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Putative deletions of the proximal heterochromatin of chromosome three in Drosophila melanogaster Baldwin, Madeline Carol 1969

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PUTATIVE DELETIONS OF THE PROXIMAL HETEROCHROKATIN OF CHR0K0501-E THREE IN DROSOPHILA. MEL*NOGASTER by MADELINE CAROL RALDWIN B.S., Tufts University, 1964 M.S., University of Connecticut, 1966 A THESIS SUBMITTED IN PARTLAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY -- - IN THE DEPARTMENT OF ZOOLOGY-Y/e accept this thesis as conforming to the reouired standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a 1 lowed w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Department o f i i •TABLE OF CONTENTS Page • INTRODUCTION 1 MATERIALS AND METHODS 5 RESULTS 10 I. Synthesis of Putative Deletions 10 I I . Complementation Studies 10 I I I . Localization of Putative Deletions 11 IV. Nondisjunction of Reattached Third Chromosomes . . . . 12 V. Mutations Induced by EMS 13 VI. Length of Development 14 VII. Variegation 14 VIII. Other Lethals 15 DISCUSSION 18 LITERATURE CITED) 23 i i i LIST OF TABLES Table Page I. Third chromosome reattachments recovered after irradiation of C(3L)j C(3R) virgin females . . 11a I I . The number of male recombinants recovered during the localization study for each lethal stock tested 12a> I I I . The frequency of nondisjunction of nine deletion chromosomes along with the confidence limits on the frequency for the control chromosome 13a IV. The length of development of eleven deletion heterozygotes are presented. The t 1 values for each mutant when compared to the Oregon-R control are also given 14a iv LIST OF FIGURES Page Figure 1. A schematic representation of the method used to form the deletions, (a) Normal chromosome III and attached chromosome III. (b) The formation of attached third chromosomes, (c) The reestablish-ment of the normal gene order, resulting in duplications and deficiencies 5a>. 2. The genetic markers used in making the lethal chromosomes 6a 3. The markers used in localizing the putative deletions 7a 4. A complementation map of the fifty-three mutants. . 10a Leaf v missing. ABSTRACT' v i The term, heterochromatin, refers to those portions of a chromosome which stain very darkly early in the division cycle at a time when most of the chromosomal material stains very lightly. After the discovery of the mutagenic effects of X-rays, large numbers of mutants were recovered in Drosophila. These mutants were genetically mapped but none of them seemed to be located in the heterochromatic regions of the chromosomes. This observation led to the hypothesis that heterochromatin was geneti-cally inert. Since then the problem of the function of heterochromatin has engaged the attention of many different researchers. One of the few mutants known to reside in the proximal heterochromatin of the X chromosome is bobbed. Ritossa and Spiegelman (1965) have presented evidence that bobbed is the nucleolus organizer, the site of ribosomal RKA synthesis. Their results suggest that this is a highly redundant region of the chromosome. If heterochromatin in general is greatly redundant, this would offer one explanation for the apparent lack of genetic loci. A mutant phenotype might result only after large blocks of the chromosome have been deleted. A scheme involving the use of attached-autosomes to select for those third chromosomes which have sustained breaks in the proximal heterochromatin was devised. In general, virgin females were irradiated and attached-third chromosomes were selected for by mating these females to attached-third males. Female progeny carrying these newly synthesized attached-autosomes are, in turn, irradiated and newly reconstituted chromosomes were selected for by mating the females to males carrying normal third chromosomes. v i i Th,us, each newly reconstituted chromosome has sustained a number of breaks at different positions i n the proximal heterochromatin. Using this procedure, we recovered sixty-six homozygous lethal chromosomes. Fifty-three of these sixty-six lethals may be placed i n one of the six complementation groups, two of which appear to be multisite de-letions. In addition, these fifty-three lethals appear to be located, by the results of genetic mapping, i n the region spanning the centro-mere. These two pieces of evidence suggest that the method used here does, indeed, select for deletions i n the proximal heterochromatin. 1 INTRODUCTION Cytologically, the chromosome may be divided on the basis of stain-ing properties into two types of chromatin, heterochromatin and euchro-matin. Heterochromatin, discovered by Heitz in 1928 stains more darkly than euchromatin during early prophase, telophase, and interphase (Gold-schmidt, 1949). During interphase, heterochromatic regions form massive blocks of Feulgen positive material (Schultz, 1947), a phenomenon re-ferred to as heteropyknosis. In Drosophila large blocks of heterochro-matin are localized adjacent to the centromeres and in salivary gland nuclei these heterochromatic areas from a l l chromosomes condense into a single structure defined as the chromocenter. In Drosophila heterochromatin was found to differ genetically from euchromatin in that few, i f any, mutants were located in heterochromatin (Muller and Painter, 1932). Moreover, although in the giant salivary chromosomes the centric heterochromatin forms a very small proportion of the total chromosome mass, in mitotic chromosomes i t forms a relatively large part. Recently, Rudkin (1965) has shown that the reduced propor-tion of heterochromatin in salivary gland cells can be attributed to the fact that DNA in the chromocentral region fails to replicate soon after the initiation of polyteny. The apparent absence of genetic loci in heterochromatin and its reduced bulk in salivary chromosomes led to the theory that heterochromatin is genetically inert. A number of other properties of heterochromatin in Drosophila have become apparent during the past forty years. Considering the relative 2 physical length of heterochromatin in mitotic chromosomes, very l i t t l e meiotic crossing over occurs in this region (Hannah, 1951). Mitotic recombination, however, is believed to occur mainly in these regions (Stern, 1936; Kaplan, 1953). A second observation concerns the phenomenon of variegated-type position effects. In general, when an euchromatic gene is transposed into a heterochromatic region, the euchromatic gene variegates or gives a mottled phenotype. The extent of variegation can be suppressed by adding heterochromatin to the genome and enhanced by subtracting hetero-chromatin from i t . The opposite rules seem to apply to heterochromatic genes which are transposed to euchromatic regions (Lewis, 1950). This suggests that heterochromatin plays some role in the control of gene activity. Recently, Ritossa and Spiegelman (1965) have demonstrated that the amount of DNA complementary to ribosomal RNA (called r-DNA) in Drosophila is directly related to the number of nucleolus organizer (NO) regions present. The NO region is part of the proximal heterochromatin of the Xj the Y has an homologous region. They have also presented evidence that the NO region is highly redundant; they have calculated that a haploid genome has 130 cistrons for each of the 18s and 28s r-RNA components. This suggests that other heterochromatic regions might also be highly redundant. The Y chromosome in Drosophila is entirely heterochromatic. In addition to the NO region, i t contains two complexes of factors deter-3 -mining male fe r t i l i t y , one on each arm. The Y is necessary only for fe r t i l i t y of the malej development in XO males proceeds normally until sperm formation and XXI individuals are normal females. Cooper (1959) stresses the fact that the YT is "a specialized chromosome with a specialized and essential function. In no manner is i t to be considered 'inert*, Accessory 1, 'dispensible1 or the potential 'seat of the un-orthodox* simply because i t i s not required for the normal external phenotype of the adult male and femalei" Heterochromatization of entire chromosomes in other organisms serves as a quantitative control of gene expression. For instance, in the mealy bug the entire paternally derived chromosome set becomes heterochromatic early in development in males. These chromosomes have been shown to be genetically inert and are eliminated from the germ line during spermatogenesis (Brown, 1966). Male mealy bugs, therefore, transfer only their maternally inherited set of chromosomes to their offspring. In female offspring these chromosomes remain active and euchromatic while in male offspring they become heterochromatic (Brown, 1966). Thus, in this insect gene activity is controlled by the hetero-chromatization of large groups of genes and the paternal inheritance designates the chromosomes to be inactivated. The existance in Drosophila of large blocks of heterochromatin with distinct properties but no general functions makes this area of interest for an understanding of chromosome structure and function. If as suggested by the work with the X chromosome, heterochromatic 4 regions are highly redundant, then the use of deletions may be the best way to uncover genetic activity in these regions. A scheme involving the use of attached-autosomes was devised for producing deletions in the centromeric region of chromosome III. The results of that experiment are presented in this paper. 5 MATERIALS AND METHODS The deletions were synthesized in a two-step experimentj the first step involved the formation of attached-autosomes. An attached-autosome is a reversed metacentric compound chromosome (Figure la). Compound third chromosomes may be generated by exposing virgin females to a dose of 4000r of X-rays and then mating the irradiated females to males of a compound third stock (Figure lb). Newly synthesized attached-third chromosomes may then be recovered. The exact scheme used in this experiment to make these deletions, which are recognized as homozygous lethals, is given in Figure lc . In general, i t involves synthesizing new C(3L) chromosomes, growing up a separate stock for each new C(3L) and then irradiating C(3&), C(3R) virgin females to recover newly reattached third chromosomes. The exact procedure used is presented in Figure 2. In a l l irradiation experiments, virgin females were exposed to 4000r of tf -rays delivered from a Cobalt^ research source. The i r -radiation was kindly done by Dr. David Walker of the Chemistry Department, the University of British Columbia. Irradiated females were then mated in quarter pint milk bottles containing standard Drosophila medium in lots of thirty pairs per bottle. The newly reattached third chromosomes were maintained in stock balanced over In(3LR)Ubx13°-Each recovered chromosome was then tested for the presence of a lethal. Those chromosomes found to behave as recessive lethals were tested for their ability to complement with each other by crossing each chromosome with most of the others. Deletion of known loci was tested by crossing each lethal chromosome to known mutants in the proximal 5a. FIGURE 1: A schematic representation of the method used to form the deletions, (a) Normal chromosome III and attached chromosome III. (b) The formation of attached-third chromosomes, (c) The re-establishment of the normal gene order, resulting in duplications and deficiencies. NORMAL CHROMOSOME M ftlTflQiED QiROnOSONEM 3 L m^r'f-%om: > -—3 r\ a b d cet erf 6 region and inspecting for co-dominance. These mutants included egf, in. Dfd. roe, and x>b, a l l of which map in the region spanning the centromere. A l l complementation tests of different lethal chromosomes or of lethal chromosomes with visible mutants were carried out in vials with two pairs of flies per vial. In general, two vials were set up for each cross. Males from six different stocks, one from each complementation group, carrying the lethal balanced over TM3, were crossed to bw^ "/Cy and w1"^  females. Controls involved crosses of these females with males from the non-lethal stock, pP cu/TH3. In addition, lethal (TM3 ) and non-lethal (TM3) bearing offspring could be scored in each cross thus providing an internal control. Variegation was scored sub-jectively as changes in eye color between these two classes. Two vials were set up for each cross with two pairs of flies per vial. The lethals were localized genetically according to the scheme diagrammed in Figure 3. An i n i t i a l assumption is made that the lethal is located in the proximal heterochromatin. Mapping was then done by selecting for crossovers between the closest marker to the centromere on the left arm and a very closely linked marker on the same arm; crossovers between r i and e ^ were recovered in males. These males were then kept in stock and the recombinant chromosome was tested for lethality when made heterozygous with the original lethal chromosome. Females carrying this recombinant chromosome were used to select for crossovers between Dfd and jaP, both of which are located in the right arm. The net result is that in the original lethal chromosome as much •FIGURE 2: The genetic markers used in making the lethal chromosomes. 7 . euchromatin as possible has been replaced adjacent to the proximal heterochromatin. I f these recombinant chromosomes are s t i l l lethal, i t i s probable that the lethal i s i n the region between r i and p£ or, i n other words, the centromeric region. The genetic localiza-tion experiments were carried out i n half pint milk bottles. Ten bottles were set up for each stock and five to seven virgin females and five males were placed i n each bottle. Individual male recombin-ants were selected from these bottles and mated i n vials to females of the appropriate genotype (see Figure 3). In males, attached-third chromosomes assort at random so that approximately one half of a l l sperm w i l l be either nullosomic or disomic with respect to the third chromosome (Holm, 1968). Therefore, attached-third males may be used to assay for nondisjunction of normal third chromosomes i n females. Nondisjunction i s defined as the failure of homologous chromosomes to segregate to opposite poles during meiosis. In order to determine whether nondisjunction was increased by the lethal chromosome, thi r t y females heterozygous for a lethal chromosome were mated to t h i r t y C(3L)RM, SW-3, +; C(3R)RM, SH-18, + males i n half pint milk bottles containing the standard Drosophila medium. Nondisjunction was indicated by any surviving offspring. In the ethyl methane sulfonate (EMS) experiments 200 males from the wild-type Oregon-R stock, 12-24 hours old, were placed on f i l t e r paper saturated with a 1$ sucrose solution containing 0.025 M EMS for 24 hours (Lewis and Bacher, 1968). The treated males were then mated i n bottles to In(3LR) CxD/In(3LR) TM3 virgin females, ten pairs of f l i e s FIGURE 3: The markers used i n localizing the putative deletions. -r eg2"-*-£ 3 a s t » n r i e^g 2" s t ih e« Gl T M 3 ri" 4 - -4- T M - 3 . T M 3 , ri p P 54> 5er le+nality of 4-+j + p P 7 7 1 3 -t- 4- J t + i - -t- +• +• pi n -f- 1 T H 3 i l e + h c j i " t y o f -»- + I + + 8 per bottle. males carrying the In(3Ift)CxD) chromosome were collected and mated individually in vials to virgin females of one of the lethal stocks. A total of 1350 males was tested with four different lethal stocks or 330 males per lethal stock. The method employed by Eker (1939) was used to measure the develop-mental time of different lethal stocks. Thirty females , in the presence of males, were allowed to lay eggs at 22.5 • 1°C. for twelve hours on petri plates f i l l e d with standard fly medium. At successive four hour intervals first instar larvae were picked from the plates with a dissecting needle and placed on squares of standard fly medium. Approximately twenty-five larvae were placed on each square of food. Each square was then placed in a vial containing medium, and the larvae were allowed to develop. It was hoped that the use of 0-4 hour old fi r s t instar larvae would provide a more synchronous culture than the use of eggs that had been laid during a four hour period. The vials were examined every four hours during the twelve hour day and were left undisturbed for twelve hours at night. As each larva crawled out of the food and pupated on the side of the vial, a number was assigned to him by writing on the vial with a felt tip marker. Thus i t was possible to record the length of time necessary for each larva to reach the pupal stage and for each pupa to eclose. The standard map positions of the mutants used in this study (Lindsley and Grell, 1967) are Gl (Glued, 3-41.4), st (scarlet 3-44.0), in (inturned, 3-47), r i (radius incompletus, 3-47.0), eg2, (eagle2, 3-47.3V 9 Dfd and Dfd r (Deformed and Deformed-recessive, 3-47.5), roe (rough eye, 3-47.6), £b (proboscipedia, 3-47.7), EE (pink-peach, 3-48.0), cu (curled, 3-50.0), Sb (Stubble, 3-58.2), &L3 (glass 3, 3-65.1), and Ser (Serrate, 3-92.5). Inversion (l) white-mottled dn(l)wn1^) and brown-Variegated (bw^ -) were used in the variegation experiments. In addition, three third chromosome balancer chromosomes were used: In(3LR)GxD. In(3LR)Ubx13°. and In(3Ifi)TM3. Sb Ser (Lindsley and Grell, 1967). 10) RESULTS Synthesis of Putative Deletions A total of 162 fertile reattached chromosomes was recovered (Table I). An attached-third chromosome has a genetic order of 3R-Centromere-3R or 3I<-Centromere-3L. A reattached third chromosome is derived from two attached-third chromosomes, and the normal gene order of 3L-Centromere-3R has been restored. This does not necessarily mean that reattached third chromosomes are genetically complete, for deletions or duplications are very likely to be present. Of the 162 fertile reattached chromosomes, 96 were viable when homozygous and 66 were lethal when homozygous. The 66 lethal-bearing chromosomes were maintained as separate stocks and the 96 non-lethal chromosomes were discarded. Dr. Leonie K. Piternick kindly examined adults of 63 of the 66 lethal stocks for the presence of any visible abnormalities. She was able to detect a slight to medium Minute phenotype in 25 of the stocks, a medium to strong Minute effect in 4j the remaining 34 had no detect-able Minute characteristics. Complementation Studies During the course of these experiments, most of the lethal stocks were crossed to standard laboratory mutant stocks which map between 47.0 and 48.0 on chromosome IU; this region spans the centromere and includes a l l of the proximal heterochromatin. A l l lethals tested were 2 phenotypically wild type when heterozygous with the mutants in, eg . roes 10a FIGURE 4: A complementation map of the fifty-three complementing mutants. o P & 6 9 ^ , CO ^ 3 f I on , 1=1 : 11 and £b and were viable with the recessive lethal Dfd. Thus, none of the known loci was allelic to the lethal chromosomes. A complementation map of the 66 lethal chromosomes was construct-ed (Figure 4). This map is based upon results from approximately 1350 different pairwise crosses between lethal stocks. In these tests, two different lethals are said to complement i f trans heterozygotes for the lethals are viable as adults. Fifty-three of the 66 lethals can be placed in one of two main complementation groups. One of these is composed of five distinct complementation groups, two of which span two other groups and are probably multisite deletions. This group provides evidence of at least three indispensible genetic units in this region. The remaining 13 lethals complemented with each other as well as with a l l of the mutants in both major complementation groups. These 13 are believed to represent lethals which were induced by the irradiation. Localization of Putative Deletions The lethals were localized genetically according to the scheme diagrammed in Figure 3. The number of crossover chromosomes recovered in males for each stock and the number of these which were s t i l l lethal are given in Table 2 for each part of the experiment. Stocks, such as H-2, in which the number of chromosomes s t i l l retaining lethality and the number of crossover chromosomes do not agree: were eliminated from the second half of the experiment. There are two exceptions to this, 11-12 and 11-28. The results obtained with these two stocks were surprising since they both f i t into one of the two complementation 11a I'abJ.e I: Third chromosome reattachments recovered after irradiating C(3L); C(3R) virgin females C(3L) No.°£ No. No. No. No. Stock Irradiated Reattach. Sterile Nonlethal Lethal 0*2 795 53 13 16 24 0*4 1690 70 13 37 20 70 3 0 2 1 (fe 340 4 1 2 1 565 38 9 18 11 220 12 4 6 2 335 26 4 15 7 Total 4015 206 44 96 66 -12 groups. When the stock cultures of these two mutants were examined they were found to be contaminated. For these two reasons, one lethal-bearing male from each stock was saved and used for the second half of this experiment. When stocks derived from these males were later tested for their complementation pattern, 11-28 was found to behave in the same manner as the original chromosome but 11-12 did not. 11-12 was, therefore, discarded. The positions of the lethals determined genetically are in general agreement with the patterns of complementation. Crossover chromosomes of those mutants which belong to either of the two main complementation groups remain lethal iia both crossover experiments. However, only two of the thirteen mutants which do not f i t into either complementation group segregated with lethality after the first recombinational event. In one of these (IV-16) lethality was lost after the second crossover event. Nine other mutants in this group of thirteen had both lethal and non-lethal recombinant chromo-somes. This suggests, therefore, that those lethals falling into the main complementation groups are indeed located in the proximal area whereas the thirteen others do, in fact, map extensively throughout the chromosome arms. Nondisjunction of Reattached Third Chromosomes The frequency of nondisjunction of attached-third chromosomes in females varies with each compound third chromosome for each pair tested (Holm, 1968). This observed difference could result from differences in the amount of proximal heterochromatin included in each attached-12a "Table II: The number of male recombinants recovered during the localization study for each lethal stock tested 'Crossover I 'Crossover II No, Males No, Lethal No. Males No. Lethal Stock r i eg + r i + eg* Bfd + pP+ Dfd + pP+ I I - l 4 4 0 0 II-2 6 3 — — H-3 1 1 1 1 II-4 2 2 1 1 LT-6 11 11 0 0 II-8 6 6 0 0 11-12 4 1 0 0) 11-14 7 5 — -11-17 6 & 0) 0) 11-18 , 3 0 — — II-23 2 2 0 0 H-26 2 2 0 0) H-28 2 1 0 ® 11-32 2 2 0 0 H-335 1 1 0 0) H-34 2 22 1 DL 11-35 11 2L 2 2 11-36 4 4v 0 0 11-37 3 3 0 0) 11-42 2 2 0 0) 11-45 0 ' 0) _ — 11-46 2 2 1 31 II-52 4 4 0 0) II-53 5; 5 0) 0 17-9 ? 7 0) 0 17-10 6> 6 0 0) 17-11 3 3 0 0) 17-12 4 4 1 1 17-15 h 4: 0 0) 17-16 3 3; 1 © 17-20 1 1 — — 17-22; 1 1. 0 o 17-24 4 4 0 0) 17-25 4 0) — — 17-28 1 1 0 0) 17-32 4 4 0 0! 17-33 0 0) — — 17-45 1 1 0) 17-53 2 2. 0 0^  17-55 2 0 — — 17-58' 2 2 0 0 17-61 2 2: 0 0) Table II: Continued Crossover I Crossover II No. Males No. Lethal No. Males No. Lethal Stock n eg T n eg T Dfdt pP+ Dfd + IV-62 2 2 1 11 IV-67 0 © - -¥-2 7 7 0 0 ¥1-3 3 3 0 0) ¥11-4 0 0 - -YII-5 4 4 © V1I-7 1 1 0 e» 711-12 1 1 0) 0) ¥11-13 2 2 0 0 111-15 1 1 0 © ¥11-22 1 1 1 l ¥11-26 7 6 - -¥11-28 2 2 0 0' ¥11-33 2 2 0 0 ¥11-34 1 1 2 2 A-3 2 2 0 © A-8 3 1 - • -B-12 1 1 0 0) B-14 6 4 — -B-15 2 0 - -B-19 0 0 -B-24 0 0 - -B-25 0 0 - -B-26 0 0 — —-13 third chromosome. Crouse (i960) demonstrated the control of chromo-some disjunction by proximal heterochromatin in Sciara and Lindsley and Novitski (1958) and Ptashne (i960) showed similar effects of proximal heterochromatin in Drosophila. Since the lethals recovered in this experiment probably carry variable amountsof proximal hetero-chromatin, i t was of interest to see how the putative heterochromatic deletions would behave. The results of this experiment are presented in Table 3. The 95^ Poisson confidence interval as calculated by Stevens (1942) on the mean number of flies per bottle is also given. The control JD£ CU/TM3 fe-males yielded an average of 3.6 flies per bottle; the 95$ Poisson confidence interval on 3»6 is 0.92-8.88. Four lethal stocks (II-6, II-8, IV-24, VXE-33) had means which f e l l outside of these limits; two stocks (11-36, 17-12) had means which were close to the upper limit; three other stocks (IU-28, VII-7, B-19) had means which f e l l within this interval. We can conclude that nondisjunction was in-creased in some lethal stocks. However, i t must be noted that these stocks were not coisogenic. Therefore, we cannot eliminate the back-ground genotype as a cause of the increased rate of nondisjunction. Mutations Induced by EMS This experiment was performed as a pilot test of the feasibility of inducing lethal mutation within the region of the putative deletions. Consequently, only a small number of F^ males were tested. Nevertheless, from this limited sample, four mutants which were allelic with the tester chromosomes were recovered. Since the Oregon-R stock was not isogenic Table III: The frequency of nondisjunction of nine deletion chromosomes along with the confidence limits on the value for the control chromosome Confidence Limits 0.92; 8.88 Stock No. Bottles No. Flies Flies/Bottle pP cu 8 29 3.6 II-6 8 105 13.1 II-8 7 223 31.9 11-36 3 27 9.0 17-12 3 29 9.7 17-24 3 35 11.7 IV-28 4 7 1.8 VtfI-7 3 12 4.0 711-33 7 108 15.4 B-19 2 13 6.5 14 and since no control crosses were done, i t is possible that the four lethals recovered may not have been chemically induced but may have already existed in the stock. Length of Development Early in this study i t was observed that the stocks of some lethals seemed to take about 12 days from the time the eggs were laid to eelosion whereas other laboratory stocks required 10 days at that temperature. An experiment was designed, therefore, to determine whether this difference was real. The results of this experiment are presented in Table 4. A t test of the null hypothe-sis that the mean developmental interval of the control population is not significantly different from the mean value of the lethal population was done. Since the two populations do not have the same variance, a weighted t statistic, t 1 , was used (Ostle, 1963). The t' value for each lethal tested can be found in Table 4 along with the limits at P=0.05 at which the null hypothesis is to be accepted. In a l l cases the calculated t f value was outside these limits and, there-fore, the null hypothesis must be rejected. We can conclude that the length of de-welopment of mutant individuals is significantly longer than that of non-mutant individuals. Here again, however, i t must be emphasized that no attempt was made to make these lethal stocks coiso-genic. Ahe increased development time could conceivably be due to other background factors. Variegation Wheim an euchromatic gene is transposed next to heterochromatin, Table Tin The length of development of eleven deletion heterozygotes are presented. The t 1 values for each mutant when compared to the Oregon-R control are also given. Stock No. Flies Mean Develop. Time (Hours) 9. 0-R/TM3 245 251.0 II-1/TM3 55 276.3 -11.46 1.9995 II-3/TM3 84 281.8 -20.40 1.9866 II-8/TM3 90 279.4 -12.79 1.9857 II-17/TM3 137 282.0 -21.53 1.9815 II-23/TM3 98 292.0 -23.40 1.9822 IV-10/TM3 78 301.2 -27.89 1.9876 IV-12/TM3 59 279.2 -15.16 1.9966 IV-24/TM3 21 278.1 -6.83 2.0829 IV-32/TM3 86 298.7 -28.40 1.9847 VII-4/TM3 101 284.4 -20.88 1.9820 B-19/TM3 97 291.5 -24.85 1.9820 t' = (X\-\)^Vn^S2/riZ) Reject i f "-q• £ t l * q Where q =Ksi/n 1)(t 1) + (si/n 2)(t 2J/ ( 3 ^ 1 + sl/n 2) 15 a variegated phenotype usually results. In general, the extent of variegation, that i s , the proportion of mutant to wild type tissue, increases as heterochromatin i s removed from the genome and decreases as heterochromatin i s added to i t (Schultz, 1936). The lethals re-covered i n this experiment are believed to be deletions of proximal heterochromatin. I f this i s true and i f the deletions are sizeable, then these lethal chromosomes would be expected to increase the ex-tent of variegation. The results of this experiment, were negative. No observable VI eye color difference was evident between the bw : TM3 and the VI bw : TIC offspring i n any of the crosses. There were also no differences between TM3 and w1"4: TM3+ males i n the crosses of four different lethals. In two others, however, the w^: TM3*1" males had more red pigment than the w"^-; TM3 males. Other Lethals When the scheme for the genetic localization of the lethals was f i r s t devised, females i n cross V (see Figure 3) were to have been mated to Dfd r pP males. Dfd r i s ah al l e l e of Dfd which survives when homozygous and enhances the Deformed phenotype when heterozygous with i t . For this reason a Dfd r pP stock should have made the selection of Dfd* recombinants much easier. Since no such stock existed, one had to be made. This was done by crossing Dfd r/st i n r i pP females to st i n r i pJP males and selecting pJP male recombinants. These males were then crossed to Dfd/TM3 females and five different lines which seemed to show an enhanced Dfd phenotype were saved and made homozygous. 1 6 •Surprisingly two of these lines, DP-B and DP-C, failed to yield pro-geny homozygous for the recombinant chromosome. DP-C gave no homozy-gotes and 465 heterozygotesj DP-B gave 1 6 (15<*> l£) homozygotes and 715 heterozygotes. These 16 DP-B homozygotes had a rather unusual phenotype. The wings were abnormal in texture, venation and position; the bristles on the mesothorax and scutellum were either missing entirely or present in duplicate; the tarsi of the legs were fore-shortened and the males have sex combs on a l l three tarsi of the first leg; the eyes were abnormal in shape; the antennae were transformed in the direction of legs; the genital apparatus of some males was rotated from 10° - 90°j; and one male had a deformed sucking organ and an un-developed labial palp. In addition, DP-B stock bottles had many dead pupae which when dissected from the pupal case were found to be un-hatched, malformed homozygotes. On the other hand, DP-C stock bottles had no unhatched pupae which suggests that lethality of the homozygotes probably occurs during the egg or larval stage. It is not surprising, thereforejj that DP-B/DP-C heterozygotes are viable as adults and pheno-typically normal except in a few cases where the eye is slightly de-formed. Both chromosomes are fully viable when heterozygous with Dfd. When DP-B and DP-C virgin females were crossed to Dfd r and st in r i JJP males, no significant deviations from the expected pheno-typic ratios were observed in the offspring. This, of course, does not eliminate the possibility that these lethals occur in one or both of these stocks at a low frequency. 17 DP-B and DP-G virgin females were also crossed to the 53 lethal stocks which form the two main complementation groups and i n a l l cases viable heterozygous progeny were recovered. An attempt was made to repeat the original experiment i n order to determine whether the lethals were generated by the crossover event i t s e l f . Heterozygous Dfdf/st i n r i j£ females were again crossed to st i n r i p_£ males and jpP male recombinants were saved. Nineteen pP males were collected, and one of these, P-14, failed to give homozygous progeny. When P-14 was crossed to DP-B or DP-C, viable heterozygotes were recovered - an indication that P-14 represents a third lethal site. When P-14 females were crossed to representatives of each complementation group, viable heterozygotes were also obtained. A l l three of these lethals were recovered with a crossover event between r i and pP. This i s a very interesting region of the third chromosome since i t contains a number of homeotic mutants (Lindsley and G r e l l , 1967). Much more work should be done with these lethals. •In the f i r s t place they should be mapped to determine whether the lethal does indeed l i e between Dfd r and pJP. Secondly, an experiment should be carried out to see i f these lethals are found with low frequen-cy i n either the Dfd r or st i n r i ^  stocks or whether they are indeed the products of a crossover event i n the proximal region. DISCUSSION i s Before a deletion of the proximal heterochromatin may be synthe-sized, two main questions must be answered. First, what is the best method available for generating deletions, and second, how can such a deletion be recognized? The formation of a deletion requires two breaks ;.either within one chromosome or at different positions in homologous chromosomes followed by rehealing of the broken ends with the loss of the intervening piece; radiation is probably the most effective means of inducing such breaks. The second problem, that of recognizing the proper deletions, is a much more difficult one to solve. The selection of these chromosomes out of a random sample of treated chromosomes would be a very tedious procedure. What is needed, then, is a method which selects preferentially for breaks in the proximal heterochromatin. The method chosen here selects for those chromosomes which have sustained breaks in the proximal heterochromatin. The formation of attached-third chromosomes involves two hits, one in each third chromosome on opposite sides of their respective centromeres (see Figure l ) . This is followed by a rejoining event which results in both right arms being attached to one centromere and both left arms to the other. Reconstruction of a regular third chromosome is then effected by irradiating such compound chromosomes. Thus each newly reconstituted chromosome has sustained a number of breaks at differ-ent positions in the proximal heterochromatin. The recovery of lethals 19 in this region should provide an efficient screening procedure for the recovery of deletions. The results of these experiments suggest that selection of reattached chromosomes from attached autosomes does indeed yield chromosomes with lethals predominantly localized to the centromeric region of the third chromosome. Of the 162 fertile reattachments recovered, 41# (66) carried third chromosome lethals, and 80$ (53) of these were members of one of the two main complementation groups. This indicates that there is a functional relationship among these lethals; in fact, i t seems likely that they involve changes in the same loci. The results of the localization study corroborate this idea. These 53 lethals are most probably localized between r i and pP. the region containing the centromere. However, the 13 lethals which do not form a part of the two main complementation groups are located in other parts of the third chromosome; most of them do not appear to be linked closely to the centromeric region. In summary then, the majority of lethals selected by this scheme appear to be functionally related and located in the same proximal region of the chromosome. In addition, these proximal lethals share properties with two other classes of mutants which are thought to be heterochromatic, the bobbed (bb) alleles and the Minutes (M), which suggest that they are, in fact, heterochromatic mutants. Minutes are a class of mutants which have a similar phenotype consisting of recessive lethality and a dominant visible phenotype of short fine bristles. They are found 20 on a l l four chromosomes and map at many different sites (Lindsley and Grell, 1967). Some Minutes are associated with cytologically visible deletions; Morgan, Schultz, and Curry (1940) have reported that in mitotic metaphase in stocks which carry M(2)S2^ the right arm of the second chromosome is about three-fourths its normal size. M(2)S2^ is also associated with a deficiency for salivary band 41A; bands 41A and B are described by these authors as "heterochromatin in the strict sense" in that they form part of the chromocenter, thus showing the property of indiscriminate synapsis with other hetero-chromatic regions. M(2)S2^ also enhances variegation of white-mottled and brown-Variegated chromosomes to the same extent as the removal of a Y chromosome/ Morgan, schultz, and Curry conclude that "within this region there is a section of typically 1heterochromatic* properties." Other characteristics of Minutes in general are a decrease in viability and f e r t i l i t y and an increase in developmental time (Dunn and Mossige, 1937; Brehme, 1941). Minutes also enhance somatic recom-bination (Stern, 1936; Kaplan, 1953). The bobbed locus which is located in the proximal region of the X chromosome shares many similarities with Minutes. Phenotypically, bb flies are characterized by having' short, fine bristles and etching of the abdominal tergites. Stern (1936) reports that bb when homozygous acts as a Minute and increases somatic exchange. The developmental time of stocks carrying bb is also increased, and the length of development is directly proportional to the severity of the bb allele (Lindsley and Grell, 1967). 21 The lethals reported here have a number of similarities to Minutes and bb alleles. Phenotypically, a number of them have visible Minute characteristics, that i s , they have short, fine bristles. A l l mutants tested increased developmental time significantly as do Minutes and bobbed. Results of an experiment to see whether they increase somatic recombination were negative; however, the experiment should be repeated. The variegation experiments were also negative. An increase in variegation would only be expected i f there was a substantial de-letion of heterochromatin. It should be remembered, however, that because of the way these lethals were generated there may be large duplications accompanying the deletions. On the whole, the character-istics shares by these lethals with Minutes and bobbed indicates that they are heterochromatic mutants. Recently i t has been suggested that heterochromatic loci are highly redundant, ^he work with bb mutants (Ritossa, Atwood, and Spiegelman, 1966) suggested that the bb locus represents a series of tandem duplications which code for r-RKA. In this experiment two-thirds of the fertile, reconstituted third chromosomes did not carry lethals in the centromeric region. This fact might indicate that this region is highly redundant and that a large portion of the material must be deleted in order to result in lethality. An alternative ex-planation for this concerns the mechanism by which these reconstituted chromosomes were made. If the two attached-third chromosomes paired in the region of the centromere and adjacent heterochromatin and i f pairing were followed by a recombinational event, then a non-lethal reconstruct-22 ed third chromosome could result. Experiments are being done to see i f any light can be shed on this problem. The scheme proposed i n this paper provides a new approach to the old problem of heterochromatin. I t i s hoped that others w i l l expand this work and look i n detail at the genes which are located i n the proximal heterochromatin of both the second and third chromosomes. The deletions recovered provide a tool for the specific recovery of EMS-induced point mutations which may allow an analysis of the gene function of this region. LITERATURE CITED Brown, Spencer W. (1966) Heterochromatin. Science 151: 417-425. Brehme, Katherine S. (1941) Development of the Minute phenotype in Drosophila melanogaster. A comparative study of the growth of three Minute mutants. J. Expl. Zool. 88: 135-160. Cooper, Kenneth W. (1959) Cytogenetic analysis of major hetero-chromatic elements (especially Xh and I) in Drosophila  melanogaster. and the theory of heterochromatin. Chromosoma: 10: 535-588 Crouse, Helen V. (i960) The controlling element in sex behavior in Sciara. Genetics 4J>: 1429-1443. Dunn, L. C. and Jeanne Coyne Mossige (1937) The effects of the Minute mutations of Drosophila melanogaster on developmental rate. Hereditas 23_: 70-90. Eker, Reidar (1939) Further studies of the effect of temperature on the manifestation of the shortwing gene in Drosophila melanogaster. J. of Gen. 28: 201-227. Goldschmidt, Richard B. (1949) Heterochromatic heredity. Proc. VIII Intr. Cong. Gen. Hereditas, Suppl. Vol.: 244-255. Hannah, Aloha (1951) Localization and function of heterochromatin in Drosophila melanogaster. Advan. Genet. l±i 87-125. Holm, D. (1968) Personal conununication. Kaplan, William D. (1953) The influence of Minutes upon somatic crossing over in Drosophila melanogaster. Genetics 2&: 630-651. Lewis, E. B. (1950) The phenomenon of position effect. Advan. Genet. 73-115. Lewis, E. B. and F. Bacher (1968) Method of feeding ethyl methane sulfonate (EMS) to Drosophila males. Dros. Info. Serv. 43s 193. Lindsley, Dan L. and E. H. Grell (1967) Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627 Lindsley, D. L. and E. Novitski (1958) Localization of the genetic factors responsible for the kinetic activity of X chromosomes of Drosophila melanogaster. Genetics 43* 790-798. 24 'Morgan, T. H., Jack Schultz, and Viola Curry (1940) Investigations on the constitution of the germinal material in relation to heredity. Carnegie Inst. Wash. Yearbook 22' 251-255. Muller, H. J. and T. S. Painter (1932) The differentiation of the sex chromosomes of Drosophila into genetically active and inert regions. Zeitschr./ f. ind. Abst. u. Vererb. 62; 316-365. Ostle, Bernard (1963) Statistics in Research, 2nd edition. Iowa University Press, Ames, Iowa. pp. 119-120. Ptashne, Mark (i960) The behavior of strong and weak centromeres at second anaphase of Drosophila melanogaster. Genetics 45* 499-506. . Ritossa, F. M. and S. Spiegelman (1965) Localization of DM. complementary to ribosomal RNA in the nucleolus organizer region of Drosophila melanogaster. Nat. Acad. Sci. U.S., Proc. 21s 737-745. Ritossa, F. M., K. C. Atwood, and S. Spiegelman (1966) A molecular explanation of the bobbed mutants of Drosophila as partial deficiencies of "ribosomal" DNA. Genetics j>4.: 819-834. Rudkin, G. T. (1965) N0n replicating DNA. in giant chromosomes. Genetics J52: 470. Schultz, Jack (1936) Variegation in Drosophila and the inert chromosome regions. Nat. Acad. Sci. U.S;., Proc. 22: 27-33. • Schultz, Jack (1947) The nature of heterochromatin. Cold Spring Harbor Symp. Quant. Biol. 12: 179-191. Stern, Curt (1936) Somatic crossing over and segregation in Drosophila melanogaster. Genetics 21: 625-730. . Stevens, W. L. (1942) Accuracy of mutation rates. J. of Genet. 43: 301-307. 

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