immunochemical analysis of the effects ofimmunochemical techniques: the techniques used for...

IMMUNOCHEMICAL ANALYSIS OF THE EFFECTS OF HETEROCHROMATIC-EUCHROMATIC REARRANGEMENTS

ON A PROTEIN IN DROSOPHILA MELANOGASTER

KATHRYN E. FUSCALDO AND ALLEN S. FOX

Department of Biology, St. John’s Uniuersity, Jamaica, New York, and Department of Biochemistry, Michigan State University, East Lansing, Michigan

Received April 2, 1962

HE introduction of the term heterochromatin by HEITZ (1928) implied the Texistence of chromosomal segments with special properties. Primarily distinguished on the basis of observable heteropycnosis during mitotic and meiotic cycles ( SCHULTZ 1947; WHITE 1948), to such segments have also been attributed distinction of function. Originally this distinction was thought to consist of genetic inactivity (MULLER and FAINTER 1932); as the years have passed this original concept has been replaced by that of specialized function. Unfortunately it has not been possible to define this function in a unique fashion, and heterochromatic regions have been charged with widely vaned responsibilities in cellular metabolism and chromosomal behavior (review: HANNAH 1951 ) . In- deed, the list of imputed functions has become so extensive that some workers either have been led to reject the concept of distinctiveness itself (COOPER 1959), or have relegated the problem to the era of “pre-science” (PONTECORVO 1958).

Among hypotheses concerning the function of heterochromatin, that which suggests that such regions are concerned with the nucleic acid metabolism of the cell (SCHULTZ 1936, 1956) would seem to possess the greatest unifying value. The developments of recent years demonstrate, however, that all genetic material is concerned with nucleic acid metabolism in some sense, so that the hypothesis requires redefinition. This might best be done in terms of the mechanisms of information transfer in protein synthesis. It is now clear that genetic information coded in DNA is transferred to the sites of protein synthesis by RNA and there serves to determine protein structure. In these terms, the question of the function of heterochromatin may be formulated in the following way: (1) Do alterations of heterochromatin result in changes in the structure of proteins? (2) Given such effects on protein structure, are they fundamentally different from the effects of alterations of euchromatic genes?

Answers to these questions have been sought through an examination of the effects of two heterochromatic systems on protein structure by means of immunochemical techniques. One system has involved the Y chromosome in Dro- sophila melanoguster, and has yielded evidence of a maternal effect of this chromosome on the structure of a particular protein referred to as Y-I (FOX, YOON and MEAD 1962). The present work is concerned with the effects of physi- Genetics 47 : 999-1015 August 1962

1000 K. E. FUSCALDO A N D A. S. FOX

cal rearrangements of euchromatin and heterochromatin in the chromosomes of the same organism.

When heterochromatin is brought into the vicinity of a euchromatic locus by a chromosomal rearrangement, a mosaic phenotype (i.e., variegation) is frequently induced in which patches of mutant tissue are scattered on an otherwise normal background (see review by: LEWIS 1950). Mosaicism of this type is extremely variable. The mutant phenotype is expressed in some cell lineages but not in others, and its extent is affected to a marked degree by environmental condi- tions. A threshold seems to be involved, such that irreversible differentiation either in the normal or mutant direction takes place in any given cell lineage (SCHULTZ 1936, 1956).

It has been demonstrated that gene effects exhibiting variegation as a result of euchromatic-heterochromatic rearrangements revert to normal when the heterochromatin is removed by crossing-over (DUBININ and SIDOROV 1935; PANSHIN 1935; JUDD 1955). The decisive factor in the determination of variegation by the wm4 rearrangement in D. melanogmter was shown by SCHULTZ (1943) to be located at the junction between the white segment and the introduced heterochromatin. I t thus appears that the influence exerted by heterochromatin on the expression of euchromatic loci is primarily responsible for the induction of variegated effects.

The system used in the present work involves euchromatic-heterochromatic rearrangements with breaks in the vicinity of the white segment on the X chromosome (map position, 1.5). This is a pseudoallelic segment marked by 21 mutants, in which four loci have been identified by recombination (information compiled by K. B. WARREN). The whole segment is located in the region of bands 3C1-3C3 of the salivary X chromosome.

MATERIALS A N D METHODS

Description of stocks: The demonstration that euchromatic-heterochromatic rearrangements have an effect on protein structure depends, in the first place, on the establishment of a standard pattern of proteins detectable by the immunochemical techniques employed. Two related wild-type stocks were used for this purpose:

1. Oregon-R-1.-An isogenic wild-type stock, originally isogenized by J. SCHULTZ and maintained in this laboratory through 230 generations of brother- sister matings at the start of this work.

2. Oregon-R-I (S) .-A subline of Oregon-R-I maintained in the laboratory of J. SCHULTZ. The two lines of Oregon-R-I have been maintained separately approximately since generation 50 of brother-sister mating. They provided a check on the effects of genetic drift on the standard protein pattern.

The following stock provided material for the observation of effects of the introduction of heterochromatin into the vicinity of the white segment:

3. Zn(l)wm4.-An inversion, originally described by MULLER (1930), resulting from two breaks and subsequent rearrangement of the X chromosome

ALTERED PROTEIN 1001

(BRIDGES and BREHME 1944). The proximal break occurred in chromocentric heterochromatin of section 20 of the salivary chromosome map of the X chromosome. The distal, euchromatic break occurred between bands 3C1 and 3C2 to the left of white (w) . The result of the inversion is to place the white segment in close proximity to chromocentric heterochromatin. The phenotypic expression of the rearrangement is variegation of eye color, some of the ommatidia exhibiting the mutant white phenotype. Prior to use, the stock was cleared of extra Y chromosomes as confirmed by cytological examination of larval cerebral ganglia.

The following stock provided a check on the specificity of the effects of In(1) wm4:

4. Zn(l)wm4".-A derivative of Zn(l)wm4, exhibiting more extreme variegation ( SCHULTZ 1943). No change in the rearrangement is detected cytologically. The absence of extra Y chromosomes in the stock was confirmed by cytological examination.

Since the Y chromosome is heterochromatic, has a modifying effect on the variegation induced by euchromatic-heterochromatic rearrangements (cf. LEWIS 1950), and has been demonstrated to affect the structure of the protein Y-I (Fox et al. 1962), the following stocks were subjected to immunochemical examination:

5. Z ~ ( ~ ) W ~ ~ ~ ; Y ~ ~ - ~ . - A stock possessing the same rearranged X chromosome as in number 4, but with an altered Y chromosome which suppresses variegation in the male (J. SCHULTZ, personal communication). The removal of extra Y's from the stock was confirmed cytologically.

6. Zn(l)wm4";Df (Y)Y-bb.-Possesses the same X chromosome as stock number 4, but with a Y chromosome deficient for the bobbed locus (J. SCHULTZ, personal communication). Cytological examination revealed no extra Y chromosomes.

The generality of the effect of the introduction of heterochromatin into the proximity of the white segment was tested by use of the following stock:

7. T(1;4)wm5.-A stock which is homozygous for a reciprocal translocation between the X and fourth chromosomes (MULLER 1930; BRIDGES and BREHME 1944; additional information compiled by K. B. WARREN). The break in the X chromosome is to the right of,the white locus, between it and the roughest (rst) locus (1.7) on the genetic map and between bands 3C2 and 3C3 (BRIDGES and BREHME) or 3C3 and 3C4 (LEWIS 1950) in the salivary chromosome map. The break in the fourth chromosome is located in heterochromatin between the cubitus interruptus (ci) locus and the kinetochore, more exactly between bands IOlFl and 101F2 in the salivary map and between the bent (bt) and cubitus interruptus (ci) loci on the genetic map. Reciprocal translocation of the products of these breaks puts the tip of the X including the white locus onto the chromocentric portion of the fourth chromosome; the cubitus interruptus locus and almost all of the fourth chromosome is placed on the end of the X chromosome. This rearrangement places the chromocentric heterochromatin of the fourth chromosome to the right of the white locus (see below) in contrast with Zn(l)wm4


and Z ~ ( I ) Z U ~ ~ ~ , where heterochromatin is placed to the left of white. The translocation produces variegation of white and a position effect of cubitus interruptus. GRIFFEN and STONE (1940) have shown that the white-variegated phenotype exhibited by T( 1 ;4) wm5 reverts to wild type when the white locus is transferred to a new euchromatic position by subsequent, X-ray-induced rearrangements. The variegation is therefore attributable to the proximity of heterochromatin to the white locus rather than to mutation within the locus itself.

Finally, the following stock was used to compare the effects of mutational modification of the white segment itself with the effects of the introduction of heterochromatin into its vicinity:

8. white (w) .-The mutant for which the white region is named. Localized at the third locus from the distal (left) end of the four sites identified in the white segment (information compiled by K. B. WARREN). Eye color nearly snow white.

Immunochemical techniques: The techniques used for immunochemical analysis have been adapted from those described by Fox (1958, 1959) and Fox et al. (1962), involving application of the agar-diffusion methods devised by OUCH- TERLONY (1949) and BJORKLUND (1952).

In brief, lyophilized flies from mass cultures of a given stock (sexes not separated) were homogenized in buffered saline (2.0 g dry weight/lOO ml) . Intraperitoneal injections of the whole homogenates served to immunize rabbits, yielding multivalent antisera. Homogenates were centrifuged at 3000 x g to obtain the complex protein extracts (0.09 mg protein/ml) which were used in the immunochemical tests.

Resolution of these complex systems and identification of individual antigenic components was achieved by means of OUCHTERLONY tests, utilizing the geometry given in Figure 1. Each well in these agar plates holds 0.05 ml of test solution. Antigen extracts were placed in the six outer wells (1, 2, 3, 4, 5, 6) and undiluted antiserum in the central well (An). The wells were all filled and refilled simultaneously, five doses being sufficient for the development of definitive patterns of precipitate lines in the agar. The 0.25 ml of each antigen extract used in such a test contained 0.023 mg of protein.

Further resolution was accomplished by use of the BJORKLUND inhibition technique. For this purpose, inhibiting antigen (i.e. concentrated protein extract from a given stock) was allowed to diffuse into the agar from the central well of an OUCHTERLONY plate prior to the performance of the test itself. Inhibiting antigens were prepared by lyophilizing the nondialyzable portion of the original extracts and then reconstituting the resulting material in one tenth the original volume of buffered saline (0.85% NaCl plus 0.05 M phosphate, pH 7.4). The amount of protein used to inhibit the plates varied from 0.135 mg to 0.45 mg (three to ten doses of 0.05 ml each).

RESULTS

Table 1 lists the antisera cited in the present report. One antiserum against each of the genotypes described above is included in the list. Additional antisera were tested. These either yielded results in agreement with those reported here,


P 1

FIGURE 1.-Geometry of OUCHTERLONY plates used in this investigation. An well: antiserum well. Wells 1, 2, 3, 4, 5, 6: wells for antigenic (protein) extracts. Each well holds 0.05 ml of test solution. All wells are filled or refilled simultaneously.

or lacked the pertinent antibodies. It is common experience that antisera produced in response to complex antigenic mixtures frequently lack antibodies to one or more components of the mixtures.

OUCHTERLONY tests with uninhibited antisera produced plates exhibiting four to seven precipitate lines (Figure 2), depending on the particular serum tested. Each such line represents a minimum of one antigen-antibody system (OUDIN 1952). Most of the lines are of no special interest since they were continuous between antigen wells, and consequently represented antigens common to all of

TABLE 1

List of antisera cited in this report

Antiserum Stock providing immunizing antigens - S-314 In(ljwm4W;Df (Y)Y-bb S-336 In(l)wm4*;Ysu-v s-337 In(1 jwm41U s-338 In(1) wm4 s-339 T( 1 ;4) wm5 s-340 Oregon-R-I S-341 Oregon-R-I (S) S-342 white

A total of 28 antisera have been examined. Twenty contained W-l antibodies; eight exhibited no demonstrable W-1 antibodies.

1004 K. E. FUSCALDO AND A. S . FOX

0 /-4

3

5' FIGURE 2.-Photograph and diagram of OUCHTERLONY plate. No inhibition. Serum (An well) :

S-337, anti-Zn(I)w"'A''". Protein extracts: Well 1, Zn(l)w"'4; Well 2, In(l)w"'4'0;Df (Y)Y-bb; Well 3, Z n ( l ) w ' ~ ~ ~ ~ ~ ; Y ~ ~ ~ - V ; Well 4, Oregon-R-I; Well 5, Oregon-R-I (S) ; Well 6, Z n ( l ) ~ " ' 4 ' ~ .

the stocks. These antigens are presumably products of those portions of the genotypes which are the same in all of the 4tocks. One antigen, however, exhibited differences among the stocks, correlated either with the constitution of the white segment or with its relationship to introduced heterochromatin. This antigen will be referred to as W-1, and its behavior in OUCHTERLONY and BJORKLUND tests will be illustrated largely by reference to the results obtained with a typical antiserum, S-337. It should be noted that this antigen has been designated H(w)-1 in preliminary reports (FUSCALDO and Fox 1961a,b,c). The present designation replaces that used originally.

Examination of Figure 2 discloses marked differences in the characteristics of the W-1 line opposite wells 4 and 5 (containing extracts of Oregon-R-I and Oregon-R-I (S) ) as compared with its characteristics opposite the other wells (containing extracts of stocks possessing In(Z)wn'4 or its derivative I n ( 1 ) ~ " ' ~ ' ~ ) . In the latter case it is well formed, distinct, and is located at a position approximately two thirds of the distance from the An well in close juxtaposition to line 2. As it approaches wells 4 and 5, however, it extends out to a distance farther from the An well, loses its distinctness, bends inward on either side, and is lost from view as it reaches lines 1 a and 1 b.

With other antisera the W-I line is distinguishable opposite wells containing extracts of Oregon-R-I and Oregon-R-I( S), but displaced outward as in the previous case and exhibiting a distinct decrease in density. This is illustrated in Figure 3, where the Oregon-R extracts are contained in wells 4 and 5. Opposite these wells the W-I line is displaced to a position just within line I and is dis- tinctly less dense than it is opposite wells containing extracts of stocks with In(l)wm4 or In(2 )~" '4 '~ .

These results are completely repeatable. In the case of the antiserum S-337, extracts prepared from the various stocks at different times yielded OUCHTERLONY


2a S 2 -la S Ib

. W - l

FIGURE 3.-Photograph and diagram of OUCHTERL~NY plate. No inhibition. Serum (An well): S-338, anti-In(l)w~l’~. Protein extracts: Wells 1,2,3,4,5, and 6 as in Figure 2.

patterns not differing in essential features from that illustrated in Figure 2, and the antiserum S-338 repeatedly yielded plates like that illustrated in Figure 3. In all cases the W-1 line developed by the extracts of Oregon-R-I and Oregon- R-I (S) was displaced outwards toward the antigen wells and exhibited reduced density relative to the line developed by extracts of the inversion stocks. Beyond this, similar differences in the characteristics of the W-1 line have been observed with every antiserum, now totaling 20 in number, which has possessed anti-W-1 antibody, regardless of the genotype providing the immunizing extract.

In other words, the distinction between the Oregon-R stocks on the one hand and the inversion stocks on the other is not attributable to accidents of extract preparation, nor to peculiarities of particular anti-W-1 antibodies, but reflects a difference in the W-1 antigen itself. For purposes of convenience we shall refer to the characteristics of Oregon-R W-1 as “normal” and to those of the W-1 in the inversion stocks as “modified.” This terminology is dictated by the genetic convention which refers to wild type as normal and to mutants as having under- gone modification. For the moment, no implication is intended as to the nature of the difference between normal and modified W-1.

The condition of W-1 in the translocation stock, T(1;4)wm5, is illustrated in Figure 4. This figure also illustrates the condition of W-1 in the mutant w, which will be discussed below, but it was specifically chosen to illustrate in addition a difficulty which occasionally is encountered in tests of this sort. It will be noted that line 1 is poorly developed opposite wells 1,3,4, and 5. Since both the serum and the extracts used in this test yielded normal development of line 1 in other plates, its faulty development in the present instance must be attributed to artifact. The W-1 line, however, is not disturbed and is clearly evident opposite wells 1,2, 3, and 4. The first three of these wells contained extracts of inversion stocks; well 4 contained T( 1 ;4) wm5 extract. While the W-1 line lies immediately outside line 2 opposite well 4, its position and density correspond more closely to its development with the inversion extracts (wells 1, 2, and 3) than with the Oregon-R-I extract (well 5). In other plates with the same serum (S-337), and


I la 6 Ib

1;rc;uiti: 4.--Photogriiph and diagram of OUCHTERLONY plate. No inhibition. Serum (An well) : S-337, anti-In(l)w””“‘. Protein extracts: Well 1, In(l)w”‘4; Well 2. In(l)w”’4‘0; Well 3, In(l)wJ1’410;Y~l+~; Well 4, T( 1 ; 4 ) ~ ~ ~ ~ ~ ; Well 5, Oregon-R-I; Well 6, w.

with other antisera (i.e., S-314 and S-339), the position and density of the W-1 line in the T(1;4)w1lt5 field are not detectably different from its position and density in the inversion fields. There can be no doubt, therefore, that T( 1;4)wIn5 is responsible for a modification of W-1 which is similar to that observed in stocks possessing In(Z)w”’$ or In(Z)wl’‘~lr. The evidence, however, does not provide a demonstration that the modification is identical in all cases.

Figure 5 provides a comparison of the condition of W-1 in the mutant white with its condition in Oregon-R and the inversion stocks. The position and density of the W-1 line opposite well 4 (w) clearly correspond to its condition opposite well 3 (Oregon-R-I (S) ) . Figure 4 yields the same observation (well 6 contains

I

I

w - l

P’1c;um .i.--Photograph ilnd diagram of OUCHTERL~NY plate. No inhibition. Serum (An well): S-337, anti-In(l)w”I:“‘. Protein extracts: Well 1, In(l)w’l’G; Well 2, In(l)w’n4w; Well 3, Oregon- R-I(S); Well 4. w; Well 5, In(l)w”’41r;Y“l-’; Well 6, In(Z)w’”l’O;Df(Y)Y-bb.


w) , but in this case the artifact responsible for the absence of line 1 in the Oregon- R-I field (well 5 ) appears also to have resulted in the absence of the W-1 line in the same region. Other antisera (S-344, S-336, S-338, S-339, S-342) confirm the conclusion that the W-I of white behaves in a manner similar to that of Oregon- R-I and Oregon-R-I (S) in uninhibited OUCHTERLONY tests. It will therefore be regarded as normal for the moment, although the results of BJORKLUND inhibition tests given below demonstrate that it is not identical with that of the wild stocks.

Table 2 summarizes the characteristics of the W-1 line formed by extracts from the different stocks with uninhibited antisera. The table contains docu- mentation of the statement made above that the W-1 contained in the extracts of each genotype behaves in a similar manner in OUCHTERLONY tests with all antisera possessing anti-W-1 antibody regardless of the genotype which provided the extract used to induce antibody formation. It therefore is possible to characterize the W-1 of Oregon-R-I, Oregon-R-I(S), and white as normal (with the qualification that the results of BJORKLUND inhibition tests given below serve to distin- guish between the W-I of white and that of the wild stocks), and to characterize the W-1 of Zn(l)wm4, Zn(l)wm4", Zn(l)wm4";Df (Y)Y-bb, Z n ( l ) ~ ~ 4 ~ ; F ~ - ~ , and T( 1;4) wm5 as modified (with the qualification that the modification in T( 1 ;4) wm5 may not be identical with that in the inversion stocks). It is also possible to state that the W-1 of all stocks except Oregon-R-I and Oregon-R-I (S) , regardless of whether it is normal or modified, induces the formation of antibodies which are indistinguishable in OUCHTERLONY tests with uninhibited antisera. As we shall see, however, the antibodies formed in response to the W-1 of white are distinguishable in BJORKLUND inhibition tests.

We have thus far failed to find anti-W-I antibodies in antisera to Oregon-R-I or Oregon-R-I (S) , but only two such antisera have been tested, namely S-340 and S-341. It therefore is not possible to know whether the W-1's of Oregon-R-I and Oregon-R-I (S) are poor antibody inducers or whether the particular rabbits used were refractory. Antisera lacking anti-W-1 have also been obtained from

TABLE 2

Characteristics of W-l precipitate line formed with uninhibited antisera by antigens from different stocks

Antiserum

Stocks S-314 S-336 S-337 S-338 S-339 S-340 S-341 S-34%

Oregon-R-I N N N N N 0 0 N Oregon-R-I (S) N N N N N 0 0 N In(1) wm4 M M M M M 0 0 M In(1) wm4w M M M M M 0 0 M In(l)wm4w;Y~~-V M M M M M 0 0 M In(l)wm4W;Df ( Y ) Y-bb M M M M M 0 (E M T( 1 ;4) wm5 M - M - M 0 0 white N N N N N 0 0 N

-

N=nornial. Mzmodified. O=no anbW-1 antibody. -=not tested.

1008 K. E. FUSCALDO AND A. S . FOX

rabbits immunized with extracts from the other genotypes, but only in a frequency of about one in three.

The results obtained with uninhibited antisera are confirmed and extended by BJORKLUND inhibition analysis. Figure 6 is a photograph of a plate obtained with S-337 after inhibition with 0.225 mg of Oregon-R-I (S) protein, incorporated into the agar by diffusion from the center well. This is to be compared with Figure 2, which is a plate obtained with uninhibited S-337. Of the five lines visible in Figure 2, only one remains in the inhibited plate. Since Oregon-R-I (S) resembles Z n ( 1 ) ~ ” ’ ~ ~ ~ (the extract used to induce antibody formation) in all respects except with regard to W-1, the remaining line must represent precipitate formed by the union of W-I with antibody not affected by the inhibition. This conclusion is supported by the distribution of the line in the various extract fields, but special note should be taken of its complete absence in the Oregon-R-I and Oregon-R-I (S) fields. A slight shift of the line toward the An well may also be detectable.

This slight shift in position and the absence of the W-I line in the Oregon-R-I and Oregon-R-I(S) fields suggests that some inhibition of the anti-W-I antibody has occurred, but that inhibition is incomplete with only 0.225 mg of Oregon-R-I (S) protein. Indeed, complete inhibition of the W-1 line is achieved with 0.45 mg of Oregon-R-I (S) protein, i.e. twice the amount used in Figure 6.

The same inhibition behavior is exhibited by extracts of Oregon-R-I: incomplete inhibition is achieved by 0.225 mg of protein, while 0.45 mg result in com-

FIGURE 6.-Photograph of BJORKLUND inhibition plate. Inhibiting extract: Oregon-R-I(S), 0.225 mg protein. Serum (An well): S-337. Protein extracts: Well 1, In(l)w”t4; Well 2, In(l)wm4w; Well 3, In(l)w”l/l~;Y*“-~‘; Well 4, Oregon-R-I; Well 5, Oregon-R-I (S) ; Well 6, In(1) wm4to;Df (Y) Y-ab.


plete inhibition of the W-1 line. This behavior is in marked contrast, however, with the results of inhibition by extracts of the inversion stocks. Figure 7 demon- strates that the incorporation of 0.225 mg of In(I)wm4lo protein into the agar results in complete inhibition of the W-1 line, i.e. in its complete absence. The extracts of In(l)w1"4, I ~ ( I ) W " ' ~ ' ~ ; Y ~ " - ~ , and In( I )~" '4~~jDf (Y)Y-** behave in the same way: 0.225 mg of protein results in complete inhibition of the W-1 line in each case. It therefore appears that the modification of W-1 in the inversion stocks results not merely in a shift in position and an increase in density of the W-1 line on uninhibited plates, but also in an increase of inhibitory power of approximately twofold. The inhibitory power of T( 1;4) wms extracts has not been tested.

Although the W-1 of white behaves in uninhibited plates like that of the wild stocks, i.e. as if it were normal, its inhibitory powers resemble those of the inversion stocks: 0.225 mg of white protein result in complete inhibition of the W-I line in tests with S-337. A distinction can therefore be made between the W-1 of white and that of Oregon-R-I and Oregon-R-I(S). The W-1 of white is not identical with that of the wild stocks.

Table 3 summarizes the results of BJORKLUND inhibition tests of additional antisera. Antisera to the extracts of all of the inversion stocks behave in a similar manner. Inhibition with 0.225 mg of protein from any of the inversion stocks or from white results in complete disappearance of the W-1 line in tests with any of these antisera (S-314, S-336, S-337, S-338). Complete inhibition by Oregon-R-I or Oregon-R-I (S) extracts on the other hand, requires 0.45 mg of protein. The

FIGURE 7.-Photograph of BJORKLUND inhibition plate. Inhibiting extract: I n ( 2 ) ~ ~ 4 ' ~ , 0.225 mg protein. Serum (An well): S-337. Protein extracts: Wells 1, 2, 3, 4, 5, and 6 as in Figure 6.


TABLE 3

Summary of behavior of W-I precipitate line following inhibition of antisera

Presence or absence of W-1 line when tested with extracts from

Oregon-I-I, Inhihiting Oregon-I-I (S) , 1 ~ ( 1 ) ~ " 4 ~ . i n ( i ) w m 4 w .

Antiserum antigen' or vhite I n ( i j w " 4 I n ( l ) w m h W Df(Y)Y-bb' YSu-" '

Oregon-R-I Oregon-R-I (S )

S-314, S-336, In(l)wm4 S-337, S-338 In(f)wn'4w

In(1) wmht*;Df (Y)Y-bb ~ ~ ( l ) ~ m i w ; y s n - ~ white

Oregon-R-I Oregon-R-I (S ) In(l)wm4

In(l)wm4to;Df (Y)Y-bb S-342 1nC1) ~ u m 4 w

In(1) w m 4 w ; y s -

+ + + + + + + +

~~

Inhibition performed with 0.225 mg of inhibiting protein. Inhibition with 0.45 mg nf protein is complete in all cases. + =W-l line present. - = W-1 line absent.

following conclusions are derived from these observations: (1) The W-1 of the four inversion stocks induces the formation in rabbits of antibodies of indistinguishable specificity in BJORKLUND inhibition tests as well as in ordinary OUCHTERLONY tests. (2) The inhibitory power of the W-1 of the four inversion stocks is indistinguishable in BJORKLUND tests. (3) Since the anti-Oregon-R-I and anti-Oregon-R-I (S) sera that have been tested have lacked antibody to W-1, no conclusion can be drawn regarding the specificity of antibody induced by the normal W-l of these stocks. (4) The distinction between normal and modified W-l is confirmed by the observation that the inhibitory power of both Oregon- R-I and Oregon-R-I (S) extracts is approximately half that of the extracts of the extracts of the inversion stocks. (5) The inhibitory power of the W-I of white is similar to that of the inversion stocks, even though it behaves like the W-1 of Oregon-R-I and Oregon-R-I (S) in ordinary OUCHTERLONY tests.

The results of BJORKLUND inhibition tests of anti-white serum (Table 3; S-342) yield further information regarding the W-1 of white. In this case, the W-1 antibody is inhibited by similar amounts of inversion, wild, and white protein, i.e. 0.225 mg. It therefore appears that the specificity of the antibodies induced by the W-1 of white is different from that of the antibodies induced by the W-1 of the inversion stocks, reflecting in turn a difference in the antigens themselves. This means that the W-1 of white can be distinguished both from the normal VV-1 of the Oregon-R stocks (on the basis of a difference in inhibitory power), and from the W-l of the inversion stocks (on the basis of differences in the specificity of induced antibody and the relatively peripheral position and low density of the

ALTERED PROTEIN 101 1

line that it forms in uninhibited OUCHTERLONY plates). It therefore will be referred to as “variant” W-I.

Table 4 summarizes the distribution of the three forms of W-I among the various stocks. The term normal refers to the W-I of the Oregon-R stocks, characterized by the relatively peripheral position and low density of the line that it forms in OUCHTERLONY plates, and its relatively low inhibitory power. Antibodies to normal W-I have not yet been encountered. The term modified refers to the W-I of the rearrangement stocks characterized by the relatively central position and high density of the line that it forms in OUCHTERLONY plates, and an inhibitory power about double of that of normal W-I. The term uariant refers to the W-I of white, characterized by the formation of an OUCHTER- LONY line similar in position and density to that of normal W-1, but exhibiting an inhibitory power similar to that of modified W-I. Modified and variant W-I are also distinguishable on the basis of the specificity of the antibodies that they induce. The characteristics of the three forms of W-I are summarized in Table 5.

DISCUSSION

The following circumstances strongly indicate that W-I is a protein: (1 ) It is nondialyzable; (2) it is precipitated by 50% ethanol and 10% trichloroacetic acid; ( 3 ) its antigenic properties are destroyed by heating at 100°C for five min- utes; (4) on the basis of the frequency with which it induces antibody formation,

TABLE 4

Distribution of the three forms of W-I among the genotypes examined

Genotype

Oregon-R-I Oregon-R-I (S) In(1)wmA In(1)wmAw Zn(l)wm4m;Df(Y)Y-bb Zn(l)wm4w;Ysu-~’ T( 1;4) W m 5

white

~

Form of W-t

Normal Normal Modified Modified Modified Modified Modified Variant

TABLE 5

Immunochemical characteristics of the three forms of W-I

Form of W-t

Charactenstic Normal Modified Variant

Position of line in O u c H n m L o N Y plates Peripheral Central Peripheral Density of line in OUCHTERLONY plates Light Heavy Light Inhibitory power with antibody to Low High High

Inhibitory power with antibody to High High High modified W-I

variant W-I

1012 K. E. FUSCALDO AND A. S. FOX

it is a relatively good antigen. Nucleic acids are notoriously poor antigens; ( 5 ) Twelve other gene-affected antigens of D. melanogaster have been studied and all have been demonstrated to be proteins (Fox 1958). Indeed, no antibodies have ever been found in rabbit antisera tc Drosophila antigens not destroyed by proteolytic enzymes. In particular, no polysaccharide antigens have been encountered. While it has not yet been possible to do definitive chemistry, the following discussion will be based on the assumption that W-1 is a protein.

On this basis, it is appropriate to inquire into the nature of the differences among normal, modified, and variant W-1. A possibility to be considered is that the differences in the immunochemical behavior of W-1 in the various extracts are attributable simply to differences in its concentration. This possibility, however, is not consistent with the facts. In particular, the difference between the (normal) W-1 of Oregon-R extracts and the (variant) W-1 of white extracts is almost certainly not simply quantitative. This conclusion follows from the discrepancy between the observation that the position and density of the lines formed by normal and variant W-1 in uninhibited OUCHTERLONY plates are similar, and the observation that their inhibitory powers are markedly different. Similarly, the fact that the inhibitory powers of variant and modified W-1 are similar while the position and density of the lines that they form in uninhibited plates are different, militates strongly against the suggestion that the difference between the W-1 of white and the rearrangement stocks is simply quantitative. Finally, the results obtained with antibody induced by variant W-l serve tG indicate that the difference between normal and modified W-1 is not simply quantitative. In this case, the position of the pertinent lines in uninhibited plates is different (Serum S-342, Table 2) while the inhibitory power of the extracts is the same (Serum S-342, Table 2 and Table 5).

Turning to genetic analysis, the following conclusions emerge: (1) Mutational alteration of the white segment, as in the white stock, results

in alteration of the immunochemical behavior of W-1 . Preliminary analysis of a number of other mutants in this segment (wa7 dz7 wbf2, wCh7 wcoz, we7 UP, and wSut) indicates that W-l is altered by mutational change at any of the sites in the segment.

(2) Alterations of the immunochemical behavior of W-1 result not only from mutational change within the white locus, but also as a consequence of rearrangements which bring heterochromatin into its vicinity. In the first place, the modified W-1’s of In(l)wmA7 of Zn(1)~”’4~, and of the I n ( l ) ~ ~ 4 ~ ~ stocks possessing Y-’” or Ysu-v are immunochemically indistinguishable. This means that the alteration responsible for the difference between normal and modified W-l is specifically attributable to the rearrangement common to all of these stocks. Secondly, T( 1 ;4) wm5 results in a modification of the behavior of W-l similar to that induced by the inversion. This observation suggests that it is not the particular position of the breaks which is important, but rather the introduction of heterochromatin into the immediate vicinity of the white segment.

The effects of mutation in the white segment on the immunochemical behavior of W-1 are different however from those of heterochromatic rearrangement.


Variant W-1, when compared with normal W-1, exhibits no shift in the position of the line that it forms in uninhibited plates, indicating no change in the equivalence ratio of antigen to antibody, but it does exhibit increased inhibitory power. Modified W-1 exhibits a similar increase in inhibitory power, but the line that it forms in uninhibited plates exhibits a centripetal shift in position, indicating a reduction in the ratio of antigen to antibody at equivalence. In addition, the specificity of antibodies to variant W-1 is different from that of antibodies to modified W- 1.

In fact, the effects of heterochromatic rearrangements on W-1 resemble closely the maternal effects of the heterochromatic Y chromosome on the immunochemical properties of the protein Y-l (FOX et al. 1962). While we have not examined the present material for maternal effects of the rearrangements on W-1 , maternal effects of white-mottled rearrangements on variegation are well- known (NOUJDIN 1944; SPOFFORD 1959, 1961; HESSLER 1961). In the former case, it was concluded that Y-1 is the product of one or more autosomal loci and that its immunochemical properties were modified by the maternal effect of the heterochromatic Y chromosome. In the present case, the data suggest that W-I is a product of the white segment, and that its immunochemical properties are modified by the relationship of that segment to chromocentric heterochromatin. In both cases, the results suggest that alterations of heterochromatin result in changes in the immunochemical properties of proteins that are different from those induced by alterations of euchromatic genes.

SUMMARY

(1 ) The immunochemical properties of a protein, W-1, are altered by mutational substitution in the white segment and by rearrangements which bring heterochromatin into its proximity. (2) Two wild stocks, Oregon-R-I and Oregon- R-I(S), possess “normal” W-1. (3) Stocks with In(l)wm4 or Z n ( l ) ~ ~ 4 ~ possess a form of W-1 referred to as “modified.” (4) The W-I of a T(1;4)wm5 stock is similar to that of the inversion stocks. (5) No difference is found between the W-1’s of In(l)w”4* stocks possessing either a normal Y chromosome, or or Df(Y)Y-ba. (6) The W-1 of a white (w) stock is distinguishable from normal and modified W-1, and is referred to as “variant.” (7) The equivalence ratio for the union of variant W-1 with specific antibody is the same as that for normal W-1, but the antibody inhibiting power of the former is higher. (8) Modified W-1 exhibits a similar enhancement of inhibitory power, but the ratio of antigen to antibody at equivalence is lower than with variant or normal W-1 . (9) The specificity of antibodies to variant W-1 is distinguishable from the specificity of antibodies to modified W-1. (10) The differences in the immunochemicd behavior of the three forms of W-l are not attributable to simple differences in concentration. (1 1 ) The immunochemical behavior of W-I is altered both by mutational change in the white segment and by rearrangements of the relationship of that segment to chromocentric heterochromatin. (12) The nature of the changes induced by mutation in the euchromatic segment is different from those induced by heterochromatic rearrangement.

1014 K. E. FUSCALDO AND A. S. FOX

ACKNOWLEDGMENTS

This work was supported by research grants No. C-2440 and No. A-5376 from the National Institutes of Health. Part of this work was done while one of the authors (K.E.F.) was a guest investigator at the Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, N.Y. The author wishes to grate- fully acknowledge the kind assistance and encouragement of DR. BERWIND P. KAUFMANN and his staff. The manuscript was prepared in part while one of us (A.S.F.) was a Fulbright Research Fellow at the C.S.I.R.O. Animal Genetics Division, Sydney, Australia; grateful acknowledgment is given to the hospitality of all in that Division. Journal Article No. 3043 from the Michigan Agricultural Experiment Station.

LITERATURE CITED

BJORKLUND, B., 1952 Specific inhibition of precipitation as an aid in antigen analysis with the gel diffusion method. Proc. Soc. Exptl. Biol. Med. 79: 319-324.

Wash. Publ. 552. BRIDGES, C., and K. BREHME, 1944 The Mutants of Drosophila melanogaster. Carnegie Inst.

COOPER, K. W., 1959 Cytogenetic analysis of major heterochromatic elements (especially Xh and Y) in Drosophila melanogaster and the theory of “heterochromatin.” Chromosoma 10: 535-588.

DUBININ, N. P., and E. N. SIDOROV, 1935 The position effect of the hairy gene. Biol. Zh. 4: 555-568.

Fox, A. S., 1958

Fox, A. S., S. B. YOON, and C. G. MEAD, 1962

Genetics of tissue specificity. Ann. N. Y. Acad. Sci. 73: 611-634. 1959 Genetic determination of sex-specific antigens. J. Natl. Cancer Inst. 23: 1297-1311.

Evidence for the persistence in protein synthesis of an information transfer mechanism after the removal of genes. Proc. Natl. Acad. Sci. U. S. 48: 5ffi-561.

Immunogenetic studies of white-variegated position

An immunogenetic analysis of white-variegated position effects in D. melanogaster.

An immunogenetic analysis of variegation in Drosophila. Carnegie Inst. Wash. Year-

FUSCALDO, K. E., and A. S. Fox, 1961a effects. Droslophila Inform. Serv. 35: 84.

1961 b Records Genet. Soc. Am. 30: 7 6 7 7 and Genetics 46: 866-867.

1961c book 60 : 486-487.

GRIFFEN, A. B., and W. S. STONE, 1940 190-200.

HANNAH, A., 1951

HEITZ, E., 1928

HESSLER, A. Y., 1961 46: 463-484.

JUDD, B. H., 1955

LEWIS, E. B., 1950

The wn’5 and its derivatives. Univ. Texas Publ. 4032:

Localization and function of heterochromatin, Advan. Genet. 4: 87-125.

Das heteromchromatin der moose. I. Jahrb. Wiss. Botan. 69: 7 6 H 1 8 .

A study of parental modifications of variegated position effects. Genetics

Direct proof of a variegated type position effect a t the white locus in Drosophila melanogaster. Genetics 40 : 739-744.

The phenomenon of position effect. Advan. Genet. 3: 73-115.

MULLER, H. J., 1930 Type of visible variations induced by X-rays in Drosophila. J. Genet. 22 (3) : 320-334.


MULLER, H. J., and T. S. PAINTER, 1932 The differentiation of the sex chromosomes of Dro- sophila into genetically active and inert regions. Z. Induktive Abstammungs-Vererbungslehre 62: 316365.

NOUJDIN, N. O., 1944 The regularities of heterochromatin influence in mosaicism. J. Gen. Biol. (USSR) 5: 357-388.

OUCHTEFLLONY, ti., 1949 Antigen-antibody reactions in gels. Acta Pathol. Microbial. Scand. 26:

OUDIN, J., 1952 Specific precipitation in gels and its application to immunochemical analysis.

PANSHIN, L. B., 1935 New evidence for the position effect hypothesis. C. R. (Dokl.) Akad.

PONTECORVO, G., 1958 Trends in Genetic Analysis. Columbia University Press. New York. SCHULTZ, J., 1936 in MORGAN, T. H., C. B. BRIDGES, and J. SCHULTZ, The constitution of the

germinal material in relation to heredity. Carnegie Inst. Wash. Yearbook 35: 289-297. A derivative of In(l)2@4 showing a different reactiton to heterochromatin. Drosophila

Inform. S e w . 17: 64. The nature of heterochromatin. Cold Spring Harbor Symp. Quant. Biol. 12: 179-191. The relation of the heterochromatic chromosome regions to the nucleic acids of the

cell. Cold Spring Harbor Symp. Quant. Biol. 21 : 307-328. SPOFFORD, J. B., 1959 Parental control of position effect variegation. I. Parental hetero-

chromatin and expression of the white locus in compound-X Drosophila melanogaster. Proc. Natl. Acad. Sci. US. 45: 1003-1007.

Parental control af position effect variegation. 11. Effect of sex of parent contributing white-mottled rearrangement in Drosophila melamgaster. Genetics 46 : 1 151-1 167.

Animal Cytology and Evolution. Cambridge University Press. Cam- bridge, England.

507-515.

Methods Med. Res. 5: 335-378.

Sci. URSS N. S. 4: 85-88.

1943

1947 1956

1961

WHITE, M. J. D., 1948

immunochemical analysis of the effects ofimmunochemical techniques: the techniques used for...

Documents