In Drosophila Melanogaster

POSITION EFFECTS AT THE HAIRY LOCUS IN DROSOPHILA MELANOGASTER

DUANE E. JEFFERYI

Department of Zoology, University of California, Berkeley, Calif.94720 Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 Manuscript received July 3,1975 Revised copy received June 26,1978

ABSTRACT Radiation-induced chromosomal rearrangements of h+ have given rise to several Drosophila stocks that exhibit apparent position-effect inactivation; i.e., flies carrying the rearranged chromosomes heterozygously with h show varying degrees of hairiness. The numbers of hairy chaetae produce a quan- tifiable index of position effect. Six such “position-allele’’ stocks are here discussed, both as to their basic expressions and in all possible pair-wise combinations with each other. Such crosses reveal complex interactions between the respective position alleles; little evidence is seen for clear-cut dominance or recessiveness. The stocks appear not to conform unequivocally to classical distinctions between variegated and stable types of position effects, nor to usual dicta relating the degree of inactivity to the proximity to heterochromatin. Indeed, these stocks appear to suggest additional dimensions to several of the principles to which position effects usually subscribe. The evidence additionally suggests that the hairy locus itself is associated with a tissue-specific suppressor effect on an otherwise polygenic system that produces the chaetae associated with the hairy phenotype.

POSITION EFFECT continues as one of the longest-studied, unresolved prob- lems in genetics. Volumes of data have now been collected; see LEWIS(1950), BAKER(1968) and SPOFFORD(1976) for critical reviews. The causal mechanisms underlying the phenomenon, however, remain obscure. Research has long been focused primarily on eye-pigment systems, which, though readily studied, are not easily reduced to a fully satisfactory system of quantification of gene expression. The present study aims at the development of a genetic system for the study of position effects that affords simple and direct quantification of the phenotype. For these stated purposes, the hairy system appears ideal. Its susceptibility to position effect was first described by DUBININand SIDOROV(1935), but no further studies exploiting it have been published. This paper describes some beginning studies of the hairy system, and summarizes relevant data upon which further work can be based.

MATERIALS AND METHODS

Production of stocks: A marked stock carrying sepia (se) and hairy (h)was utilized. These two genes map on 3L at 26.0 and 26.5 units, respectively. There are no data reported that provide

Present address: Department of Zoology, Brigham Young University, Provo, Utah 84602.

Genetics 91 : 105-125 January, 1979. 106 D. E. JEFFERY information on the salivary gland chromosome location of se; h lies in the 13-band area bounded by 66D2 and 66E1 ( LINDSLEYand GRELL1968). Canton-S wild-type males were X rayed with doses varying between 5000 and 7000 r acute radiation. The irradiated males were mated immediately to se h/se h females, and the resulting heterozygous F, progeny were screened visually for those with microchaetae on either the scutellum or the mesopleurae. The presence of such hairs was taken as an indication of possible position effect on the h+ gene, thus inactivating it to a degree and permitting the growth of

hairs on the heterozygous fly. No evidence was found of any inactivation of se, so that the putative Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 position-effect flies have wild-type eyes, but express varying degrees of h. Such F, flies were bred back to the se h/se h stock. This produced a progeny, half of whose individuals are homozygous se h; the remaining half express the phenotype of the original F, parents. These latter flies suggest the presence of a position effect, and are hereafter designated R (f) /se h, to indicate the presumed rearrangement of the wild-type gene (s). Since the study concerns primarily the hairy trait, this notation may be shortened to R(f)/h, with the understanding that the sepia trait was used as the critical marker in all crosses. It must be further emphasized that the R(+) notation designates “rearrangement” as originally used by STERNand HEIDENTHAL(1944), and is not to be confused with the R notation of ring chromosomes. The se h/se h stock is a relatively inbred one, having been cultured without outbreeding for several years in the laboratory. To insure a comparable genetic background for all experiments, the position-effect stocks were not balanced, being bred every generation back to se h/se h. Six stocks of independently derived presumptive “position alleles” (STERNand HEIDENTHAL 1944) were obtained by the above methods. They are numbered R06, R15, R38, R40, R44, and R47. Genetic analysis involved first a characterization of the phenotype expressed by each position allele in heterozygous condition with h, i.e., R(+)/h. In its basic homozygous condition, h adds hairs to the top of the head, the wings, mesonotum, scutellum, and mesopleurae. The position alleles also express added hairs in the above-listed areas, but only the latter two locations (i.e., scutellum and mesopleurae) were selected for detailed study. Counts of the numbers of hairs present on these surfaces afford a quantitative index of the level of inactivation imposed on the h+ gene by its rearranged location. In addition to characterizing each position allele separately in the R*/h arrangement (Ro6/h, R’5/h, etc.), a study was made of all possible pair-wise combinations of the alleles with each other, that is, R*/RV arrangements (ROS/R‘5, etc.).The cross Rx/h x Rv/h predicts se h homozygotes, Rx/h, RV/h, and RS/RV in approximately 1: 1: 1 : 1 ratios, the latter three classes all having wild-type eye color. Ascertainment of genotype among the three was by single-pair progeny tests back to the se h/se h stock: R*/h and RV/h produce sepia and nonsepia (wild-type) progeny in a 1:1 ratio; R*/RV produces all nonsepia offspring. Production of ten wild-type offspring and no sepia was taken as the minimal acceptable assay for a parent carrying the R*/RY genotype. Each position allele was also studied in the homozygous condition: R*/h x Rz/h yielding se h homozygotes, Rx/h, and RX/P in a predicted 1:2: 1 ratio. Again, single-pair progeny tests back to se h/se h were used to distinguish the latter two genotypes, thus isolating the homozygote for study. Cytological analysis of the chromosomal structural rearrangements was carried out with standard lacto-aceto-orcein techniques for salivary gland chromosome work, coupled with phase- contrast microscopy.

RESULTS Description of obtained stocks: Though yield vaned with the radiation dose, flies carrying a putative hairy position effect occurred with an approximate fre- quency of one per 12.000 F, progeny screened. Table 1 summarizes the critical data describing the six position-allele stocks found. All stocks exhibit good viability and fertility with the exception of R06 males, which are sterile. POSITION EFFECTS OF HAIRY 107

TABLE 1

Summary drrta of the six porition allele stocks, excluding data on chaetae

Percent Stock Formal Chromosomal homozygote No. designation breaks between: New order lethality R06 T(1;3)h+o6 1B9-10& 1C1; 61-66D/I C-20; -

66D14& El-2 1AB/66E-100 Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 RI 5 ln(3LR)h+15 66D14 & El-2; 6 1-66D/85B-66E/ 100 85B4-5 & C12 85C-100

R38 In(3L)h+ 38 66D14 & El-2; 6 1-66D/80F-66E/ 85 in 80F 81F-100 R40 T(2;3)h+40 30B7 & Cl-2; 61-66D/30C-60; 100 66D14 & El-2; 21-30B/73B-66E/ 73B7 & Cl-2 73C-100 R44 T(3;4)h+44 63A3 & B1-2; 61-63A/67-100; 73 66F1-2 & 67A1-2; 101/63B-66/102 right end 101F R47 T(2;3)h+47 23A1-2 & B1-2; 60-34C/99-100; 95 34C1-2 & (24-5; 21-23A/66D-98/ 66D5-6&D10-11; 34C-23B/66D-61 98F11-12& 99A1-2

Chaetae counts: No attempt was made to distinguish heavy thick hairs from thin fine ones. Such differences do exist, but the variation is continuous, and intermixture of types can occur even on a single fly-in short, such a distinction offers no diagnostic criteria of gene action. On the mesopleurae, all chaetae observed were scored, regardless of their size or specific ‘‘sublocation,’’ and the scores from the two mesopleurae are lumped together as a single total. It was noted very early in the study that the two mesopleurae were highly correlated in expression; lumping of data to give one index for mesopleural expression thus seemed appropriate. The scutellar counts include all chaetae whose bases were observable from a dorsal/oblique view; chaetae located on the scutellar sides are included if their bases were visible Irom above the level of the scutellum proper. The scutellar macrochaetae (bristles) were not included in the data since they are not an integral part of the hairy system. (The chaetae of the hairy system are formally called microchaetae, and are part of a four-celled bristle organ made of a trichogen, a tormogen, a nerve cell and a nerve sheath cell. In keeping with the name of the mutant responsible for their expression, they are also called hairs, but they must not be confused with the minute, nonsocketed cell trichomes that appear on the cuticle and wing. This discussion will use the terms microchaetae, chaetae, and hairs to refer to the products of the hairy system; other specific bristles or chaetae will be referred to by their formal names if needed.) Counts of the chaeta expression of the six stocks in heterozygous condition with se h are based uniformly on a random sample of 20 males and 20 females. Those for the LLcombination”genotypes, i.e.,RxRY, RxRx, etc., are based on a varying sample size, depending on the number identified in the single-pair mating 108 D. E. JEFFERY progeny tests. Such tests were uniformly set up in trays of 120 vials, 60 for males, 60 for females of the combination being assayed. In a cross of Rx/h X RY/h, using only the nonsepia Flfor single-pair testing, one expects ca. 20 females, 20 males to be of the R"Ry type. In practice, this was seldom attained. Such attain- ment is expected only if all of the following criteria (and possibly others) apply: (1) there are no differences in developmental time between R"RY, Rx/h,and Rv/h, so that the sample of F, used for parents in the assay is truly random; (2) all Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 three genotypes have equal viability; (3) all three genotypes have equal fertility. Since each R stock is the product of at least two independent chromosome breaks and subsequent rearrangement, it would seem likely that these rigorous criteria would frequently be violated, and this is indeed reflected in the lower numbers of combination genotypes observed. Table 2 presents the data on chaetae counts for each of the basic stocks and for all obtainable combinations. Data for males carrying R06 are often lacking since these males are sterile and their identity cannot always be reliably demonstrated in combinations other than in the basic Ro6/h.

TABLE 2 Chaetae count data for basic stocks and pair-wise combinations*

Xz-values in comparison with: Genotype Parameter N z sw h/h RZ/h RV/h h/h MSC 20 18.70 0.40 MMS 20 13.20 0.54 self self self FSC 20 25.15 0.64 FMS 20 13.75 0.70

RO6/R15 FSC 19 25.53 0.66 0.04 3.40 13.37 FMS 19 9.21 0.33 18.01 4.82 0.03

R06/h MSC 20 19.90 0.56 0.75 MMS 20 6.40 0.15 47.18 self same as FSC 20 28.60 0.39 4.43 h/h FMS 20 7.20 0.37 40.96

RO6/R38 FSC 10 17.10 0.80 18.78 35.60 57.17 FMS 10 5.80 0.83 37.96 1.94 5.31

ROb/R40 FSC 18 30.94 0.46 11.45 1.75 15.73 FMS 18 16.72 0.68 5.46 73.35 3.75

ROb/R44 FSC 15 3.87 0.27 243.61 291.30 0.02 FMS 15 0.00 0.00 206.69 108.00 0.00

ROb/R47 FSC 15 14.40 2.51 7.64 21.41 20.40 FMS 15 15.53 0.69 1.81 55.26 5.92

R15/R15 lethal POSITION EFFECTS OF HAIRY 109 TABLE 2-Continued

X2-values in comparison with: Genotype Parameter N r s5 h/h R=/h Rg/h R15/h MSC 20 15.35 0.39 6.59 MMS 20 10.35 0.54 6.90 self same as FSC 20 19.95 0.34 11.99 h/h FMS 20 9.05 0.36 19.38 Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021

R15/R38 MSC 15 8.53 0.48 61.58 32.05 5.89 MMS 15 5.40 0.27 114.32 25.52 5.24 FSC 17 10.24 0.39 111.63 56.00 5.84 FMS 17 5.71 0.38 59.27 13.68 6.34

R15/R40 lethal

RI 5/ R44 MSC 5 3.60 0.51 57.62 42.48 0.20 MMS 5 0.20 0.20 63.77 4Q.53 0.01 FSC 13 3.77 0.28 209.94 151.95 0.07 FMS 13 0.00 0.00 175.15 117.65 0.00

R15/R47 MSC 14 13.43 0.36 14.21 2.09 1.98 MMS 14 8.64 0.29 15.47 2.49 1.23 FSC 12 15.83 0.35 30.16 6.91 1.19 FMS 12 9.33 0.58 12.01 0.07 6.46

R38/h MSC 20 6.30 0.33 123.01 MMS 20 3.75 0.23 105.37 self same as FSC 20 7.85 0.37 181.39 h/h FMS U) 3.90 0.34 109.94

R38/R38 MSC 1 4.00 0.00 11.43 0.81 MMS 1 4.00 0.00 6.55 0.02 same as FSC 3 10.67 1.33 23.73 2.52 Rz/h FMS 3 11.67 0.33 1.01 32.03

R38/R40 MSC 14 10.71 0.M 34.31 19.77 53.22 MMS 14 7.43 0.45 25.82 21.17 66.46 FSC 25 13.56 0.38 80.05 32.87 68.78 FMS 25 9.52 0.60 17.44 49.98 77.97

R38/R44 MSC 9 0.89 0.46 150.38 39.33 13.35 MMS 9 0.00 0.00 118.95 33.75 1.45 FSC 8 1.00 0.27 182.61 45.50 16.01 FMS 8 0.00 0.00 110.84 31.20 0.00

R38/R47 MSC 12 4.83 0.39 106.82 2.81 38.75 MMS 12 6.75 0.57 28.53 13.85 0.66 FSC 16 7.13 0.33 170.05 0.62 41.18 FMS 16 9.94 0.44 10.79 49.22 4.95

R40/h MSC 20 21.15 0.47 3.01 MMS U) 17.90 0.41 14.21 self same as FSC 20 24.20 0.59 0.37 h/h FMS 20 19.40 0.56 19.26 110 D. E. JEFFERY TABLE 2-Continued

X2-values in comparison with: Genotype Parameter N I Sm h/h Rx/h Rg/h R40/R40 lethal

R40/R44 MSC 18 3.67 0.28 186.37 225.03 0.60 MMS 18 0.00 0.00 237.41 322.20 3.67 FSC 17 3.47 0.36 286.45 269.06 0.57 Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 FMS 17 0.00 0.00 232.21 329.80 0.00

R40/R47 MSC 12 16.08 0.79 3.01 10.00 10.80 MMS 12 15.58 1.26 3.07 2.36 45.82 FSC 16 18.31 0.64 18.93 14.28 8.90 FMS 16 19.94 1.58 25.01 0.13 31.59

R44/h MSC 20 3.20 0.25 219.41 MMS 20 0.30 0.13 246.53 self same as FSC 20 3.95 0.38 308.89 h/h FMS 20 0.00 0.00 275.00

R44/R44 MSC 3 0.00 0.00 56.39 9.60 MMS 3 0.00 0.00 40.35 0.12 sameas FSC 8 0.00 0.00 201.76 31.60 Rz/h FMS 8 0.00 0.00 110.84 0.00

R44/R47 MSC 14 1.07 0.17 223.22 16.06 127.03 MMS 14 0.00 0.00 185.66 2.67 105.70 FSC 11 1.36 0.24 240.73 15.66 122.32 FMS 11 0.00 0.00 152.26 0.00 136.95

R47/h MSC 20 11.70 0.45 32.23 MMS 20 7.55 0.39 30.77 self same as FSC 20 14.30 0.48 59.68 h/h FMS 20 12.45 0.71 1.29

R47/R47 MSC 0 lethal MMS 0 FSC 1 11.oo 0.00 8.24 0.73 same as FMS 1 23.00 0.00 6.07 8.18 Rz/h

* Legend: iMSC = male scutellar count, MMS = male mesopleurae count, FSC = female scutellar count, FMS = female mesopleurae count. N = sample size, Z = sample mean, s2.= standard error of mean. x2 values were computed by homogeneity tests. All comparisons have 1 d.f.; significance = 3.84 for the 5% level, 6.63 for the 1% level.

Figures 1 to 14 present the same data as shown in the foregoing table, but arranged so as to facilitate comparison of each basic stock in all its combinations with other genotypes. The degree of expression of each genotype is represented as proportional to that of the hairy homozygote, h/h, i.e., the homozygote’s expression is considered as 100%. Males and females are treated separately. POSITION EFFECTS OF HAIRY 111 FIGURES1 to 14.-Closed bars = scutellar expression; open bars = mesopleural expression; L = lethal; 0 = no hair, wild-type expression; S = sterility renders genotype either not obtainable or not critically identifiable.

140 .$I .$I 120 I Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 100 L 0 8 80 In 0 c 60 .- U) 40 F: 20

0 ---.. _.. hhhhhhh FIGURE1.-Comparison of the phenotypic expression of females carrying the hairy gene in homozygous condition, and heterozygously with each of the six position alleles.

140

120

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80 60

FIGURE2.-Comparison of the phenotypic expression of males carrying the hairy gene in homozygous condition, and heterozygously with each of the six position alleles. 112 D. E. JEFFERY

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0644 47E h 06 15 38 i FIGURE3.-Comparison of the phenotypic expression Iof R06 females in differing genotypes. 120

FIGURE4.-Comparison of the phenotypic expression of RO6 males in differing genotypes.

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O 21_5EQl!5Efi h 06 15 38 40 44 47 FIGURE5.-Comparison of the phenotypic expression of RI5 females in differing genotypes. 113

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FIGURE6.-Comparison of the phenotypic expression of RI5 males in differing genotypes.

140

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20 -38 3E P-d 31 -38 hE15 i5 FIGURE7.-Comparison of the phenotypic expression of R38 females in differing genotypes.

FIGURE8.-Comparison of the phenotypic expression of R38 males in differing genotypes. 140-

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OL CO 4-0 40 44 FIGURE9.-Comparison of the phenotypic expression of 2340 females in differing genotypes.

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h 06 15 38 40 44 4 FIGURE10.-Comparison of the phenotypic expression of R40 males in differing genotypes.

20/10lo mo Bo mo O4444344*44%- lo 0 h 5 15 38 40 44 47 FIGURE11 .-Comparison of the plenotypic expression of R44 females in differing genotypes 140-

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h 06 15 38 40 44 47 FIGURE12.-Comparison of the phenotypic expression of R44 males in differing genotypes.

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h 06 15 38 40 44 47 FIGURE13.--Comparison of the phenotypic expression of R47 females in differing genotypes.

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h 06 15 38 40 44 47 FIGURE14.-Comparison of the phenotypic expression of R47 males in differing genotypes. 116 D. E. JEFFERY

DISCUSSION Structural relationships between the rearranged (h+) and recognizable het- erchromatin: BAKER(1953,1968) indicated that a rearranged euchromatic gene, to exhibit position effect, must be placed next to a heterochromatic segment which is itself broken. Since breakpoint localization in heterochromatin is problematic at best, this principle is difficult to apply with precision, at least to the present data. JEFFERY(1972) reviewed all available data pertaining to heterochromatin Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 localization in the D. melanogaster genome, permitting the following analyses of the present hairy position alleles. Only two of the position alleles, R38 and €244, are rearranged such as to place the h+ gene into or adjacent to regions that can confidently be called heterochromatic. RO6 and perhaps R40 are associated with areas that have in different studies produced’varying and somewhat ambivalent evidence of heterochromatin, whereas R15 and R47 are associated with regions virtually free from evidence for heterochromatin, and which would be best categorized as euchromatic in nature. In at least these two latter cases, then, and possibly R06 and €240 as well, the usual association with broken heterochromatin appears to have been violated. BAKER( 1968) concluded that only five of the 3 12 position effect rearrangements reported in the literature failed to show this association, and that they were likely the result of inaccurate localization. Such a possibility does not appear likely with R15 and R47. One cannot, of course, negate the possibility that “undiscovered” heterochromatin may be associated with them, but such would of necessity have to be a tiny segment of the chromosome. Map location of hairy: More precise localization of the hairy locus was attempted on the basis of the present data. Only one position allele, R47, is of such a nature as to be useful; it provides slight though inconclusive evidence that the locus lies in the section 66 D7 to E 1-2 on the salivary gland chromosome map. Relation of heterochromatin proximity to degree of position effect: In general, rearranged euchromatic genes show greater variegation the closer their proximity to heterochromatin. It has been demonstrated that heterochromatic regions vary in their ability to elicit variegation, however, so that distance alone is not the deciding factor for the amount of variegation shown in any given case. In the present study, however, the distance generalization seems to be somewhat re- versed (cf., Table 2; Figures 1 and 2). R38 and R44, the two position alleles rearranged into undisputably heterochromatic regions, show the least variegation (fewest hairs). R06 and R40, the two alleles whose relations with heterochromatin are somewhat equivocal, show the most variegation. And the two “euchromatic” alleles, R15 and R47, express an intermediate phenotype. R38 and R44 are located in centromeric heterochromatin, the position usually most produc- tive of variegation, yet fail to respond in the expected manner. Such exceptions to generalizations are of course not unprecedented (e.g., HESSLER1958), but the reasons for their action are by no means obvious. STERNand KODANI(1955) found some rather unexpected relationships involving centromeric and pericen- POSITION EFFECTS OF HAIRY 117 tromeric heterochromatin and position effects of cubitus interruptus. They found a consistent interpretation, however, by assuming that the general peri- centromeric heterochromatin was physiologically essentially the same as that surrounding ci in its normal position, and thus elicited only slight position effect. However, that heterochromatin immediately adjacent to the centromeres was considered to be different, giving a more pronounced position effect. Euchromatic areas, much different from the ci resident chromatin, gave the most pronounced Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 position effect of all. Such a relationship appears not to exist with the h alleles, however. h resides in a region that appears to be euchromatic, though KAUFMANN and IDDLES(1963) do report two cases of ectopic pairing involving bands 6631-3. These bands, it will be remembered, form the extreme right boundary of the area in which the h gene normally resides, so that they are certainly within a distance of 13 bands from h, and possibly even immediately adjacent to it. Sec- tion 67 (further to the right) seems to constitute the closest major block of prob- able heterochromatin. In terms of the six position alleles, then, the resident neighborhood of h would appear to be most closely approximated in RO6 and R40, and one would predict that they should elicit the least extreme position effect of the six alleles. The reverse is true: they are the most extreme. The two alleles whose rearranged locations appear to be most different from the h point of origin, R38 and R44, give the least position effect, again contrary to expectations. Homozygote-heterozygote comparisons: Comparisons between Rx/Rxhomozy- gotes and their respective Rx/h heterozygotes are hampered, in one case (RO6) by male sterility, and otherwise by the high lethality associated with these rearrangements in the homozygous condition. Those comparisons that are obtainable (Table 2) are usually characterized in statistical analysis by small sample sizes. Only one homozygous stock (R44) has been observed in sufficient numbers to merit confidence in its response. R44 is the least hairy of all the position alleles, and in homozygous condition invariably shows a wild-type phenotype. Such a response runs counter to an early dictum in position effect literature (LEWIS 1950), but it has ample precedence from more recent studies. It is clear, however, (compare other homozygous expressions, Table 2) that the response of R44 is not to be considered as representative of the other stocks; the response is not general. “Dominance” patterns among the position alleles: It is of interest to learn if the expression of any given allele can control the phenotype when in heterozygous combinations with other alleles. Scrutiny of the Rx/RV combinations reveals that, with one exception, no individual stock manifests any clear-cut “dominance” or “recessiveness” with respect to the others. Table 3 presents a summary of the responses of each stock in heterozygous combination with the other position alleles. All parameters of all obtainable combinations were tested by a homogeneity test, and all results are statistically significant as shown, with the exception of one indicated comparison. Placement of a particular interaction in the intermediate column indicates that the phenotype of a given Rx/R” combination is intermediate in expression between those of the Rx/h and Rv/h parents. In other combinations, the Rx/R”phenotype is not significantly different from one 118 D. E. JEFFERY TABLE 3 Summary of numbers of types of interactions shown by the four basic parameters (MSC, MMS, FSC, FMS) of indiuidual stocks when in heterozygous combination wiih other position alleles

Not Complementation Stock Lethal obtainable Dominant Recessive Intermediate f - N RO6 0 10 3 4 2 0 1 20 RI5 4 2 2 6 6* 0 0 20 Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 R38 0 2 2 4 10 2 0 20 R40 4 2 3 5 6 0 0 20 R44 0 2 14 0 0 4 0 20 R47 0 2 2 7 6 2 1 20

* Two of these six intermediates are not significantly different from either parent. parental type (e.g.,R”/h) , but is statistically different from the other, in which case R” is said to be the dominant allele, and D the recessive. The term complementation is used to describe those cases in which the Rz/RV phenotype differs significantly from that of both parents, but is either more, or less, extreme in expression than both parents, i.e., it is not intermediate. Negative complementation indicates a phenotype more extreme than either parent (i.e.,more hairs) ; positive complementation indicates one that is less extreme, or more nearly wild type. It is emphasized that these terms (dominance, complementation, etc.) are used here to imply only the definitions above, and do not necessarily carry their usual conaotations from standard genetic parlance. The “not obtainable” combinations indicated below are invariably those involving males carrying Rob, which are sterile. Since positive identification is made via progeny tests, these males cannot be conclusively identified and are thus excluded from the main comparison columns of the table. It is evident that only R44 manifests a consistent pattern of interaction: it is dominant over all other alleles. Since R44 is also the least variegated, most nearly wild-type allele, this dominance is logically consistent, and fits well with the concept of position effect acting through some sort of inactivation process. One thus is not surprised that a slightly inactivated allele controls the phenotype (is “dominant”) when heterozygous with a more inactivated homologue. But if such a simple relationship were active for the other alleles also, we should observe a hierarchy of dominance among the alleles. Such is not evident in the data, either by pooling parameters and considering each stock as a unit against the other stocks, or by considering individual parameters alone. R38 will illustrate this matter: in all parameters, it is second only to R44 in closeness to wild type. But it does not exert any simple form of dominance, as attested by the data in Figures 7 and 8. These data show primarily intermediate dominance, but with clear-cut cases of complete dominance (R38/R47 FSC) ,complete recessiveness (R38/R47 MMS) , negative complementation (R38/R44 FSC, MSC) , and positive complementation (R38/R38 FMS) . Similarly, the scheme above would predict that the hairiest of alleles (= the most inactivated) should thus be completely recessive in POSITION EFFECTS OF HAIRY 119 heterozygous combinations. Except for FSC, R40 is the hairiest of all alleles, exceeding even the h/h homozygote (Figures 1 and 2). But the data of Figures 9 and 10 demonstrate that this allele is by no means characterized by recessiveness; it is usually either dominant or intermediate. It becomes evident, then, that a scheme of simple dominance determined by relative inactivation is not workable. SLATIS(1955) found additivity in heterozygotes of position alleles at the bw locus, and that the amount of pigmentation Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 produced in a heterozygous compound could be predicted from the amounts produced by each allele separately. The concept does not hold with the hairy alleles. Other authors have interpreted their data in terms of accumulation of various gene products and competitive actions of the alleles. This idea has strength because of its generality, but it does not afford predictive value. The identification of specific gene products, and resultant interactions, are precisely the heart of the problem and cannot yet be accomplished. In the case of the hairy alleles, we may be able to make a partial approach via gene action, to be discussed next. Genetic action of h+, h, and the position alleles: In contemplating the h system, one is struck quickly with the fact that the presence of hairs is due to the recessive allele, whereas the dominant wild-type allele produces an absence of hairs. The simplest interpretation is that h+ produces a suppressor molecule of some type. But it is highly unlikely that h+ makes a suppressor molecule and that h directs the synthesis of hairs; such action seems too disparate for a pair of allelic genes. If, however, h is interpreted as a hypomorph action, so that it produces either (a) a lessened amount of suppressor, (b) an altered suppressor molecule with lessened activity, or (c) a combination of (a) and (b) , the inconsistency is resolved. (We cannot consider h an amorph, since it does not represent the extreme end o€ the phenotypic spectrum; it is exceeded in hairiness by R06 and R40). This idea also presents a further question: if the h locus is merely the site of a suppressor cistron, from whence comes the genetic information to make the hairs even when per- mitted? If such elicitation comes from a single gene or a small number of genes (oligogenes), then one would expect to see mutants that should be visible in h/h stocks. Such have never been directly identified and reported, though of course that does not conclusively indicate their nonexistence. A more feasible idea, however, is that the hairs are produced by the action of polygenes scattered thwhigh- out the genome, and whose mutational effects would be subtle and likely missed unless specifically studied. A working hypothesis can now be formulated: the hairs are elicited by polygenes, but their expression is controlled by the epistatic h locus, which produces a tissue-specific suppressor. Several types of supporting evidence for this concept exist, as follows. It is possible that the action of a rearranged h+ gene has been observed when opposite a deficiency. DUBININand SIDOROV(1935) ,in their study of an R (-I-)/h position effect stock (“translocation #6$4”) placed the R (+) chromosome in heterozygous combination with a purported Df(h+). They report that the result- their judgment is correct, the hairs must have been evoked by genes elsewhere in 120 D. E. JEFFERY ant flies did produce hairs, but much more feebly than the R (+)/hcompound. If the genome than at the h locus, since the of(+)could not make them, h+ does not, and it is not likely that R(h+) could. It should be noted that DUBININand SIDOROVhad no compelling proof that their stock really represented a deficiency -it was known to be a hairy offspring from a cross between h/h females (111-ple) and irradiated wild-type males, and the stock was homozygous lethal. On that basis, they designated it a deficiency, but it is at least equally likely to have been Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 a position effect rearrangement, R(h+)/h. In that case, their heterozygous flies would have been R"(h+)/RV(h+), but the origin of the hairs still would seem to be best relegated to genes other than those of the h locus. The viable R"RY com- pounds in the present study underscore the point: all such combinations still manufacture hairs; the phenomenon is most easily understood if the hairs are elicited by polygenes. If this polygene concept is correct, one should be able to select for higher and lower numbers of hairs. SCOWCROFT(1968) has done just that, and showed very definite responses. Further, it is likely that h/h stocks carried independently of each other over a number of years would progressively diverge in their respective inventories of polygenes associated with the hairy system. And differences do indeed exist in the phenotypes of different h/h stocks. The studies herein reported uniformly used a se h/se h stock maintained in the Zovrlogy Department at Berkeley. Comparisons of the relevant phenotypes of this stock with those of the se h/se h stock from the Pasadena stock center, with x2 values for homogeneity test, are shown in Table 4. All parameters show highly significant differences. It has been observed that when the position-allele stocks are bred to the Pasadena homozygote, the predicted upward shift in phenotype occurs. Another possible line of evidence comes from the allele h2,though the evidence alsa invokes another corollary to the basic hypothesis. Early in the study, it was noticed that a particular pattern of expression seems to exist among the position- allele stocks. All stocks had scutellar hairs; not all of them had hairs on the mesopleurae. Those with low numbers of mesopleural chaetae (or none) also had low scutellar numbers. When one compared individual flies of the low expression stocks, it became evident that one never found mesopleural hairs unless a fairly high number also existed on the scutellum. It became further evident that the scutellum showed no hairs unless a certain number had also been produced on the wing, usually around the bases of the veins. A definite impression results:

TABLE 4

Comparisons of average number of chaetae in homozygous flies of two different se h/se h stocks

Stock Parameter -. and x' N MSC MMS FSC FMS Pasadena 10 28.4 17.0 34.8 23.7 Berkeley 20 18.7 13.2 25.2 13.8 X2 28.92 6.65 22.37 39.53 POSITION EFFECTS OF HAIRY 121 that a type of threshold phenomenon exists. The weakest expression of hairiness affects only the wings; as hairiness becomes more extreme, the hairs are produced on the scutellum and finally on the mesopleurae. Further, they always appear in a specific topographic pattern on the scutellum and mesopleurae. On the mesopleurae, they appear first on the hind portion, but appear in the anterior portion only after the posterior region is quite well covered. Last of all, the mid-section of the mesopleurae develops hairs. Chaetae appear first on the scutellum at the Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 anterior corners, slightly lateral and forward of the anterior scutellar bristles. Only after the anterior corners are well endowed do hairs appear on the lateral margins between the anterior and posterior scutellars, between the post-scutellars themselves, and finally in the interior. Specific exceptions to these patterns do exist, but the general impression still seems to hold. The question thus arises: since the h2 allele reportedly shows chaetae only on the wing (LINDSLEYand GRELL1968), is it possible that it is an allele of high threshold, producing a suppressor only slightly less active than that of h+? Could one alter the threshold so as to produce hairs on the scutellum and finally on the mesopleurae, by either (a) selecting for modifiers that directly reduce the effectiveness of the suppressor, or (b) selecting for an increased “potency” of polygenes, which could then somehow “flood the threshold” and produce hairs in the previously prohibited places? The basic question is obviously multi-faceted, but of interest. One wonders further if, in the process of selecting parents with a good expression of the trait to continue the stock, a curator might not unconsciously generate just such a selection experi- ment as outlined above. An examination of the h”/h”stock from the Pasadena stock center revealed that such an occurrence had likely happened: virtually every fly in the stock now has at least two scutellar hairs, and up to five have been observed. Interestingly, the hairs expressed are invariably on the anterior corners. None have been observed on the mesopleurae, nor has any attempt yet been made to select for them. The genetic basis for this change in phenotype is of course unknown; it is only consistent with, not proof of, the polygene hypothesis. If the basic hypothesis is true, however, that h+ is a suppressor locus, a multitude of passible experiments come to mind. But first will have to come further selection experiments to demonstrate actions of specific chromosomes and chromosome regions, etc. Such experiments have been done, of course, with other chaetae systems. Sternopleural and abdominal bristles have been the subject of more individual reparts than any other system. Polygenes have been shown to be active in all these systems (e.g., RASMUSON1952; THODAY,GIBSON and SPICKETT1964; SPICKETTand THODAY1966; RENDEL 1969; BEARDMORE1970; LATTERand SCOWCROFT1970; MACBEAN,MCKENZIE and PARSONS1971). Polygenes and oligogenes (minor and major genes, in other terminology) interact in very complex fashion; see RENDEL(1967) for a provocative and thoughtful approach at synthesis. 122 D. E. JEFFERY The hairy system is known to have developmental affinities with some of the other chaetae systems, a fact also quite suggestive of a polygene relationship. Thus, Hw is known to increase the numbers of h microchaetae (STEINBERG 1942). Though h and pyd are both recessive and not allelic, when both are present in the heterozygous condition +/h +/pyd, they result in the formation of microchaetae (NEEL1941, 1943). The diminutive apical chaetae of the scute system (LATTERand SCOWCROFT1970) may well be essentially the same chaetae Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 that appear in the more extreme hairy phenotypes. FALK(1963) has proposed that scute, achaetae, hairy and Hairy-wing comprise a control system somewhat comparable to an operon with a regulator gene, though later studies have made the suggestion increasingly tenuous. However the controls actually operate, it is clear that the above relationships have significance for an understanding of the action of position effects on the hf gene. A given rearrangement must be contemplated not only with respect to its association with h+, but with other possible influencing factors as well. It seems evident that a rearrangement resulting in position effects on the hairy locus may also produce effects on other oligogenes associated with the hairy system, and on a variety of polygenes, There appears to be adequate rationale for the complexity of the interactions evident in the present data. Stable vs. variegated position eflects: LEWIS’(1950) distinction between S-type and V-type position effects was a major contribution. Central to the distinction between the two are three basic facts: (1) stability versus variegation in phenotype, (2) V-type position effects are associated with definite rearrangements of the chromosome structure, whereas S-types, such as Star-asteroid and Bar, appear to involve cis-trans relationships without major structural aberrations and (3) V-types are characterized by the introduction of a given gene into a foreign type of chromatin, e.g., euchromatic genes into heterochromatic regions. Though the only previously reported case of hairy position effect (DUBININ and SIDOROV1935) has long been considered a classical V-type, the hairy position alleles of this current study do not fall cleanly into either group. Using both standard deviations and variance, we have attempted comparisons of phenotypic variability both of the position alleles to the h/h homozygote, and among the position alleles themselves. The only generalization deriving from such comparisons is that the position alleles are as a rule more stable in phenotypic expression than is the h/h homozygote, contrary to what would be expected from V-type position effects. This marked stability is also seen in comparisons of the number of chaetae on the left and the right mesopleurae. Counts for the two sides were originally kept separate, but they were so consistent that it was decided to lump them together as a total score. The RxZ@combinations were at that time the major projected focus of the study, and the single figure would suffice for those comparisons. Existing data are thus too incomplete to effect a comparison of meaningful left/ right correlation coefficients, but such data would without question only verify the stability already evident in the comparison of standard deviations and vari- POSITION EFFECTS OF HAIRY 123 ances. The hairy position alleles are stable, not variegated, as judged by their phenotypes. In terms of concept (2) above, that of associated rearrangements, the six position alleles all involve major rearrangements, and are thus classed by this criterion as variegated. [Not all investigators would accept this requirement as a valid criterion, but LEWIS(1950) so derives it.] Characteristic (3) could hardly be more indecisive, since of the six associated “new residences” for the h+ gene, Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 two are clearly heterochromatic, two are euchromatic, and two “intermediate”, or at least equivocal. It would thus appear that the R(h+) position-alleles occupy a category that is so far distinctly their own. Mechanisms of position efect: EPHRUSIand SUTTON(1944) summarized the basic “explanations” of position effect offered up to that time and divided them into major groups: kinetic, or chemical diffusion, theories, and structural theories. Some later workers have attempted to add refinements, but the data available far outstrip the synthetic power of any proposal made to date. Indeed, it has been characteristic of authors during the past two decades or so to avoid the subject, since the earlier syntheses were obviously premature. Recent workers have been more cautious, attempting to define the dimensions of the problem, to stake out its bounds, before hazarding another major effort at full-scale synthesis. SPOFFORD (1976), however, is to be credited with a well-reasoned and provocative new attempt. It is almost self-evident, from the discussion to this pint, that the present paper has no new synthesis to demonstrate. It has demonstrated instead an extension of some concepts that were in the process of congealing, and while it may delay resolution of certain points, such as the necessity of juxtaposition to broken heterochromatin in variegated position effects, it should also provide further evidence that final syntheses must extend to a wide battery of cellular phenomena. Current thinking revolves primarily around transcriptional controls and tightness of chromosome packaging. The present study provides evidence that we should perhaps expand our considerations a bit. We have tended to ask, in the case of rearranged euchromatic genes, “What is the new area of residence doing to the euchrolmatic gene?” and we have found no satisfying answer. The reverse question must also be posed: “What is the euchromatic gene doing to its neighbors in the new area of residence, and can they produce some of the effects leading to the apparent conflicts observed?” Since the euchromatic gene under scrutiny in any given study is usually placed in a whole spectrum of new positions in the genome, each position allele may well be eliciting essentially unique responses from its neighbors, thereby setting up a chemical milieu quite unique to itself. It has long been evident that “heterochromatin” is not a singular and single substance, but a multitude of materials. For example, it is known that some heterochromatin is rich in repetitive DNA sequences, and that some of these sequences are rDNA, and others are DNA for coding tRNA (cf., COMINGS1972, for a superlative review, plus later sources). Will a euchromatic gene juxtaposed to one of these types produce a chemical milieu in the cell, hence a phenotype, 124 D. E. JEFFERY comparable to the other-understandable in terms of such crude measurements as distance of the affected gene from the “heterochromatin”? It hardly seems likely. It is the entire cellular environment with which we must be concerned. Rather than looking at different position alleles of a given gene whose rearrangements scatter them into variant new residences all over the genome, it may be time that we focus onto different position alleles, all of which are associated with essentially the same region of rearranged material. Once we have such an area Downloaded from https://academic.oup.com/genetics/article/91/1/105/5993315 by guest on 23 September 2021 well characterized in as precise terms as possible, then let us move to a cluster of alleles in another localized area, and compare the two groups. The present study indicates that we may need to focus on modifiers and/or polygenes, all of which can themselves be altered in any given rearrangement. Techniques and experimental design must be devised to measure these ancillary effects as well as the major ones on our chosen gene. All must be reduced to quantitative terms for any final effective analysis. I am indebted to GERALDBRAVER, who originally produced the R06 and RI5 stocks and gen- erously made them available for my use. I thank PRISCILLAHUTCHINSON and DON HELMfor technical assistance. Lastly, I express deep and special gratitude to CURTSTERN, for his initial suggestion of this study and for the unique education and encouragement that he provided in supervising my graduate program. The research was supported by Public Health Service grant 2T1-GM-367-06 to the University of California Genetics Department.

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