modification of v-type position effects in · 2003. 7. 19. · variegated position effects 27...

MODIFICATION OF V-TYPE POSITION EFFECTS IN DROSOPHILA VIRILISJ

IMOGENE SCHNEIDER3

Department of Zoology, University of Chicago, Chicago 37, Illinois

Received July 21, 1961

HE phenomenon of variegated (V type) position effects, although of intrinsic Tint erest in themselves because of their bearing on structural organization within the chromosomes, can also be used as a tool for studying the problem of functional differentiation of genetically identical cells. The majority of variegated position effects thus far studied in Drosophila melanogaster have dealt with genes whose loci are in euchromatin and which have been transposed through various types of chromosomal rearrangements in or next to interrupted heterochromatin (see review by LEWIS 1950). A limited number of genes normally located in heterochromatin and evoking a position effect when placed next to euchromatin or to foreign heterochromatin have also been discovered (DUBI- NIN and SIDOROV 1934; SCHULTZ 1936; BAKER 1953).

The mosaic phenotypes exhibited by eye color genes as a result of V-type position effects are particularly suitable for developmental studies, since not only can the genetic background be controlled but it is also possible to determine quantitatively, both visually and by chromatographic analysis, the amount of pigmentation in the compound eye.

The coloration of wild-type eyes in Drosophila is due to a combination of two distinct groups of pigments, the ommochromes or brown pigments ( BUTENANDT 1957) and the drosopterins or red pigments (ALBERT 1954; FORREST and MIT- CHELL 1954a). In the more advanced species of Drosophila, the drosopterins are confined to the eyes, whereas in the more primitive species, these pigments are also found in the testis sheaths of the males (HUBBY and THROCKMORTON 1960).

Numerous environmental and genetic factors have been found to modify the extent of variegation in V-type position effects. Temperature (GOWEN and GAY 1933), addition or subtraction of heterochromatin in the form of Y chromosomes (GOWEN and GAY 1934; SCHULTZ 1936; GRELL 1959), as well as different Y chromosome fragments (BAKER and SPOFFORD 1959) have all been shown to alter the variegated phenotype.

'This investigation was supported in part by U. S. Atomic Energy Commission Contract No. AT( 11-1)-431 and by a Traineeship from the Public Health Service Training Grant 2G-150 from the National Institutes of Health, United States Public Health Service.

2 Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy in Zoology at the University of Chicago.

3 Present address: Zoologisch-vergl. anatomisches Institut der Universitat Zurich, Zurich, Switzerland.

Genetics 47 : 2544 January 1962.

26 IMOGENE SCHNEIDER

In addition, cases have been reported in which modification of the extent of variegation has been attributed to maternal and/or paternal effects (NOUJDIN 19M; PROKOFYEVA-BELGOVSKAYA 1947; LUNING 1954). The more recent studies of SPOFFORD ( 1959, 1961 ) and HESSLER ( 1961 ) employing a rearrangement of the w+ allele, Dp(wm)264-58a, have also shown that the phenotypic expression of variegation is dependent not only upon the individual’s own genotype, but is influenced “residually” by the genotype of the individual’s parents. Factors such as (1 ) which parent contributes the duplication, (2) whether the female parent is homozygous or heterozygous for the duplication, and (3) the Y chromosome constitution of the mother, have all been implicated as affecting the amount of pigmentation and penetrance with respect to this allele.

The experiments reported in the present paper were designed to test whether such ‘Lresidual” effects are of a more general occurrence or whether they are limited to the above-mentioned cases. Specifically, chromatographic analyses of the drosopterins and their presumed pteridine precursors were carried out on a number of rearrangements of the peach locus in Drosophila uirilis which evoke position-eff ect variegation in the eyes and testis sheaths of this organism. By using a more primitive species of Drosophila, it was assumed that some evidence might also accrue which would help to clarify the metabolic interrelationships of these pteridines in the two tissues.

MATERIALS AND METHODS

The six translocation stocks of D. uirilis employed were among those described by BAKER (1953) who obtained them by irradiation of the sperm of Pasadena (wild-type) males. Each of the rearrangements when heterozygous with the recessive allele of the gene peach produces a mottling of the eye and, in some instances, of the testis sheaths also. The normal locus of this gene (pe = peach, 5-203) is in the distal end of the centromeric heterochromatin of chromosome 5.

A brief description of the various translocation stocks and their phenotypic expressions is given below; the breakage points of the rearrangements are taken from BAKER (1953).

(2) T ( Y ; 5 ) peach-mottled 2: The eyes of both male and female flies are ex- tremely mottled with areas of peach predominating over that of wild-type pigmentation. In general, the males show more extreme mottling than do the females. The testis sheaths of the males are mottled, but for the most part have the very light orange-brown color characteristic of peach males. No rearrangement is discernible in the salivary gland chromosomes. Genetic tests indicated a Y;5 translocation with a break in 5 distal to the pe locus. The rearranged chromosome may have resulted from an insertional rather than a reciprocal translocation, e.g., pe+ may be inserted into the Y (BAKER 1956).

(2) T ( 4 ; 5 ) peach-mottled 3: Mottling of the eyes in both sexes is very apparent although not as extreme as is the case with pees*. The testis sheaths are also well mottled. No rearrangement is visible in the salivary gland chromosomes. Genetic analysis indicated a 455 translocation with the break in chromosome 5 being distal to the p e locus and the break in chromosome 4 in the basal

VARIEGATED POSITION EFFECTS 27

heterochromatin. The 45 chromosome may therefore be the result of a reciprocal translocation or it may have resulted from an insertion of pe+ into the heterochromatin of chromosome 4.

( 3 ) T(3;5) peach-mottled 4 : Mottling of the eyes is less apparent than in either pemz or pem3. Some mottling is apparent in the testis sheaths but it is quite restricted and for the most part the color approximates that of wild-type sheaths. The breakage point in 3 is between 3B2e and h and the break in 5 is proximal to pe, between 5H2d and the centromere [see chromosome map of D. uirilis by GRIFFEN (PATTERSON, STONE and GRIFFEN 1940) 1.

(4) T ( Y ; 5 ) peach-mottled 2 5 : Mottling of the eyes is quite restricted with the areas of peach pigmentation being few and small. The testis sheaths are a deep orange in color and visually appear to be darker than those of the Pasadena strain. There is no mottling in the sheath. No rearrangement is visible in the salivary gland chromosomes. This translocation resulted from a break in chromosome 5 proximal to pe and subsequent attachment to the distal end of the Y from which the tip had been broken. Males of the stock used carry the Y5 chromosome but lack the small 5y chromosome. They are viable and fertile indicating, as BAKER (1953) noted, that the tip of the Y does not contain any factor(s) essential for male fertility.

( 5 ) T ( 2 ; 5 ) peach-mottled 39: Mottling of the eyes in both sexes is confined to a few facets containing peach pigmentation. The testis sheaths appear to be darker than those of Pasadena males and no mottling is visible. The breakage point in 2 is between 2D2g and 2D3a and in 5 between 5H2d and the centromere.

( 6 ) T ( 3 ; 5 ) peach-mottled 5 2 : Mottling of the eyes is very prominent with large patches of peach pigmentation in evidence. The testis sheaths are also well mottled. The breakage point in 3 is between 3B2c and d, and in 5 it is between 5H2d and the centromere.

Using chromatographic techniques, the following pteridines were isolated and measured in both the eyes and testis sheaths: drosopterins (DP) , sepia pteridine (SP) , and the complex of 2-amino-4-hydroxypteridine (HB,) and biopterin (HB,) . Isoxanthopterin (IX) is also found in both tissues but as it is present in relatively minute amounts in the eyes, measurements of this pteridine were confined to the testis sheaths.

The translocation under study was introduced by a single male or female, either homozygous or heterozygous for the rearrangement. Unless stated to the contrary. the other parent was homozygous for the peach gene. With respect to the comparison of off spring from homozygous us. heterozygous mothers, the following mating plan was used: a single R ( p e + ) / R ( p e + ) female was mated to a peach male and the resulting variegated offspring chromatographed. A single F, R(pe+) /pe female selected at random from among the progeny was mated to a peach male and the F, variegated offspring were chromatographed. The reciprocal procedure was used for comparing offspring from homozygous us. heterozygous fathers. The cultures were maintained at a temperature of 2321°C. Four to six replicates of each mating were made, the parents being transferred to fresh food every second day. For purposes of


comparing extremes in pigmentation, homozygous peach and Pasadena flies were also chromatographed.

All flies to be chromatographed were collected within four hours after emer- gence from the puparium. They were then aged ten days before being subjected to chromatography in order to assure completion of pigment deposition in the eye. The chromatographic procedure used was essentially the same as that described by HESSLER (1961 ) . The fluorescent solutions were measured with a Farrand (Type A) photofluorometer. A Corning No. 9863 filter was used as the primary filter between the UV lamp and the sample. Between the sample and the phototube, a secondary filter, Corning No. 3389 was used in conjunction with the following Bausch and Lomb interference filters: No. 33-78-55 for measurements of DP and SP; No. 33-7845 for measurements of the HB complex; and No. 33-7841 for measurements of IX. An arbitrary fluorescent standard of 1 ,ug of anthranilic acid per 1 cc solvent was used for all readings. Since the standard was found to decompose at room temperature it was kept frozen until two hours prior to use. The fluorometer was adjusted to give a galvanometer reading of 20 (1-1 00 scale) for 1 ml of standard before readings were taken on all of the substances with the exception of IX. For the latter pteridine, a reading of 10 was used. Fluorescence readings of paper blanks cut from the filter paper at the same level as the samples were subtracted from all experimental readings.

RESULTS

Before describing in detail the diverse effects found with respect to the parental source of the translocation in each of the peach-mottled stocks, a few general comments can be made concerning the relative amounts of pteridines found in these flies as compared with homozygous Pasadena and peach strains. On a quantitative basis, homozygous peach flies contain lesser amounts of the drosopterins in their eyes as well as lower amounts of all of the pteridines measured in the testis sheaths of the males (Table 1). However, the values for SP and the HB complex in the eyes are exceeded only by the corresponding pteridine values of pe". At the other extreme of pigmentation the Pasadena strain and pem39 contain the highest amounts of DP in their eyes. The other stocks in order corresponding to decreased amounts of DP, both in the eyes and testes, between these extremes are pem3, pem4, pem15, pemsl and peml. Comparative measurements indicate that all of the translocation stocks accumulate the other pteridines found in the eyes and testes to a greater extent than is found in the Pasadena strain, with the exception of IX in pe".

For convenience in presentation, the results obtained from those translocations involving the Y and 5th chromosomes, e.g., pe" and pem15 will be considered first and individually; the results obtained with pe"", p"4, pem39 and pemS1 will be considered together.

To facilitate recognition of the parental source of the rearrangement under study, the following notation is used: R ( p e + ) written to the left of the diagonal indicates that the translocation was transmitted by the female parent; R ( p e + )


coa "1c? +I +I h 9 2 2 U)*

3 c o bi'4 +I +I od- zz g p

2 2

3 3

+I +I m a

a a

d O U ) z m n u ,

3 3

+I 2 d-

00 '4 +I ? 8 a o?

+I '4 * $

W M. +I 5 3

M o?

+I ro

a 09 +I o? 3

8 Q, c? +I 3

W

2 M

Q, h +I 3

2 3

o a c?a +I +I

2s? O h .IT

* Q '4'4

+I +I "a 2 2 o m

W h "1"

+I +I " '4 83 W Q ,

m m m c o

-. '0

E ,--.a L i j -7 E S E 2


written to the right of the diagonal indicates the translocation was inherited through the male parent, e.g., R (pe+ ) / p e or p e / R (p+ ) respectively.

T(Y;5 ) peach-mottled I : Table 2 summarizes the chromatographic data of mottled offspring from three crosses involving this translocation. If the male offspring from the first two crosses are compared (columns 1 and 3 ) , it is im- mediately apparent that the values for all of the pteridines, with the exception of the HB complex in the eyes, are much higher in the first cross than in the second. These differences are significant at the one percent level ( t test).

Three possible interpretations for these results are as follows: (1) this difference is due to a parental-source effect, e.g., mottled sons receiving R ( p e + ) from the mother have more pigment than if R(pe+) is transmitted by the father, (2) the mother in the first cross has a Y chromosome whereas in the second cross the mother does not, or ( 3 ) this difference may be due to the fact that when the mother is Y:pe+/X/X; pe/pe, all of the mottled sons will carry a second Y chromosome contributed by the father.

To distinguish between these three possibilities, the following matings were made. The same u/Y:pe+; pe/pe male was mated to (1) Y:pe+/X/X; pe/pe females and (2) X/X; p e / p e females. From mating (1) the following mottled males arose: (a) X/Y:pe+; pe/pe, (b) Y:pe+/X/Y:pe+; p e / p e and (c) Y:pe+/v; pe/pe. From mating (2) one type of mottled male resulted, namely, (d) X/Y:pe+; pe/pe. Thus a comparison between males (a) and (c) will indicate the presence or absence of a parental-source effect; between males (a) and (b) the effect of a second Y chromosome; and between males (a) and (d) the effect of female parents with and without a Y chromosome. [ U = vermilion, 1-255. used to mark the X chromosome of the male parent, blocks one of the steps in the formation of the ommochromes, but presumably has no effect on the synthetic pathway leading to the formation of the drosopterins. However, HADORN and MITCHELL (1951) state that the U mutant in D. melanogaster which is homologous to the same mutant in D. uirilis (HOWLAND, GLANCY and

TABLE 2

Pteridine annlysis of variegated offspring from crosses inuoluing T(Y;5 ) peach-mottled 1. Values represent mean fluorometric readingkstandard error

1' Pe+/X/X, pe/Qe 0 P X/V, w / P e d d

X/X p C / w P P W X , w / p e P P Ci mseh

X/Y PP+, PP/W d 0" WID pet, w / p e d 0"

Offspring Y pe+/X/Y, p e / p e Y pef/X/X, p e / p e X D pef, p e / p e X/X/Y pr+, p e / p P No YL 67 04 b6

Pteridine (eyes) DP 13.50-C .55 18.61% 3 0 8.922 .I1 17.78% .64 SP 26.82-C .57 32.72% .74 23.702 .69 35.482 .53 HB 20.152 .43 23.14% .36 20.45% .39 27.912 .47

DP 6.90C .23 5.92% .16 . . . . . . . . SP 18.742 .67 . . 14.16% .35 . . . . . . . HB 12.00% .32 . . . . 9.734 .20 . . . . . . ,

IX 28.072 .62 20.20% .36 . . . . . . . .

Pteridine (testes)


SONNENBLICK 1937) has a slight lowering effect on some of the pteridines]. Table 3 summarizes the results from these crosses. Since secondary nondis-

junction in X/X/Y females of D. uirilis is very low, reported values ranging from 0.53 percent (KIKKAWA 1932) to 0.89 percent (DEMEREC and FARROW 1930), only eight Y:pe+/v; pe/pe males appeared in a total of 4123 offspring. A comparison of values from X/Y: pe+ ; pe/pe (a) males and Y : pe+/u; pe/pe (c) males may not be too meaningful since the number of individuals of the latter genotype is very small. Furthermore, no tests were made to evaluate what effect the U mutant is having on the pteridine values of (c) males. Nonetheless, if a comparison is made, it may be seen that (c) males have lesser amounts of DP, SP and the HB complex in the eyes and IX in the testes than do (a) males, whereas the values for the other pteridines in the testes do not differ appreciably. This suggests the possibility of a maternal-source effect.

Since it is not always possible to distinguish visually males having one Y chromosome (a) from those having two (b) , each male was progeny tested by mating to three X / X ; pe /pe females. Those males having two Y chromosomes produced some variegated daughters whereas those males having a single Y produced only peach females. A comparison of the values obtained from a pteridine analysis of these males indicates that those males having two Y chromosomes contain greater amounts of all of the pteridines except for the HB complex (eyes) than do males having a single Y . A comparison of males (b) with those of the genotype Y:pe+/X/Y; pe/pe (Table 2, column 1) indicates that there are significant differences in the values for DP (five percent level) but no differences for the other eye pteridines. However, the differences between the pteridines in the testis sheaths of these two types of males are highly significant.

Finally, a comparison of (a) and (d) males (Table 3 ) indicates the effect which a Y chromosome in the genome of the mother has on the phenotypic expression of mottling in her sons, even though they do not receive this chromo-

TABLE 3

Pteridine analysis of variegated male offspring from crosses inuoluing T(Y;5) peach-mottled 1. Comparison of parental source of translocation, Y chromosome constitution of female

parent and effect of an extra Y chromosome. Values represent mean fluorometric readingkstandard error

Crosses Y : p e + / X / X ; p e / w P 0 x u / Y : p e + ; w / p e d x X/X; p e / w P P X / Y : p e + ; p e / p e

(d)

NO. 102 91 8 a7

(a) (b) (C) Offspring X / Y : p e + ; p e / p e Y : p e + / X / Y : p e + ; p e / p e Y : p e + / u ; p e / p e

Pteridine (eyes) DP 10.15% .36 14.95% .52 7.622 .82 8.472 .35 SP 24.532 .51 27.97% .64 22.482 .65 23.522 .52 HB 19.622 .33 19.302 .50 18.532 .90 21.12f .34

DP 5.73% .29 8 .382 .36 5.502 .42 6 .272 .24 SP 14.872 .34 24.332 .77 14.5221.56 14.152 .46 HB 8.40f .26 13.232 .51 8.6221.24 9 .352 .55 IX 19.762 .43 31.922 .83 18.0421.78 19.702 .55

Pteridine (testes)


some. Differences in the values for DP (eyes) and the HB complex (eyes and testes) are significant at the one percent level. It is interesting to note, however, that the values for DP are higher in (a) males whose mothers had a Y chromosome, than in (d) males, whose mothers did not, whereas the reverse is true for the HB values.

A comparison of the parental-source effect of the rearrangement on variegated daughters was made through the following crosses: X / X ; pe/peO Q X X / Y / Y : p e + ; p e / p e 8 8 and Y : p e + / X / X ; p e / p e 0 0 x X / Y ; p e / p e 8 8 . Mottled daughters from the former cross received R ( p e + ) from the male parent; in the latter cross, from the female parent. It should be noted, however, that a complicating factor in this comparison is the fact that in the first cross the mothers did not contain a Y chromosome, whereas in the second cross they did. Female offspring of the genotype Y : p e + / X / X ; p e / p e have somewhat higher values for DP than do X / X / Y : p e + ; p e / p e Eemales (Table 2, columns 2 and 4) but the differences are not significant. However, the latter females contain significantly higher (one per cent level) amounts of both SP and the HB complex.

T ( Y;5) peach-mottled 15 : The results of reciprocal crosses involving this translocation are given in Table 4. Both male and female offspring have more pigment in their eyes as well as higher amounts of all pteridines, with the exception of IX in the testis sheaths of the males, when the rearrangement is transmitted by the male rather than the female parent. As with pe"', the three most likely explana- tions are that (1 ) there is a parental-source effect; here, however, it would be a paternal source which produced the most pteridines, (2) the presence or absence of a Y chromosome in the female parent is affecting the amount of pigmentation in the offspring, or ( 3 ) the differences. with respect to males only, may result from the fact that all of the male offspring will have an additional Y chromosome when the female parent contributes R ( p e + ) .

To resolve this problem, Y ' . p e + / X / X / p e females were crossed to X / Y / Y 5 . p e + /

TABLE 4

Pteridine analysis of variegated offspring from crosses involving T(Y;5) peach-mottled 15. Values represent mean fluorometric readingestandard error

Pteridine (eyes) DP 21.76zk .69 21.462 .39 33.68% .86 23.52e1 .I9 SP 15.322 .32 16.942 .36 28.42-r- .70 25.00zk1.59 HB 10.45k .28 11.59% .2l 13.245 .31 16.20% .67

Pteridine (testes) DP 12.23% .43 . . . . . 14.80zk .47 . . . .

SP 38.11% .96 . . 47.29e1 .I8 . . HB 20.47-C .54 . . . 23.52e .58 . . . . . . IX 49.34e1.06 . . . 48.25-C-1 .I 7 . . . . . .


BSpe males. ( B S = Branched 3,5-141, used to mark the normal 5 chromosome of the male parents, affects the venation of the wing; although described as a dominant, it was recessive to its wild-type allele in the stock used). From this cross, the following mottled off spring resulted: (a) X / X / Y 5 - p + / p e females, (b) Y 5 . p e + / X / X / B S p e females, (c) Y 5 . p e + / X / X / Y 5 . p e + females, (d) X/Y/ Y 5 - p e + / p e males and (e) Y 5 p e + / X / Y / B s p e males. A comparison between (a) and (b) females and (d) and (e) males will indicate whether the parental source of R ( p e + ) has any effect on pigmentation in the offspring. Females (a) and (b) compared with (c) females will assess the influence of a second Y chromosome and/or two doses of R ( p e + ) as compared with one. Likewise, a comparison can be made between males (d) and (e) and those from the cross X / X ; p e / p e ? ? x X / y 5 p e + / p e 8 8 (Table 4, column 3) which have a single Y chromosome. The results of this cross are given in Table 5.

To ascertain the genotypes of the flies to be chromatographed, the female offspring were individually mated to X / Y ; BSpe/B8pe males; the male offspring to X / X ; BSpe/B3pe females. Females of the genotype Y 5 - p e + / X / X / y s . p e + produced mottled off spring only; females X / X / Y 5 . p e + / p e and Y 5 p e + / X / X / B 3 p s produced peach and mottled offspring in approximately equal numbers. These latter two types of females were distinguished from each other in that most of the peach off spring from Y 5 . p e + / X / X / B 3 p e females had branched venation in their wings. The genotypes of the male offspring were determined in a similar manner.

The data in Table 5 show no significant differences if the values for DP and SP obtained from X / X / Y ” . p e + / p e (a) and Y 5 . p e + / X / X / B s p e (b) females are compared. The discrepancy between these results and those obtained in the original crosses (Table 4) with respect to the SP values is great. Since the DP and SP values for Y 5 . p e + / X / X / p e females (Table 4, column 2) and for Y 5 p e + / X / X / Bspe females (Table 5 , column 2) are similar, the most likely explanation of this discrepancy is that a Y5 chromosome in the mother suppresses pigment produc-

TABLE 5

Pteridine analysis of uariegated offspring from crosses involving T ( Y ; 5 ) peach-mottled 15. Comparison of parental source effect versus the addition of a Y chromosome to the

genome. Values represent mean fluorometric reading-tstandard error

Pteridine (eyes) DP 21.74f .84 21.662 .58 17.91f1.22 21.91 2 1.33 20.9621.34 SP 17.22+- .61 17.322 .40 23.39-Cl.68 17.182 .74 15.00+1.22 HB 15.87% .33 13.632 .38 17.41-C .92 10.682 .64 10.56+ .49

Pteridine (testes) DP . . . . . . . . . . . . . . . . . . . . . . 11.772 .68 13.432 .83 SP . . . . . . . . . . . . . . . . . . . . . . 37.6422.02 43.2221.94. HB . . . . . . . . . . . . . . . . . . . . . . . . 18.8621.46 20.22.21 .I 5 IX . . . . . . . . . . . . . . . . . . . . . . . . 42.41k1.96 48.7822.43


tion in the offspring. Although very few Y s . p e + / X / X / Y 5 p e + (c) females were obtained, it is nonetheless apparent thai these females have far lesser amounts of DP and greater amounts of SP and the HB complex than do either (a) or (b) females.

If X / Y / F . p e + / p e (d) males are compared with Y 5 . p e + / X / Y / B 3 p e (e) males it is evident that there are no differences in the values for DP and the HB complex (eyes). However, (d) males have somewhat higher values for SP (eyes) whereas the reverse is true for the pteridines in the testis sheaths. Here, higher values are recorded for all of the pteridines, the differences attaining considerable magnitude with respect to the values €or SP and I X .

Finally, it should be noted that X/Y”pe+/pe males (Table 4) which have a single Y chromosome contain greater amounts of all the pteridines with but one exception ( I X ) than do either (d) or (e) males (Table 5 ) , each of which has two Y chromosomes. Therefore, the extent of mottling is increased in both male and female pem15 offspring by the addition of extra Y chromosomes to the genome, although part of this difference may be attributed to the fact that the mothers of the former males do not carry a Yj chromosome.

T ( 4 ; 5 ) peach-mottled 3 , T ( 3 ; 5 ) peach-mottled 4, T(2;5) peach-mottled 39 and T ( 3 ; 5 ) peach-mottled 51: These translocations will be dealt with as a unit since they possess one important attribute in common: the homozygotes, e.g., R ( p e + ) / R ( p e + ) , of both sexes are viable and fertile. It is therefore possible to compare heterozygous mottled off spring having ( 1 ) homozygous female us. homozygous male parents, (2) homozygous us. heterozygous female parents, ( 3 ) homozygous us. heterozygous male parents, and (4) heterozygous female us. heterozygous male parents. A comparison of the effects obtained with respect to the different parental sources of R ( p e + ) can be made by referring to Table 6. the actual data being given in Table 7.

Efjlect of homozygous female vs. homozygous male pcrrent in contributing R(pe+) : considering first the visible pigments (DP) in the heterozygous mottled offspring of both sexes, it may be seen that there is a definite effect as to which parent contributes R ( p e + ) . With each of the four translocations the effect is in the same direction; namely, if a homozygous mother rather than a homozygous father contributes R ( p e + ) , the values for DP in the eyes of both sexes, and in the testis sheaths of the males, are significantly higher. The values for I X are also higher if R (pe+ ) is transmitted by a homozygous mother. This effect, however, is not always carried over to the other pteridines. In this respect the four translocations are seen to differ with each other. Furthermore, the values for these pteridines in male and female off spring heterozygous for the same translocation may show differences as to the parental source of R ( p e + ) . This same dichotomy is also reflected in the eyes and testis sheaths of the males.

Effect of homozygous vs. heterozygous female parent in contributing R (pe+ ) : Off spring heterozygous for either pem3 or pen’ 39 have significantly higher amounts of all pteridines (both eyes and testes) if R ( p e + ) is contributed by a homozygous rather than a heterozygous mother. This same effect is present in offspring heterozygous for pemr, but only for values of DP, the HB complex and I X . In the


TABLE 6

General trends of pteridine values for different parental sources of the translocation in heterozygous variegated offspring of peach-mottled 3, 4 , 39 and 51 (compiled from Table 7)

Source (a)Homozygous mother (a)Homozygous mother of us. us.

R(pe+) (b)Homozygous father (b)Heterozynous mother

Eyes Testes __.__

T(4;Fj)pemns ? ? dd dd DP > > >

> SP _ _ > HB - -.

IX >

DP > > > SP < = < HB > > = IX >

DP > > > SP _ _ - HB < = > IX >

DP > > > SP = > = HB = > > IX >

_ _ - _

T (3;5) pe”4

T (2;5 ) pem3-9

- - -

T (3;5)pem51

Eyes Testes ~~

? ? dd dd > > > > > > > > >

>

> > = < < = > > =

>

> > > > > > > > >

>

(a j Homozygous father

(b)Heterozygous father (b)Heterozygous father

(a j Heterozygous mother us. us.

~~~ - Eyes Testes - _ _ Eyes Testes

-___ ~

P P dd dd 9 9 dd dd > = = , > > = = < = < < = < = = < < = - - - -

> indicates that (a) values are significantly higher (1% level) than (b) valuec <indicates that (a ) values are significantly lower than (b) values - indicates that there are no sigmficant differences betaeen (a) and (b) values

eyes of both male and female offspring, the reverse holds true for values of SP. In the testis sheaths, there is no effect with respect to the values for DP, SP and the HB complex.

Female off spring heterozygous for pem51 have significantly higher values for DP and the HB complex if R ( p e + ) is transmitted by a homozygous rather than a heterozygous mother. However, there are no significant differences in the values for SP. Heterozygous pem5’ males differ to a much greater extent in that there is no effect as to the parental source of R ( p e + ) for any of the pteridines with the exception of SP and the HB complex (testes) which are significantly higher if R ( p e + ) is transmitted by a homozygous mother.

Effect of homozygous vs. heterozygous male parent in contributing R(pe+) : Variegated daughters of pem4 and pems3 have significantly higher amounts of all three eye pteridines if R ( p e + ) is transmitted by a homozygous father. This effect is also present for pem3 and pem51 females, but only with respect to values for DP. The paternal genotype in pems females has no effect on the values for SP; the values for the HB complex are significant but in a direction opposite to that obtained with values of DP. The reverse of this is found in pemsf females.

36 1 IMOGENE SC HNEIDER

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With the exception of pen$39 male offspring, there is little evidence as to any consistent effect of the parental genotype. In these males, the values for all of the pteridines with the exception of IX are higher if R ( p e + ) is transmitted by a homozygous father. In pem3, only the values for SP (eyes) are significantly higher if R ( p e + ) is transmitted by a heterozygous father. With pem4, values for the HB complex (eyes and testes) are higher if R ( p e + ) is transmitted by a homozygous father. No differences are in evidence for the other pteridines in both the eyes and testis sheaths.

Male offspring of pemsl show quite an anomalous behavior. Values for all of the eye pteridines and for the HB complex and IX in the testes are significantly higher if a heterozygous rather than a homozygous male contributes R ( p e + ) . The reverse holds true for values of SP (testes) whereas there is no effect on the amount of DP in the testes.

Effect of heterozygous female vs. heterozygous male parent in contributing R(pe+) : There is no effect as to the parental source of R ( p e + ) in pem3g offspring. The differences in the pteridine values are well below the level of significance for both male and female offspring.

In contrast, pem4 off spring have significantly higher values for all pteridines with the exception of SP (male eyes and testes) and IX if R ( p e + ) is transmitted by a heterozygous mother. The values for DP in the eyes of pe3'L3 offspring are higher if R ( p e + ) is contributed by a heterozygous mother. The opposite holds true for values of SP and the HB complex. There is no effect on the testis sheath pteridines.

With respect to pem5' offspring, the values for DP, both eyes and testes, are higher if R ( p e + ) is transmitted by a heterozygous mother. In the female offspring, the reverse holds true for values of SP. There is no effect as to the parental source of R ( p e + ) for the other pteridines.

DISCUSSION

The principal modifying factor in suppressing the extent of variegation in pe" males of the genotype X / Y / Y : p e + ; pe/pe and X / Y : p e + / Y : p e + ; pe/pe as compared to X / Y : p e + ; pe/pe males is that of a second Y chromosome. If a parental-source effect is present with respect to the transmission of R ( p e + ) , an effect neither proven nor disproven in the present study, it is of minor importance insofar as its influence in modifying thc extent of variegation in the mottled male offspring is concerned. This effect of an additional Y chromosome is not consistent with the generalization based on the behavior of the light gene in D. melanogaster ( SCHULTZ 1936; MORGAN and SCHULTZ 1942) that the addition of extra heterochromatin to the genome enhances the variegation of heterochromati- cally located genes. However, studies on the modification of position effects of the cubitus interruptus locus in D. melanogaster, due to addition or subtraction of heterochromatin to the genome, indicate that the case of the light gene may, in fact, be somewhat anomalous. ALTORFER (1952) found an enhancement in the ci-index of XO, R(c i ) /c i males when compared with X/Y, R(ci ) /c i males. Unfortunately she did not make similar comparisons with respect to R(c i+) / c i .


GRELL (1959) showed that the degree of cubitus interruption in R(ci+)/ci females was suppressed in direct proportion to the number of Y chromosomes present; e.g., X / X / Y / Y females showed greater suppression than did X / X / Y females, which in turn showed more suppression than did X / X females.

These findings in conjunction with those of this paper, indicate that the dis- tinction between position effects involving genes situated in euchromatin and heterochromatin with respect to their behavior toward addition of extra heterochromatin to the genome may no longer be a completely valid one.

The parental source of R ( p e + ) has no effect on the amount of pigmentation in the eyes of peml female offspring. This finding more or less parallels the results with respect to pe" males. Since the presence of a Y chromosome in the genome of the mother led to a suppression of variegation in her mottled male offspring, it was of interest to know if pe" female offspring were affected in the same way. In an attempt to determine this, X / Y : p e + ; pe /pe males were crossed to ( 1 ) Y : p e + / u / v ; pe /pe females and ( 2 ) X / X ; pe /pe females. A comparison of mottled daughters from these two crosses could not be made, however, since no excep- tional females were found in a progeny count exceeding 2000 individuals from the first cross.

Inasmuch as the translocation stocks which are viable in the homozygote, R (p"+ ) / R (pe+ ) , have more pigment in the eyes than the corresponding hetero- zygote, R ( p e + ) / p e , it should not be surprising that X / Y : p e + / Y : p e + ; pe /pe males of peml contain higher quantities of the pteridines than do X / Y / Y : p e + ; pe /pe males. If two Y : p e + chromosomes suppress the variegation to a greater extent than does one Y : p e + chromosome plus an unaltered Y , it is interesting to note that this effect does not encompass the eyes and testis sheaths to the same extent although it may possibly be a reflection of their different embryonic origins (POULSON 1950). (Although measurements of the pteridines were confined to the eyes and testis sheaths, visual observation of larval Malpighian tubes indicates that the color of the tubes in offspring of male or female R ( p e + ) / R ( p e + ) flies may be somewhat darker than those from the offspring of R ( p e + ) / p e flies, but the difference is not striking. It should be noted that this contrasts with the findings for the light gene in D. melanogaster [BRIDGES and BREHME 19441 ) .

With respect to the other Y;5 translocation, pem15, the parental source of R (pe+ ) apparently has no effect on the amount of pigmentation in the variegated offspring. It is also probable that the addition of extra Y chromosomes to the genome of these flies results in an enhancement of variegation, a finding dia- metrically opposite to that obtained with pe". Since in a number of instances, the pteridine values were averaged from genotypic classes consisting of a very few individuals, these statements must remain tentative until further experimenta- tion supports or negates them.

One valid criticism can be leveled against any interpretation made concerning the results obtained with respect to pe", p e n a 4 , pena3$ and pem5'. Since these translocations were maintained as homozygotes prior to the present study, it is possible that a number of genetic modifiers of variegation have been incorporated into


these stocks. However, descriptions as to the extent of mottling in the eyes of these four translocation stocks when they were first obtained (BAKER, personal communication) are equally applicable to the present stocks. This would indicate that if modifiers have arisen, their effect on the phenotypic expression of mottling in the eye is not visually perceptible. However, the possibility of different modifiers of variegation being present in the homozygous translocation stocks and in the peach stock should be kept in mind in the discussion which follows.

The effect of the parental source of R ( p e f ) will be discussed only with respect to the amount of visible pigmentation present in the mottled offspring. The significance of the other pteridine values will be deferred until later.

Without exception, heterozygous variegated off spring of pem3, pem4, pem39 and pem5* are more highly pigmented if R ( p e + ) is transmitted by a homozygous female rather than a homozygous male parent. Since, in essence, the male parent contributes only nuclear material to the zygote, the most obvious mechanism by which this difference could be explained would be that of incorporation of maternal products into the egg. Since the contents of the accessory nurse cells are emptied into the cytoplasm of the developing eggs before fertilization takes place, it is not implausible to assume that some of the products of these nurse cells are in one way or another associated with the metabolic pathways leading to eventual pigment formation.

A similar mechanism could also be used to explain the parental-source effect of R ( p e + ) from heterozygous female us. heterozygous male parents. Heterozygous mottled offspring of pem?, pem4 and all have more pigmentation if R ( p e + ) is transmitted by a heterozygous mother. The offspring of pemJ9 remain a puzzle in that there is no effect on any of the pteridines with respect to the parental source of R ( p e + ) .

A maternal effect may also be applicable in explaining the fact that heterozygous mottled offspring receiving R ( p e + ) from a homozygous mother have more pigmentation than comparable off spring receiving R ( p e + ) from heterozygous mothers. Assuming that the same number of nurse cells contribute their contents to the eggs of both mothers, the further assumption must be made that the substances produced in homozygous mothers are more competent in their influence on the pigmentary system or the same substances are produced in greater quantity. However, with this scheme it is difficult to reconcile the fact that in contrast to heterozygous pemS1 daughters. heterozygous pen151 sons show no differences as to the parental source of R ( p e + ) . Nor does it explain the lack of a parental-source effect on the testis sheaths of peTn4 off spring.

Finally, considering the heterozygous mottled off spring from homozygous us. heterozygous male parents, it may be seen that the paternal effect of R ( p e + ) is limited to female progeny, with the exception of pem39. In all cases, if a paternal effect is present, the offspring receiving R ( p e + ) from a homozygous father have more pigment. One possible explanation is that one or more genetic modifiers are present in these translocation stocks. Since the heterozygous male parents were obtained by crossing homozygous males to peach females, this modifier(s) could have been outcrossed and not incorporated into the genome of the heterozygous


males. However, it is difficult to explain these results with such a scheme without further postulating a differential effect of these modifiers on male and female offspring bearing the same translocation. Another possible mechanism to explain these results could be based on polyspermy since COUNCE (1959) found that 50-100 supernumerary sperms are regularly found in the fertilized eggs of D. uirilis females.

The results obtained with pem3, pem4, pen139 and pemS1 with respect to the transmission of R ( p e + ) indicate that the so-called "residual" effects of the parental genotype on the phenotypic expression of genes exhibiting V-type position effects are not restricted to the white locus in D. melanogaster. Whether or not such effects are confined to the pteridine synthetic system alone or are operating on a number of systems within the organism has yet to be determined.

It is interesting to note that pem4 and pemS1 show no greater similarities in their behavior toward the parental source of R ( p e + ) when compared with each other, as when compared with the behavior of pem3 and pe"". A more consistent behavior between the former two translocations might be expected since their respective breakage points in the euchromatin are within a few bands of each other on chromosome 3, and in each case the break in chromosome 5 is between 5H2d and the centromere. By measuring the relative lengths of the 35 and 53 chromosomes in metaphase ganglion cells, BAKER (1954) concluded that in pemS1 the break in chromosome 5 occurred in close proximity to the centromere whereas in pem4, the break was just proximal to the pe locus. Therefore, the amount of interstitial heterochromatin incorporated into the 35, pe+ chromosome was much more extensive in the case of pemS1 than in pem4. Thus some of the differences in their behavior might possibly be attributed to this fact.

The metabolic interrelationships between the simpler pteridines, e.g., isoxanthopterin and 2-amino-4-hydroxypteridine and the more complex pteridines, biopterin, sepia pteridine and the drosopterins, are as yet unknown. Although structures have been formulated for all of these compounds (FORREST and MIT- CHELL 1954a,b, 1955; VISCONTINI 1958a,b; FORREST, HATFIELD and VAN BAALEN 1959; VISCONTINI and MOHLMANN 1959), the immediate precursors to the drosopterins have yet to be discovered.

In view of the fact that both biopterin and sepia pteridine have been suggested as possible precursors in the synthetic pathway leading to the drosopterins, it is of interest to compare the mean values obtained in this study for these three pteridines. In all of the translocation stocks, with the exception of pe", wild-type tissue greatly predominates over that 01 peach tissue in the eyes. The mean values for DP are correspondingly high in these stocks when compared with pe". However, the reverse holds true for the values of SP and the HB complex. A similar comparison can be made between the pteridines in the eyes and testes of male flies. In the testis sheaths, minimal levels of DP are produced; as a conse- quence, the levels of SP and the HB complex attain considerable magnitude, suggesting an accumulation of these compounds in the tissue. More meaningful comparisons could perhaps have been drawn had separate values for the amounts of HB, and HB, been obtained. Although these two compounds can be separated

42 I M O G E N E .SCHNEIDER

by running two-way chromatograms, much of the sepia pteridine will be lost due to its extreme instability. Therefore, reliable values for each of the HB’s could be obtained but at the expense of obtaining unreliable values as to the amounts of SP present.

Although there are no exceptions to the statement that if the mean values of DP are high, those of SP and the H B complex are low and uice uersa, there is no consistent correlation, linear or otherwise, if values of DP are plotted against those of either SP or the HB’s for individual flies. This holds true regardless of whether these values are plotted for the eyes only, for the testis sheaths only, or for eyes us. testes values. The only consistent correlation found is a linear one between SP and the H B complex in the testis sheaths. The puzzling relationships of the other pteridines to each other and to the drosopterins no doubt reflect our lack of knowledge as to the metabolic pathways involved in pigment formation as well as the likelihood that there is tissue differentiation of the pteridines during ontogeny.

SUMMARY

A series of six translocations, each of which evokes a position effect of the peach locus (chromosome 5 ) in D. uirilis, were studied to determine whether the parental source of R ( p e + ) had any influence on the amount of pigmentation in the heterozygous variegated offspring. Utilizing paper chromatography. quantitative measurements were made on the drosopterins and a number of other pteridines, some of which presumably act as intermediates in the synthetic pathway leading to the formation of the red pigments. Measurements were made on the eyes of both male and female offspring, and on the testis sheaths of the males.

Without exception, heterozygous Variegated offspring are more highly pigmented if R ( p e + ) is transmitted by a homozygous female parent in contrast to a heterozygous female, homozygous or heterozygous male parent. Homozygous male parents usually produce more highly pigmented off spring than do heterozygous male parents. In addition, mottled offspring receiving R (pef ) from heterozygous mothers often have more pigment than the corresponding off spring receiving R ( p e + ) from heterozygous fathers. These statements are true only so far as the visible pigments (drosopterins) are concerned. The other pteridines, in contrast, show no consistent pattern as to the parental source of the translocation.

With respect to one of the Y;5 translocations studied, the presence of a Y chromosome in the genome of the mother suppressed the phenotypic expression of mottling in her male offspring, even though they did not receive this chromosome. Paralleling this finding is the fact that the addition of an extra Y chromosome to the genome of these flies led to an even greater suppression of variegation, a finding inconsistent with the generalization that extra heterochromatin enhances the extent of mottling in V-type position effects associated with genes located in heterochromatin.


ACKNOWLEDGMENTS

The author takes pleasure in acknowledging her gratitude to DR. WILLIAM K. BAKER for his guidance and interest throughout the course of this work, and to DRS. JANICE B. SPOFFORD and JACK L. HUBBY for their comments and criticisms of the manuscript.

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