlin and New York. 2. G o r i n i , L . , and B e c k w i t h , J . R . , 1966. S u p p r e s s i o n . Annu. Rev. Microbiol. 20, 401-421. 3. Hartman, P . E . , and Roth, J . R . , 1973. Mechanisms of Suppression. Adv. Genet. 17: 1-105. 4. 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Genetic analys i s of the centromeric heterochromatin of chromosome 2 of Drosoph iI a me Ianogaster : Deficiency mapping of EMS-Induced lethal complementation groups. Genet. 83: 765-782. 55. Parker, D . R . , 1954. Radiation induced exchanges in Drosophila females. Proc. Natl. Acad. Sc i . U.S.A. 4fl: 795-800. 56. Sokal, R.R. and Rohlf, F . J . , 1981. Biometry: the principles and practice of s tat i s t ics in biological research. 2nd ed. W.H. Freeman, San Francisco. 57. T a t t e r s a l l , P . J . , 1981. Induced recombination in the proximal regions of chromosome 2 In females of Drosoph i I a me I anogaster. M. Sc. Thesis, U.B.C. 58. Hannah, A . , 1951. Localization and function of heterochromatin in Drosoph iI a me Ianogaster. Adv. Genet. 4: 87-125. 59. Br i t tnacher , J . G . and Ganetzky, B . , 1984. On the components of segregation distortion In Drosoph ila me Ianogaster. III. Nature of Enhancer of SD. Genet. 1Q7: 423-434. 60. Sharp , C . B . , H i l l i k e r , A . J . and Holm, DG. , 1985. Further characterization of genetic elements associated with the segregation d i s t o r t i o n phenomenon In Drosoph iI a me Ianogaster. Genet. 110: 671-688. 61. Skinner, D.M., 1977. SatelIite DNA's. Bioscience 22: 790-796. 62. Welshons, W . J . , and Welshons, J . H . , 1985. Suppression of the facet-strawberry position effect in Drosoph 11 a by lesions adj'acent to Notch. Genetics 1_LQ: 465-477. 63. McCI intock, B . , 1984. The significance of responses of the genome to challenge. Science 22§: 792-801. 64. McCIIntock , B . , 1949. Mutable l o c i in maize . Carnegie Inst. Wash. Yearb. 4 § : 142-154. 65. McCI i n t o c k , B . , 1950a. Mutable l o c i in maize. Carnegie Inst. Wash. Yearb. 50: 157-168. 66. McCIintock, B . , 1950b. The origin and behaviour of mutable loci In maize. Proc. Natl. Acad. Sc i . U.S.A. 36: 344-355. 122 APPENDIX Spectrophotometric measurement of eye pigments reported in Chapter 2. S2 = TABLE.3 A495 X-X (x-x); ORR 1) 0.786 .006 0 2) 0.715 -.065 .0042 3) 0.835 .055 .0030 4) 0.866 .086 .0074 5) 0.699 -.081 ,0066 X) 0.780 .0212 C(2L)VY1,bpr; 1 ) 0.481 .086 .0074 C(2R)P,px 2) 0.368 -.027 .0007 3) 0.445 .050 .0025 4) 0.306 -.089 .0079 5) 0.377 -.018 ,0003 X) 0.395 .0188 C(2L)VF1,bpr; 1) 0.391 .090 .0081 C(2R)P,px 2) 0.399 -.008 0 3) 0.466 -.075 .0056 4) 0.387 -.004 0 5) 0.402 -.011 .0001 X) 0.391 .0138 C(2L)VF1,pr; 1) 0.318 -.065 .0042 C(2R)P,px 2) 0.243 -.010 .0001 3) 0.335 .083 .0069 4) 0.174 .079 .0062 5) 0.190 -.036 ,0013 X) 0.252 .0187 C(2L)VF1,pr b w 1) 0.253 .061 .0037 C(2R)P,px 2) 0.263 .071 .0050 3) 0.172 -.030 .0004 4) 0.131 -.031 .0009 5) 0.160 -.061 ,0037 X) 0.192 .0137 (X-X)2 n-1 percent w11d-type A495 = percent w iId-type A495 = percent w iId-type A495 = percent w iId-type A495 = percent w iId-type A495 = nz5 S 2 = s = 100 S 2 s 51 S 2 S 50 S2 S 32 S2 S 25 .0053 .0728 .0047 .0686 .0035 .0587 .0047 .0684 .0034 .0585 123 A495 X-X (X-X)2 C(2L)P,b; 1) 0.959 .074 .0055 C(2R)P,px 2) 0.921 .036 .0013 3) 0.941 -.056 .0031 4) 0.803 -.082 .0067 5) 0.799 -.086 ,0074 X) 0.885 .0240 C(2L)SH3,+; 1) 0.775 .015 .0002 C(2R)SH3,+ 2) 0.879 .119 .0142 3) 0.744 -.016 .0003 4) 0.701 -.059 .0035 5) 0.703 -.057 ,0032 X) 0.760 .0214 TABLE 4 ORR 1) 0.732 -.060 .0036 2) 0.820 .028 .0008 3) 0.837 .045 .0020 4) 0.755 -.037 .0014 5) 0.815 .023 ,Q005 X) 0.792 .0083 C(2L)VY1,bpr; 1) 0.981 -.031 .0010 C(2R)VK43,SD72 2) 0.988 -.024 .0006 cnbw 3) 0.947 -.065 .0042 4) 1.052 .040 .0016 5) 1.091 .079 ,0062 X) 1.012 .0136 C(2L)VF1,bpr; 1 ) 0.919 -.090 .0081 C(2R)VK43,SD72 2) 0.994 -.015 .0002 cnbw 3) 1.080 .071 .0050 4) 1.101 .092 .0085 5) 0.950 -.059 ,0035 X) 1.009 .0253 S 2 = (X-X) 2 n-1 percent w iId-type A495 = percent w iId-type A495 = percent w 11d-type A495 = percent w iId-type A495 = 0=5 S 2 = .0060 S = .0775 113 S 2 = S = 97 .0054 .0731 S 2 = .0021 S = .0455 100 percent w 11d-type A495 = S 2 S 128 S 2 = S = 127 .0034 .0583 .0063 .080 124 A495 XzX (X-X) : C(2L)VF1,pr; 1) 0.865 -.007 0 C(2R)VK43,SD72 2) 0.889 -.017 .0003 cnbw 3) 0.992 .120 .0144 4) 0.817 -.055 .0030 5) 0.799 -.073 .0053 X) 0.872 .0230 C(2L)VF1,prbw; 1) 0.854 .052 .0027 C(2R)VK43,SD72 2) 0.915 .113 .0127 cnbw 3) 0.740 -.062 .0038 4) 0.718 -.084 .0007 5) 0.781 -.021 .0004 X) 0.802 .0203 TABLE 5 ORR 1 ) 0.847 .059 .0035 2) 0.755 .033 .0011 3) 0.724 -.064 .0041 4) 0.843 -.055 .0030 5) 0.769 -.019 .0003 X) 0.788 .0120 C(2L)VY1,bpr; 1 ) 0.962 -.023 .0005 C(2R)VF5,$P72 2) 0.927 -.058 .0038 cnbw 3) 1.040 .055 .0030 4) 1.090 .105 .0110 5) 0.905 -.080 .0064 X) 0.985 .0243 C(2L)VF1,bpr; 1 ) 0.943 -.018 .0003 C(2R)VF5,SD72 2) 0.947 -.014 .0002 cnbw 3) 1.011 .050 .0025 4) 0.999 .038 .0014 5) 0.905 -.056 .0031 X) 0.961 .0075 S 2 = (X-X) 2 n-1 percent w iId-type A495 = percent w iId-type A495 = percent w iId-type A495 = 0^ S 2 = S = 110 .0058 .0758 S 2 = .0051 S = .0712 S 2 = .0030 S = .0548 percent w11d-type A495 = 100 percent w iId-type A495 = S 2 -S = 125 S 2 S 122 .0061 .0779 .0019 .0433 125 A495 X-X (X-X) 2 C(2L)VF1,pr; 1) 0.789 -.050 .0025 C(2R)VF5,SD72 2) 0.919 .080 .0064 cnbw 3) 0.789 -.050 .0025 4) 0.889 .050 .0025 5) 0.809 .030 .0009 X) 0.839 .0148 C(2L)VF1,prbw. 1) 0.915 .084 .0071 C(2R)VF5,SP72 2) 0.761 -.070 .0049 cnbw 3) 0.753 -.078 .0061 4) 0.925 .094 .0088 5) 0.803 -.028 ,0008 X) .0277 TABU 6 ORR 1) 0.788 .003 0 2) 0.704 -.081 .0066 3) 0.834 .049 .0024 4) 0.863 .078 .0061 5) 0.734 -.051 ,0026 X) 0.785 .0177 C(2L)VY1,bprj 1) 0.944 -.022 .0005 C(2R)VF10,SP72 2) 0.970 .004 0 cnbw 3) 1.015 .049 .0024 4) 1.003 .037 .0014 5) 0.899 -.067 ,0045 X) 0.966 .0088 C(2L)VF1,bpr; 1) 0.949 .001 0 C(2R)VF10,SP72 2) 0.903 -.047 .0022 cnbw 3) 0.995 -.045 .0020 4) 1.001 .051 .0026 5) 0.900 -.050 ,0025 X) 0.950 .0093 S 2 = (X-X) 2 n-1 percent w 11d-type A495 = percent w iId-type A495 = percent w 11d-type A495 = percent w 11d-type A495 = 11^ 5 S 2 = S = 106 .0037 .0608 S 2 = .0069 S = .0832 105 S 2 = S = 100 s 2 = s = 123 .0044 .0665 .0022 .0469 S 2 = .0023 S = .0482 percent w iId-type A495 = 1 2 1 126 A495 XzX (X-X) C(2L)VF1,pr; 1) 0.949 .084 .0071 C(2R)VF10,SD72 2) 0.864 -.001 0 cnbw 3) 0.791 -.074 .0055 4) 0.855 .020 .0004 5) 0.834 -.031 ,ooi.p X) 0.865 .0140 C(2L)VF1,prbw; 1) 0.835 .026 .0007 C(2R)VF10,SD72 2) 0.777 -.032 .0010 cnbw 3) 0.729 -.080 .0064 4) 0.823 .014 .0002 5) 0.881 .072 .0053 X) 0.809 .0135 TABLE 7 ORR 1 ) 0.802 .006 .0004 2) 0.855 .060 .0036 3) 0.715 -.080 .0064 4) 0.752 -.043 .0018 5) 0.851 .056 ,0031 X) .0153 C(2L)VY1,bpr; 1) 1.008 .039 .0015 C(2R)VF30,SP72 2) 0.998 .029 .0008 cnbw 3) 0.897 -.072 .0052 4) 0.893 -.076 .0058 5) 1.051 .082 ,0067 X) 0.969 .0200 C(2L)VF1,bpr; 1) 0.993 .031 .0010 C(2R)VF30,SD72 2) 0.943 -.019 .0004 cnbw 3) 0.962 0 0 4) 0.899 -.063 .0040 5) 1.015 .053 .0028 X) 0.962 .0082 S 2 = (X-X) 2 n-1 percent w 11d-type A495 = percent w11d-type A495 = percent w FId-type A495 = percent w iId-type A495 = 0=5 S 2 = .0035 S = .0592 110 S 2 = S = 103 .0034 .0581 S 2 = S = 100 s 2 -s = 122 S 2 = s = .0038 .0618 .0500 .0707 .0021 .0453 percent w iId-type A495 = 1 2 i 127 A495 X-X (X -X) : C(2L)VF1,pr; 1) 0.902 .043 .0018 C(2R)VF30,$P72 2) 0.781 -.078 .0061 cnbw 3) 0.799 -.060 .0036 4) 0.899 .040 .0016 5) 0.912 .053 ,0028 X) 0.853 .0159 C(2L)VF1,prbw. 1) 0.881 .063 .0040 C(2R)VF30,SD7? 2) 0.749 -.069 .0048 cnbw 3) 0.818 0 0 4) 0.753 -.065 .0042 5) 0.888 .070 .0049 X) 0.818 .0179 TABLE 8 ORR 1) 0.705 -.073 .0053 2) 0.819 .041 .0017 3) 0.819 .041 .0017 4) 0.839 .061 .0037 5) 0.707 -.071 .0050 X) 0.788 .0174 C(2L)VY1,bpr; 1) 0.475 .072 .0052 C(2R)VF12,§P72 2) 0.461 .058 .0034 cnbw 3) 0.445 .042 .0018 4) 0.321 -.082 .0067 5) 0.312 -.091 .0083 X) 0.413 .0254 C(2L)VF1,bpr; 1) 0.398 .033 .0010 C(2R)VF12,§p,72 2) 0.361 -.040 .0016 cnbw 3) 0.322 -.033 .0011 4) 0.339 -.026 .0007 5) 0.397 -.032 .0019 X) 0.365 .0055 S 2 = (X-X) 2 n-1 percent w iId-type A495 = percent w iId-type A495 = percent w iId-type A495 = percent w 11d-type A495 = S 2 = .0040 S = .0630 108 S 2 = S = 103 .0045 .0669 percent w11d-type A495 = S 2 = S = 100 s 2 s 52 S 2 s 47 .0044 .0659 .0064 .0796 .0014 .0371 128 A 4 9 5 X-X (X-X,)' C(2L)VF1,pr; 1) 0.195 -.064 .0041 C(2R)VF12,SJ2I2 2) 0.231 .028 .0017 cnbw 3) 0.291 .032 .0010 4) 0.268 .009 0 5) 0.310 .051 ,0026 X) 0.259 .0094 1) 0.161 -.044 .0019 2) 0.152 -.053 .0028 3) 0.244 .041 .0017 4) 0.271 .066 .0043 5) 0.198 .007 0 X) 0.205 .0107 S 2 = (X-X) 2 n-1 percent w iId-type A495 = percent w11d-type A495 = S 2 s 33 S 2 s 26 .0024 .0485 .0027 .0517 TABLE, ,9 ORR 1) 0.843 .048 .0023 2) 0.733 -.062 .0038 3) 0.818 .023 .0005 4) 0.816 .021 .0004 5) 0.796 -.031 .0020 X) 0.795 .0080 C(2L)VY1,bpr; 1 ) 1.089 .001 0 C(2R)VF3,?P72 2) 1.155 .067 .0045 cnbw 3) 1.061 -.027 .0007 4) 0.992 -.096 .0042 5) 1.145 .057 .0032 X) 1.088 .0176 C(2L)VF1,bpr; 1) 1.048 -.002 0 C(2R)VF3,SP7? 2) 1.090 .040 .0016 cnbw 3) 1.001 -.049 .0024 4) 1.103 .053 .0028 5) 1.010 -.040 .0016 X) 1.050 .0084 percent w iId-type A495 = percent w11d-type A495 = percent w iId-type A495 « S 2 = S = 100 s 2 s 137 S 2 = s = 132 .0020 .0447 .0044 .0663 .0021 .0458 129 A495 (X-X)2 C(2L)VF1,pr; 1) 0.898 .062 .0038 C(2R)VF3,SP72 2) 0.772 -.064 .0041 + 3) 0.786 -.050 .0025 4) 0.894 .058 .0035 5) 0.831 -.005 0 , X) 0.836 .0139 C(2L)VY1,prbw; 1) 0.775 -.027 .0007 C(2R)VF3,3P72 2) 0.854 .052 .0027 + 3) 0.729 -.073 .0053 4) 0.787 -.015 .0002 5) 0.866 .064 ,0041 X) 0.802 .0130 S 2 = (X-X) 2 n-1 percent w 11d-type A495 = percent w11d-type A495 = n=5 S2 S 105 S2 S 101 .0035 .0589 .0033 .0570 

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