ORGANIZATION OF THE ROSY LOCUS IN DROSOPHZLA MELANOGASTER: EVIDENCE FOR A CONTROL ELEMENT ADJACENT TO THE XANTHINE DEHYDROGENASE STRUCTURAL ELEMENT1 A. CHOVNICK, W. GELBART,2 M. McCARRON
AND
B. OSMOND3
Genetics and Cell Biology Section, Biological Sciences Group, The University of Connecticut, Storrs, Connecticut 06268 AND
E. P. M. CANDID0
AND
D. L. BAILLIE4
Department of Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1 W5 Manuscript received April 29, 1976 "",respectively). Prior work (GELBARTet al. 1974) has established that the genetic basis for this difference is localized to a site at the right end of the rosy structural element, e408 (Figure 2B), with ry+O bearing e408S while ry+b possesses the e408F alternative. Hence, r y f 4 J r y f individuals contain two sites of heterozygosity localized to the kar-126 interval: one concerned with level of XDH activity (High us. Normal), and the other concerned with electrophoretic mobility as noted above. Are these sites readily separable by recombination? Females of the genotype c u k a r r x + O4- 4- -t- cu kar ryas126 ~were crossed to males. Tp(3)MKBS,M34 liar rye Sb ry+4 126 Sb Ubx From this mating, one crossover class, kar+ ry+ Sb+ sons, was selected for further analysis. In addition to carrying the cu kar ryas126 paternal chromosome, such males must carry a kar+ ry+ 12&+ Sb+ chromosome of maternal origin, which arose via a crossover in the kar-126 interval. These males were crossed individually to females of the genotype cu kar, Df(3)ryS7/Tp(3)MRS,M34 ry" Sb. The offspring of this cross permit confirmation of the genetic composition of the exceptional progeny. Moreover, this test cross provides off spring carrying the recombinant chromosome heterozygous with Df(3)ryZ7,which is missing the
++
ROSY LOCUS CONTROL ELEMENT
241
entire kar-126 region. For each crossover, extracts of such individuals were subjected to electrophoresis in order to classify the recombinant in terms of level of XDH activity and mobility. A total of 123 crossovers were analyzed, and none exhibited recombination for these characteristics: 79 were XDH1so0and normal in level of XDH activity, like the ry+O parental type, while 44 were XDH1.02 and produced high levels of activity like the ry+4 parental type. In summation, the progeny of this cross reflect exchanges in the intervals kar-XDH-126 with 79 occurring in the kar-XDH interval, and 44 in the XDH-126 interval. Our failure to recover recombinants between these phenotypes in this large sample of exchanges in the kar-126 region suggests that level of X D H activity, like electrophoretic mobility, may very well be under the control of the rosy locus itself. On the assumption that these characters are, in fact separable, the maximum possible genetic distance between them may be estimated from the 95% Poisson Confidence Interval (STEVENS 1942), and equals 0.012 map units (3/123 X 0.5). Further biochemical and genetic characterization of We are now faced with the task of distinguishing whether the genetic basis for the level of activity difference resides in the X D H structural element, or is perhaps a variant of the control element. Upon reflection, this distinction is not easily resolved. It is quite conceivable that ry+4 and the other ry+ isoalleles differ in several sites. Based upon recombination analysis, some of our ry+ isoalleles differ by as many as five or six structural element sites (unpublished work of this (1976) have identified 37 isoalleles laboratory). SINGH,LEWONTIN and FELTON of the XDH structural gene from 146 randomly isolated lines of Drosophila pseudoobscura. Taken together, these observations bespeak the considerable likelihood that any two isolated lines (i.e., ry+4 and any other ry+ isoallele) will possess structural element differences. However, these site differences may or may riot be responsible for the difference in level of X D H activity. Obviously, the extensive polymorphism exhibited by the X D H structural element may be observed as well in the adjacent control element; the difference in level of XDH activity might reflect such control element variation. Characterization of the ry+4 gene product. We may well begin by examining the possibility that there are structural differences between the X D H molecules produced by r ~ and + those ~ produced by other ry+ isoalleles which are responsible for their differencesin level of activity. Three sets of observations have failed to provide evidence in favor of a structural basis for the activity difference. 1. There is no systematic relationship between level of XDH actiuity and ebctrophoretic site diflerences. Consider ry+O, ry+I, ry+# and r y + f l ,which exhibit normal levels of XDH activity. VVe know that ry+O differs from ry+l in one identified electrophoretic site (see earlier discussion), and ry+* differs from ry+4 in two known sites, e217 and e408 (Figure 2C). On the other hand, ry+’ and ry+” produce XDH molecules of the same mobility as ry+4 (Table 1). 2. There is no systematic relationship between XDH thermolability and level of activity. One might suspect that the increased level of ry+4 enzyme activity is a reflection of a greater molecular stability resulting in increased numbers of
242
A. CHOVNICK
et al.
HEAT INACTIVATION of XDH
TIME i m , n ) AT 60"
FIGURE 5.-Heat
inactivation of XDH in extracts of r y f "
(N) and r y f 4 (H) homozygotes.
XDH molecules. Such differences may be exposed by examination of XDH thermolability associated with the several r y f isoalleles. Heat inactivation experiments over a range oi temperatures have been carried out on extracts from most of our r y f lines. While there do exist differences in thermolability of XDH molecules produced by the several ry+ isoalleles, no association with level of activity is apparent. Figure 5 summarizes heat inactivation experiments carried out to compare the thermolability of the r y f 4 and i y f J JX D H s at 60°, and indicates that there is no recognizable difference between the two1 samples. The isoalleles ry+O and ryf2 show similar thermolability curves. Clearly, thermolability differences cannot account for activity level differences seen in these lines.
60
M
v'
A0D650 12 min
4c
M
20
10
2 00
loo
b]
=
500
46)
[HYPOXANTHINE].~M
FIGURE 6.-XDH activity as a function of substrate concentration in matched extracts of ry+'l ( N ) and r y f 4 (H) homozygotes.
ROSY LOCUS CONTROL ELEMENT
243
3. We may next consider the possibility that differences in level of XDH activity reflect r y f structural element variants that alter the catalytic ability of the enzyme. Figures 6 and 7 summarize the results of one experiment that demonstrates, at least for ry+4 and ryf'I, that the differencein leuel of X D H activity between these two lines cannot be attributed to differences in affinity of the X D H molecules for substrate. Reaction velocities of matched, partially purified preparations of XDH from ry+4 (El) and ry+I1 (N) are plotted against substrate concentration in Figure 6. At each substrate concentration the activity of the ry+O enzyme is approximately 4~ that of the r y f " preparation With both preparations approaching a plateau at a concentration of 200 pM of hypoxanthine. Figure 7 presents a double reciprocal plot of the data of Figure 6. The abscissa intercept estimates 1 /Vma, for each preparation, while the ordinate intercept estimates the K, of each enzyme. The reader will note that the k, of the ry+4 enzyme estimated from this experiment is slightly greater than that of the ry+ll enzyme. I n repeat experiments, the K, estimates of these two enzymes differ negligibly. If the observed difference in V,,, between these preparations were due to substrate affinity variation, then we would have expected the r y f 4 enzyme to have a K,, that is $4 that of the ry+I1 enzyme. That the K,s are identical argues that the difference in level of activity is associated with variation in number of molecules of XDH/preparation.
Euidence from immunological experiments. From a series of immunological studies, further evidence emerges in support of the view that the difference in levels of activity reflects a difference in number
FIGURE 7.-Double reciprocal plot of the data of Figure 6. Ordinate intercept = l/Vmax: Abscissa intercept = Q.
244
et al.
A. CHOVNICK
of X D H molecules. Rabbits were immunized following a protocol (MATERIALS designed to produce an anti-XDH reagent that would react broadly with Drosophila melanogaster-XDH and would minimize known structural dif ferences. Ouchterlony plate experiments were carried out with crude extracts of adult wild-type flies as antigen. Over a broad range of antigen-antiserum concentrations, such experiments reveal a single major component which represents the X D H band as identified by staining for XDH activity. In addition, several very faint precipitin lines are observed which fail to stain for XDH. For some exper:ments, unabsorbed serum is used, while for other experiments (as indicated) this serum is pre-absorbed with extracts of the genotype Dfd Df(3)kars1 ryG0JkargDf ( 3 ) ~ yThis ~ ~ extract . possesses little or no X D H cross-reacting material, but is able to remove the extraneous reactivity of the serum. The Ouchterlony experiments also demonstrate that this serum is a competent anti-Drosophila X D H reagent. In addition to precipitating wild-type X D H , it is ( 1) capable of precipitating an enzymatically inactive counterpart to wild-type X D H from maroon-like mutant extracts, (2) capable of prccipitating weakly reactive X D H molecules from lxd extracts, and (3) unable to precipitate material from ry2mutant extracts (See review by FINNERTY 1976). Utilizing this antiserum as a reagent for the quantitative assay of X D H , we carried out experiments whose results confirm our inference that homozygous r y f 4 flies possess more XDI-I molecules than do flies homozygous for our other ry+ isoalleles. Figure 8 summarizes an antiserum titration experiment in which
AND METHODS)
% XDH
ACTIVITY REMAINING
164 132
116
I8
14 r
CONCENTRATION,ANTI- XDH SERUM
FIGURE8.-Titration of unabsorbed anti-XDH serum. % of XDH activity remaining in ry+e (N) and 4
- 4
(H) extracts following incubation in the indicated dilutions of serum.
245
ROSY LOCUS CONTROL ELEMENT
I
15
20
pI of MTRACT
FIGURE 9.-Quantitative analysis of “rocket” electrophoresis experiment. Height of rocket measured from top of well to apex of rocket. Matched extracts of homozygotes run against preabsorbed, anti-XDH serum. Volume of extract in each well is adjusted to 20 pl with buffer.
a set of dilutions of the unabsorbed anti-XDH serum is tested for ability to remove XDH activity from matched extracts of ry+4 and ry+*.The titration curves are very different for the two preparations with the ry+2extract being inactivated at an antiserum dilution that is incapable of removing more than 50% of the XDH activity from r y f 4 . Figure 9 summarizes the results of an immunoelectrophoresis experiment utilizing the method of quantitative “rocket electrophoresis’’ (LAURELL 1966; WEEKE 1973) to compare the relative number of molecules of XDH in matched extracts of r y f 4 ,ry+jj and ry+$homozygotes. In this experiment, several concentrations of ry+4 extract are run, and their resultant rocket heights describe a straight line relationship. Relative number of molecules of XDH in ry+I1 and ry+* preparations are extrapolated as indicated in the Figure. Clearly, the ry+4 extract possesses more molecules of XDH than do the other tested lines. Genetic fine structure localization. I n a previous section, experiments were described which localized the genetic basis for the high level of XDH activity associated with the ryt4-bearingchromosome to the rosy locus itself. Let us now turn to two groups of large-scale fine-structure experiments which further define this localization. Table 2 presents the results of experiments involving null enzyme mutants of the ry+4 isoallele tested against a standard series of mutants of the ry+O allele. The reader will note that from just these experiments (GELBART et al. 1974; GELBART, MCCARRON and CHOVNICK1976), recombination data provided for the localization of the null enzyme mutant sites ry4OZand ry406, as well as for the localization of the electrophoretic difference between the ry+O and ry+4 isoalleles. These results are summarized in the maps of Figure 2, with
A . CHOVNICK
et al.
TABLE 2 Number and classes of r y + chromosomes recovered from progeny of crosses of the indicated females to tester males of the genotype Dfd Df (3)kar31 rycO/kar* Df (3)ryiS
Zygotes were reared on purine-supplemented medium, permitting only rare r y f progeny to survive. Crossovers
+ + +
kar ry+ 126+
Feinale parent
+ + +
13[I102N]
,Y402 __-____ kar ryhl 126
,Y402 karsry5 126
0
,Y405 kar2ry5 126
0
kart rp+ 126
0
0
15[1.00 HI
Conv. ryhoo Xar+ ry+ 126+
8[1.02 HI
0
3[1.02 HI
0'
+
ry406 picG281
kar ry23
+
+ 126
0
2[l.OOH]
2[1.02H]'
Conv. rys
---
Zygotes sampled
kar rp+ 126
( X 10-e)
6[1.00N] 2 [ 1.02 NI
1.11
1[1.00N]
1.34
4[1.00N]
0.8
2[1.00N]
0.57
7[1.00N] I [1.00 HI
1.21
* At the time of these experiments, the ry406 bearing chromosome was lethal when homozygous, presumably due to one or more unknown recessive lethal mutations resulting from the same mutagenesis that yielded ry506. Subsequent analysis revealed the presence of a lethal allele of piccolo, picQasl,which is located immediately to the right of rosy (see SCHALET, KERNACHAN and CHOVNICK1964, and Figure 1). The indicated exceptional classes carry picG231, and must be reduced in number by virtue of the fact that one of the paternal tester chromosomes, kar2 Df(3)ryTS is deficient for the pic locus as well.
the electrophoretic site, e408, located at the right end of the map. I n the earlier presectations of these data, attention was focused upon the boundaries of the XDH structural element, and all consideration of variation in level of activity was omitted to simplify the presentation. Here, we offer the total data. Consider the first row of Table 2, which summarizes test of ryAoaagainst ryA1, a site located near the right end of the XDH structural element. With respect to the flanking markers, kar and 126, three classes of ryf recombinant chromosomes were recovered. The mobility and level of activity (H = High; N = Normal) associated with each ry+ recombinant allele was determined in subsequent electrophoresis and fluorescence assay experiments. Thirteen flanking marker crossovers were recovered, all kar l y + 126+, indicating that ry402 lies well to the left of ryA1.Moreover, these were recombinant for electrophoretic mobility and level of XDH activity. All showed the mobility of the r y + : parental allele (1.02) with the level of activity of the ry+O allele (N) . From this observation, we are able to infer that the genetic basis for the difference in mobility between r y f o and ry+4 (e408) lies to the right of all crossover points between ry402 and ryhl, while the basis for the difference in activity level falls to the left of all crossover points. Note that in all experiments described in Tables 2 and 3, no intermediate or nonparental classes with respect to XDH activity level were recovered. Additionally,
24 7
ROSY LOCUS CONTROL E L E M E N T
TABLE 3 Number and classes of ry+ chromosomes recovered from progeny of crosses of the indicated females to tester males of the genotype, Tp ( 3 )MKRS, M ( 3 )S34 kar ry2 %/In (3R)PI,,kar r y 4 l Ubx e4 Zygotes were reared on purine-supplemented medium, permitting only rare r y + progeny to survive. Conv. ry4OO
Conv. ry5
kar ry+ pic+
Crossovers kar+ ry+ pic
kar ryf pic
kart ryt pic+
1[1.03 HI
0
2[1.05 H] 1[1.02 HI
1C1.03 NI
1.99
4r1.00 HI
0
1cl.02 HI
5[1.ON] 1 [1.OO H]
0.98
--.___--
Female parent
kar2 ry4ofi picGz31
+
ryps214
+
kart ry406 pic0231
___ ______
+
.PO6
+
Zygotes sampled (X
ry+ recombinants exhibiting parental flanking markers (i.e., conversions) were recovered. Thus, eight conversions of the ryhoZallele were recovered, and all exhibited parental mobility and level of XDH activity. I n contrast, the conversions of the ry4’ allele exhibit some co-conversion with e408 reflecting proximity of these markers (Figure 2). However, each conversion of ry4’ exhibits the parental (Normal) level of activity. Obviously, the site giving rise to the increased XDH activity is different and separable from e408. Let us now designate this carrying i409H,and ry+O carrying i409N. site as i409,with The data of Row 2, Table 2 summarize the results of recombination test of ry402 against ry5,an XDH- mutant site located near the left end of the structural element. These data complement the results presented in Row 1 and serve only to locate the mutant ry402 quite close to ry5. Turning to the third row of Table 2, we see a pattern of results which reinforces the inferences drawn from the first cross. The mutant ry5, located near the left end of the XDH structural element, is tested against ry405. Fifteen flanking marker crossovers serve to locate ry405 to the right of ry5. All of these crossovers were recombinant for electrophoretic mobility and level of XDH activity. All exhibited the mobility of the ry+O parental allele (1.OO) with level of activity of the ry+4 allele (H). Again, we infer that the genetic basis for the difference in mobility between ry+O and ry+4 (e408)lies to the right of all crossover points between ry5 and ry405,while the basis for the difference in activity level (209) fall to the left of all crossover points. The experiments of rows 4 and 5 focus upon the left end of the rosy structural element by selecting for ry+ recombinants involving mutant sites located on that side of the map (Figure 2). Following the argument used in analysis of the data of rows 1 and 3, the crossover classes emphatically point to the left end of the rosy locus as the location of i409, the genetic basis for level of XDH activity, while e408, the mobility difference between the ry+Qand r y + 4alleles is located at the right end of the map. ?‘he remaining two experiments of this series are summarized in Table 3. Again, we concentrate upon the left end of the XDH structural element with the mutant ry4O6, a member of the ry4O0 series of mutants (Figure 2), tested
248
A . CHOVNICK
et aZ.
against iyPSzi4and ry606.As with Table 2, part of these data are presented elsewhere (GELBART, MCCARRON and CHOVNICK 1976). The first cross of Table 3 describes the results of recombination in heterozygotes, ry4°6JryPsg14. The reader will recall ( GELBART et al. 1974) that the electrophoretic difference between iy+" and ry+O is resident in a site, e217, located in the left portion of the XDH structural element, while the electrophoretic difference between ry+4 and ry+O relates to a site, e408, h a t e d at the right end of the map. Crosses between derivatives of the iy+4 and r y f 2isoalleles differ at both sites. Thus, the cross of Row 1, Table 3 involves assay of recombination in heterozygous females of the genotype kaP 4- 406 e217S e408F picGgss1 - , where the relative positions of all rosy -I- ps214 -I- e217F e408S f locus sites are seen from Figure 2. The single flanking marker crossover recovered in this cross (Table 3, Row 1) carried i409H, and exhibited the mobility of the ry+" parental isoallele (XDH1.03),and thus must carry e217F and e408S. Of interest is the proximity of ry406 to the e217 site (Figure 2). This fact is reflected in the observation that two of the three conversions of ry4O6 exhibit co-conversion f o r the adjacent electrophoretic site as well. This results in a ry+ allele carrying i409H, e217F, e408F, which produces an XDH1.05with the level of activity of the iy+4 parent allele. The second cross of Table 3 involves the XDH-, allele complementing mutant, ry606,which is the leftmost known unambiguous structural mutant (Figure 2). Data presented elsewhere (GELBART 1976) argues that there is a single electrophoretic site difference responsible for the mobility difference between ry+4 and r ~ +This ~ . site is located on the right side of the structural element, and may = e408S). Thus, in the ry4°6/iy606 cross well be e408 (ry+4 = e408F: (Table 3, Row 2), the single electrophoretic site difference between these alleles serves merely as a marker €or the right side of the XDH structural element. Following the previous logic, all of the i y + recombinants associated with flanking marker exchange exhibited recombination for the i409 site and e408 (Table 3, Row 2). All exhibited XDH1.OOwith the high level of activity associated with i409H. All of the crossovers of Table 3 place the genetic basis for the observed difference in level of XDH activity to the left of all crossover points. Pooling the data of Tables 2 ar?d 3, the crossovers place i409 to the left of all observed exchange points within the XDH structural element. An additional feature of these data emerges from examination of the results involving i y Z s (Table 2) and rySo6(Table 3 ) . These mutants serve to mark the present left border of the XDH structural element. The crossover data would place 2 0 9 either to the left of both these mutants, or very close to the right, presumably just inside the structural element (Figure 2). If the latter position were correct, conversions of i y Z s ,iy606,iy4O2, i y 4 0 6 and ry5 would often be accompanied by co-conversion for the i409 site (CHOVNICK et al. 1974). However, both ryZ3and ry606show a low frequency of co-conversion with i409, while iy402, ry406 and ry5 have failed to exhibit co-conversion with i409. Taken together, interpretation of both crossover and co-conversion data would place i409 well to the left of both i y Z 3and ry606,and hence, beyond the left border of the XDH structural element.
249
ROSY LOCUS CONTROL E L E M E N T
XDH STRUCTURAL ELEMENT A06
!(3)S12
i409
r
402
FIGURE 10.-Map location of i409 site relative to 2(3)S12 and the XDH structural element.
Confirmation of the localization of the i409 site. Figure 10 summarizes the previous section’s localization of 2 0 9 relative to the left end of the XDH structural element. The mutants ry4OSand ry406 are represented as distinctive sites just inside that border. Although no effort has been made to separate those rosy mutants from each other, a pattern of comparative recombination data consistently places 402 just to the left of 406 (Figure 2). Note also, the relative position of 1(3)S12 (Figure 1). This heretolfore unreported mutant was recovered from an X-ray treatment of a karPry+ll chromosome. It was positioned between mes (SCHALET, KERNAGHANand CHOVICK1964) and ry by complementation and recombination tests (SCHALET, Personal Communication). Preliminary experiments revealed that the ka? 1(3)S12 ry+ll chromosome is associated with normal levels of XDH1.02 activity, ar?d is thus designated kar3 1(3)S12 i409N ~ y + ” . Despite its proximity to the rosy locus (< 0.01 map units in preliminary experiments), there is no evidence to consider 1(3)S12 as an alteration within the control element of the rosy locus. Rather, we believe it to be a site mutant in the very next genetic unit to the left of rosy. Figure 11 presents the genotypes of two classes of females which were crossed
+
M(3)S34 kar2 1 ( 3 ) S 1 2 i409N
(A)
+
+
+
i409H ry406 p i c
+
M(3)S34 kar2 1 ( 3 ) S 1 2 i409N (B)
+
+
+
+
i409H r y 4 0 2
Sb
females
G2 3 1
+
Sb
+
+
females
X
c u kar Df ( 3 ) r ~ [1(3)S12~ ~
r y - pic-]Sb+
Ubx e 4
males Tp
( 3 ) MKRS, M(3)S34
kar
ry2
Sb
FIGURE11.-Parents of large-scale selective recombination experiments. Resultant ry+ crossover chromosomes are described in Table 4.
250
A . CHOVNICK
et al.
to the indicated males in large scale matings carried out on purine-supplemented medium. I n the absence of crossing over in the region from M ( 3 ) S 3 4 S b , all zygote classes will die due to the homozygous or hemizygous lethality of M(3)S34,1(3)S12, ry, pic, and Sb either singly o r in combination. Of particular interest are single crossovers between 1(3)S12 and r y which produce a kar+ lS12+ r y + pic+ Sb recombination product. Zygotes receiving this product, as well as a cu kar Df(3)ryS7Sb+ Ubx e5 paternal chromosome, survive and will be kar+ + y + Sb Ubx in phenotype. What about the 209 site associated with such crossovers? I n the absence of the data of the previous section, three possibilities are apparent: (1) If i409 were inside the XDH structural element and located either to the right of ry402 and/or ry406 or just to the left of one o r both rosy mutants, then all (or almost all) of these crossovers would be i409N. (2) If i409 were located to the left of the structural element border, and somewhere between 1(3)S12 and both ryGoz and ry606, then the exchange classes should include ry+ chromosomes with i409H and i409N in proportions relative to the location of i409. (3) Finally, if i409 were, in fact, located to the left of 1(3)S12 or possibly just to the right, then all (or almost all) of this exchange class should be i409H. The pertinent results of the crosses outlined in Figure 11 are summarized in Table 4. A total of 16 crossover progeny were recovered, which upon further test were shown to fall into two classes with respect to the maternally derived exchange chromosome. These chromosomes were cytologically normal. The results are entirely consistent with the second alternative described above, and thus permit the conclusion that i409 is located well to the left of the structural element border. The relative proportions of the i409H and i409N classes permit location of i409 somewhat closer to 1(3)S12 than to the left border of the XDH structural element (Figure 10). The results of these crosses, therefore, provide a very strong confirmation of the localization of i409 inferred from the recombination data presented in the previous section. TABLE 4 Number and classes of ry f crossover chromosomes recovered from progeny of crosses described in Figure 11 Zygotes were reared on purine-supplementedmedium, permitting only rare r y + progeny to survive. Crossoversf
Cross
kar+ 1(3)S12+ i409H ry+ pic+ Sb
A B'
3
ZYgOteS
kar+ l ( 3 JS12-i i409N ry+ pic+ Sb
7
4 2
sampled x 10.0)
(
0.36 0.82
* 1 kar 2(3)5'12+ i409N r y + pic+ Sb chromosome was recovered and classified as a conversion, l(3)SlZ + 1(3)S12+. 16(4) x IO2 f Map distance, l(3)SlZ-ry = -___ = 0.0054 1.18 x 106 -f Map distance, i409-ry
lO(4)
x x
102
= ___--1.18
IO6
- 0.0034 -
ROSY I.OCUS CONTIWI. ICI.IXMI.NT
25 1
Of course. several additional distinguishable classes of survivors were recovered. These include double crossovers, a variety of triploids. aneuploids. and rare tandem duplicr\tions resulting from unequal exchanges in the rosv region. All of these classes survive by virtue of the lethals being "covered" in the duplicated region or rcmoved by Crossovers. .Might i409H rrprcscnt n tnndem duplication of tlic XDH structural clcment ? Ample precedence for such consideration exists in the several well-known cases W GREEN1959. 1963). On this notion, in Drosophila (Sec, for example. ~ ~ . A F I 1967; the r y f ' isoallclc would be considered to possess two functional XDH structural elements in tandem, presumably resulting from a n unequal exchange event. Such a model is precluded on several counts: ( I ) Cytological examination of polytene chromosomes revcals no such tandem duplication. (2) Tanclem duplications arc characterized by instability in homozygotes due to increased incidence of unequal exchange events. The ry+z stock has been quite stable. (3) Fine structure recombination expcrimcnts involving tests of ry:')''series mutants against other XDH- mutants hare been characterized by regular exchange events. and the complete absence of unequal crossing over. ( 4 ) The r y f ' allcle is associated with a single XDH electrophoretic band (Figure 3) of the mobility class, XDH'."' (Table 1). XDH is a homodimcr. and the presence of two electrophoretically distinct structural elements will produce individuals possessing three XDH moieties (Figure 12). The tandem duplication model then requires that the r y + J allele possess two XDH structural elements whose peptide products are indistinguishable, and of the mobility class, XDH'.O?.
A
B
FIGURE 12.-XDH rlectropherogram indicating the rrlatiw amounts of XDH"."", XDH"."G n n d XDH'."?prrsrnt in mntchrd rxtrncts of flies of the genotypes (A) i40YN ry+'/i409N ry+" ancl (R) i409H r y + J / i 4 0 9 N r y + l O .
252
A. CHOVNICK
et al.
Thus, i409H should be associated with an XDH1.02. On this point, the model fails. In all experiments which recombine i409H with other electrophoretically distinct structural elements, there is no evidence of the production of an XDH1.02 moiety. We see from all of the arguments presented above that i409H may not be considered to mark a duplication of the XDH structural element. Functional relationship between i409 and the XDH structural element. It is apppropriate at this point to consider that i409 marks a genetic element that serves, in one way or another, to regulate XDH. On the basis of evidence thus far presented, a broad array of regulatory roles are possible, and further specification would be premature. However, by a simple experiment, we are able to describe a key feature of this regulatory function. Under one class of regulatory roles, dominance-recessiveness, or incomplete dominance in heterozygotes of i409 may obtain. In still another class of roles, the regulatory function of a specific i409 element would be restricted to the specific XDH structural element adjoining it on the chromosome. We shall refer to this as a “cis-acting” reaplator. Consider the heterozygote i409N ry+’/i409N ry+lz. Such individuals should approximately equal quantities of the ry+I and ry+I2peptides. Assume further that random union of monomers takes place to yield active XDH dimers. Electrophoresis of extracts of such heterozygotes should reveal the presence of three XDH moieties, XDH0.Oo(ry+ldenzyme) , XDHo.QG (hybrid dimer) and XDH1.02 (ry+’ enzyme) in a ratio of 1:2: 1. Figure 12 is a photograph of an electropherogram which is printed as a negative in order to enhance the contrast between XDH banded regions and the acrylamide slab gel. Figure 12A presents the XDH molecular composition of extracts of i409N ry+’/i409N ry+Iz, and is entirely consistent with the expectation and its underlying assumptions. Consider next the XDH composition of an extract of the genotype i409H r ~ + ~ / i409N ry+’%(Figure 12B). The XDH structural element of the ry+4 allele is associated with an XDH possessing a mobility identical to that of ry+’ (XDH1.02). Thus, in terms of XDH moieties, this genotype should possess the same three mobility classes as seen in Figure l2A. However, this genotype differs from the former in that it is heterozygous, i409H/i409N. Under one class of regulatory models, dominance of one or the other form of these alternatives, or even incomplete dominance, may be expected. In such event, we should find the same 1:2: 1 ratio of the three mobility classes. However, all would be produced in high, standard or intermediate quantities depending upon the specific dominance relationship that obtains. Alternatively. each i409 element may operate to regulate only the specific XDH structural element (or product thereof) located adjacent to it on the same chromosome (i.e., a “cis-acting” regulator). Thus, in the heterozygote, i409H ry+4/i409N ry+‘z, this model would predict that greater quantities of the ry+4 polypeptide than ry+lapeptide would be produced. Random union of monomers would then result in increased quantities of XDH1.02 (ry+4 enzyme) and XDHo.96(hybrid dimer) at the expense of XDHo.90(ry+’$ enzyme). Examination of Figure 12B reveals that just such result obtains, and we are led to conclude that i409 operates as a “cis-acting” regulator.
ROSY LOCUS CONTROL ELEMENT
253
DISCUSSION
We have shown that i409H and i409N represent alternative forms of a genetic element associated with variation in number of molecules of XDH/fly. We have been unable to relate this variation to alteration in the structure of the XDH polypeptide or to a tandem duplication of the XDH structural element. Finestructure recombination studies locate this element very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Moreover, these experiments have produced exchange products (Tables 2 and 3) that carry new combinations of the 209 element with electrophoretically distinct structural elements. While previously (Table 1) our array of wild-type stocks carried i409H only in combination with an XDH structural element whose product belonged to the mobility class, XDHl 02, we now possess i409H with structural elements producing XDH1.OO,XDH1 O3 and XDH1.05molieties as well. Indeed, we believe that 2 0 9 is completely separable from the XDH structural element, and that the construction of every possible combination of XDH structural alleles and 2 0 9 alternatives would be a simple genetic exercise. Taken together, these observations provide a compelling argument in favor of a regulatory role for i409.Moreover, evidence is presented that limits our consideration of the specific function of the 209 segment to that of a “&-acting” regulator. We may eliminate several broad categories of regulatory roles which are characterized by “trans-acting” regulation and which should exhibit dominancerecessiveness or intermediate phenotypes in heterozygotes. Thus, we may eliminate all regulatory models which associate i409 with the synthesis of a negative or positive acting diffusible regulatory molecule (GILBERTand MULLER-HILL 1970; DICKSON et al. 1975). GOLDBERGER (1974) has recently reviewed the evidence for autogenous regulation, largely from prokaryote studies, and has suggested that similar cases may be found in higher organisms. The possibility that the XDH peptide serves a second function as an autogenous regulatory macromolecule has not been explored. Nonetheless, we note that the “cis-acting” nature of the i409 element also precludes the possibility that i409H and i409N are structure variants in a region of the XDH peptide that serves as an autogenous regulator. Rather, we are drawn to the possibility that i409 marks the 5t controll element of the rosy locus. Variants of this element would possess alterations in DNA sequences concerned with functions generally ascribed to such elements (see Introduction). The size of the control element may be estimated from the recombination data of Table 4. We believe that these data to produce underestimates by virtue of the fact that all of the relevant crossover individuals are Ubz in phenotype. Such individuals suffer an increased likelihood of “drowning” in the food shortly after eclosion, and thus not being scored. Ignoring this problem, we take the map distance estimated from the crossovers in the interval 209-ry (Table 4) as a minimap units), mal estimate of the size of the XDH control element (3.4 x while the crossovers in the interval Z(3)SIZ-ry provide a maximum estimate (5.4 x map units). In the accompanying report (GELBART, MCCARRON and CHOVNICK 1976), we have estimated the size of the XDH structural element at
254
A. CHOVNICK
et al.
5.0 X map units and related this to a DNA length of 3,000 base pairs from the XDH peptide size (160,000 daltons) . From this, we may estimate the size of the i409 segment to range from a minimum of 2,040 base pairs [3.4 ( 3 x IO3)/ 5.01 to a maximum of 3,240 base pairs C5.4 (3 X 103)/5.0]. At a maximum, the 2 0 9 control element is no larger than the XDH structural element. We recognize that the possibility of control sequences at the other end of the rosy locus remains to be explored. Nevertheless, we note that the present results lend little support to solutions of the chromomere paradox that postulate control elements an order of magnitude larger than their structural components (See review, BEERMAN 1972). We wish to acknowledge the skilled technical assistance of FLORA GRABOWSKA, HARRY LEVINE and FLORENCE JOHNSTON. LITERATURE CITED
ANDRES, R. Y., 1976 Aldehyde oxidase and xanthine dehydrogenase from wild-type Drosophila melanogaster and immunologically cross-reacting material from ma-l mutants. Eur. J. Biochem. 62: 591-600. BAHN, E., 1967 Crossing over in the chromosomal region determining amylase isozymes in Drosophila melanogaster. Hereditas 58: 1-12. BEERMANN, W., 1972 Chromomeres and Genes. Cell Differentiation 4: 1-33. BRAVERMAN, A. S., 1973 The Thalassemia Syndromes: Genetically Determined Disorders of the Regulation of Protein Synthesis in Eukaryotic Cells. In: Progress in Molecular and Subcellular Biology 3: 183-202. Edited by F. E. HAHN. Springer-Verlag Publishers, New York. CANDIDO, E. P. M., D. L. BAILLIEand A. CHOVNICK,1974 A rapid purification of xanthine dehydrogenase for genetic studies. Genetics 77: S9. CHOVNICK, A., 1966 Genetic organization in higher organisms. Proc. Roy. Soc. London B 164: 198-208. -, 1973 Gene conversion and transfer of genetic information within the inverted region of inversion heterozygotes. Genetics 75: 123-131. and D. G. HOLM, 1971 Studies on gene conversion and its CHOVNICK, A., G. H. BALLANTYNE relationship to linked exchange in Drosophila melanogaster. Genetics 69 : 179-209. D. L. BAILLIEand D. G. HOLM, 1970 Gene conversion in CHOVNICK, A., G. H. BALLANTYNE, higher organisms: Half-tetrad analysis of recombination within the rosy cistron of Drosophila melanogaster. Genetics 66: 315-329. CHOVNICK, A., W. GELBART, M. MCCARRON and J. PANDEY, 1974 Studies o n recombination in higher organisms, pp. 351-363. In: Mechanisms in Recombination. Edited by R. F. GRELL. Plenum, New York. DAVIDSON, E. H. and R. J. BRITTEN,1973 Organization, transcription and regulation in the animal genome. Quart. Rev. Biol. 48: 565-613. W. M. BARNES and W. S. REZNIKOFF,1975 Genetics regulation: DICKSON,R. C., J. ABELSON, The Lac control region. Science 187:27-35
FINNERTY, V., 1976 The simple cistrons: rosy and maroon-like. In: Genetics and Biology of Drosophila, Vol. 1. Edited by E. NOVITSKI and M. ASHBURNER. Academic Press, New York. and A. CHOVNICK,1976 Extension of the limits of the XDH GELBART, W., M. MCCARRON structural element in Drosophila melanogaster. Genetics 84.: - . GELBART, W. M., M. MCCARRON, J. PANDEY and A. CHOVNICK,1974 Genetic limits of the xanthine dehydrogenase structural element within the rosy locus in Drosophila melanogaster. Genetics 78: 869-886.
ROSY LOCUS CONTROL ELEMENT
25 5
GILBERT,W. and B. MULLER-HILL,1970 The Lactose Repressor, pp. 93-109. In: The Lactose Operon. Edited by J. R. BECKWITHand D. ZIPSER.Pub. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. GLASSMAN, E. and H. K. MITCHELL,1959 Mutants of Drosophila melanogaster deficient in xanthine dehydrogenase. Genetics 44: 153-162. GLASSMAN, E., J. D. KARAMand E. C. KELLER,1962 Differential response to gene dosage experiments involving the two loci which control xanthine dehydrogenase of Drosophila melanogaster. Z. Vererb. 93: 399-403. GOLDBERGER, R. F., 1974 Autogenous regulation of gene expression. Science 183: 810-816. GREEN,M. M., 1959 Non-homologous pairing and crossing over in Drosophila melanogaster. Genetics 4-4:1243-1256. -, 1963 Unequal crossing over and the genetical organization of the white locus of Drosophila melanogaster. Z. Vererb. 94: 200-214. GRELL,E. H., 1962 The dose effect of ma-Z+ and ry+ on xanthine dehydrogenase activity in Drosophila melanogaster. Z. Vererb. 93 : 371-377. LAURELL, C.-B., 1966 Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15: 45-52, LEWIN,B., 1975 Units of transcription and translation: the relationship between heterogeneous nuclear RNA and messenger RNA. Cell 4: 11-20. LINDSLEY, D. L. and E. H. GRELL,1968 Genetic variations of Drosophila melanogmter. Publs. Carnegie Inst. No. 627. M., W. GELBART and A. CHOVNICK, 1974 Intracistronic mapping of electrophoretic MCCARRON, sites in Drosophila melanogaster: Fidelity of information transfer by gene conversion. Genetics 76: 289-299. MCCLINTOCK, B., 1951 Chromosome organization and genic expression. Cold Spring Harbor Symp. Quant. Biol. 16: 13-47. -, 1956 Controlling elements and the gene. Cold Spring Harbor Symp. Quant. Biol. 21: 197-216. J. and F. JACOB, 1961 General conclusions: Teleonomic mechanisms in cellular metabMONOD, olism, growth and differentiation. Cold Spring Harbor Symp. Quant. Biol. 26: 389-401. o., 1958 Diffusion-in-gel methods for immunological analysis. Progress Allergy OUCHTERLONY, 5: 1-77. PAIGEN, K., 1971 The Genetics of Enzyme Realization. pp. 1-46. In: Enzyme Synthesis and Degradation in Mammalian Systems. Edited by M. RECHCIGL. S. KARGER, Basel, Switzerland. G. M. RAMIREZ,F., C. NATTA,J. V. O’DONNELL,V. CANALE,G. BAILEY,T. SANGUENSERMSRI, MANIATIS,P. A. MARKSand A. BANK, 1975 Relative numbers of human globin genes assayed with purified ru and p complementary human DNA. Proc. Nat. Acad. Sci. U.S.A. 72: 1550-1554. SCHALET, A., R. P. KERNAGHAN and A. CHOVNICK, 1964 Structural and phenotypic definition of the rosy cistron in Drosophila melanogaster. Genetics 50: 1261-1268. and A. A. FELTON, 1976 “Alleles” of xanthine dehydrogenase in SINGH,R. S., R. C. LEWONTIN Drosophila pseudoobscura. Genetics in press. W. L., 1942 Accuracy of mutation rates. J. Genet. 43: 301-307. STEVENS,
SWANK,R. T., K. PAIGEN and R. E. GANSCHOW, 1973 Genetic control of glucuronidase induction in mice. J. Mol. Biol. 82 :225-243.
WEEKE,B., 1973 Rocket Immunoelectrophoresis. In: A Manual of Quantitative Immunoelectrophoresis: Methods and Applications. Edited by N. H. AXELSON, J. KROLLand B. WEEKE. Scand. J. Immunol. 2(Suppl. 1): 37-46. YEN, T. T. T. and E. GLASSMAN, 1965 Electrophoretic variants of xanthine dehydrogenase in Drosophila melanogaster. Genetics 52 : 977-981. Corresponding editor: D. R. STADLER
AND
B. OSMOND3
Genetics and Cell Biology Section, Biological Sciences Group, The University of Connecticut, Storrs, Connecticut 06268 AND
E. P. M. CANDID0
AND
D. L. BAILLIE4
Department of Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1 W5 Manuscript received April 29, 1976 "",respectively). Prior work (GELBARTet al. 1974) has established that the genetic basis for this difference is localized to a site at the right end of the rosy structural element, e408 (Figure 2B), with ry+O bearing e408S while ry+b possesses the e408F alternative. Hence, r y f 4 J r y f individuals contain two sites of heterozygosity localized to the kar-126 interval: one concerned with level of XDH activity (High us. Normal), and the other concerned with electrophoretic mobility as noted above. Are these sites readily separable by recombination? Females of the genotype c u k a r r x + O4- 4- -t- cu kar ryas126 ~were crossed to males. Tp(3)MKBS,M34 liar rye Sb ry+4 126 Sb Ubx From this mating, one crossover class, kar+ ry+ Sb+ sons, was selected for further analysis. In addition to carrying the cu kar ryas126 paternal chromosome, such males must carry a kar+ ry+ 12&+ Sb+ chromosome of maternal origin, which arose via a crossover in the kar-126 interval. These males were crossed individually to females of the genotype cu kar, Df(3)ryS7/Tp(3)MRS,M34 ry" Sb. The offspring of this cross permit confirmation of the genetic composition of the exceptional progeny. Moreover, this test cross provides off spring carrying the recombinant chromosome heterozygous with Df(3)ryZ7,which is missing the
++
ROSY LOCUS CONTROL ELEMENT
241
entire kar-126 region. For each crossover, extracts of such individuals were subjected to electrophoresis in order to classify the recombinant in terms of level of XDH activity and mobility. A total of 123 crossovers were analyzed, and none exhibited recombination for these characteristics: 79 were XDH1so0and normal in level of XDH activity, like the ry+O parental type, while 44 were XDH1.02 and produced high levels of activity like the ry+4 parental type. In summation, the progeny of this cross reflect exchanges in the intervals kar-XDH-126 with 79 occurring in the kar-XDH interval, and 44 in the XDH-126 interval. Our failure to recover recombinants between these phenotypes in this large sample of exchanges in the kar-126 region suggests that level of X D H activity, like electrophoretic mobility, may very well be under the control of the rosy locus itself. On the assumption that these characters are, in fact separable, the maximum possible genetic distance between them may be estimated from the 95% Poisson Confidence Interval (STEVENS 1942), and equals 0.012 map units (3/123 X 0.5). Further biochemical and genetic characterization of We are now faced with the task of distinguishing whether the genetic basis for the level of activity difference resides in the X D H structural element, or is perhaps a variant of the control element. Upon reflection, this distinction is not easily resolved. It is quite conceivable that ry+4 and the other ry+ isoalleles differ in several sites. Based upon recombination analysis, some of our ry+ isoalleles differ by as many as five or six structural element sites (unpublished work of this (1976) have identified 37 isoalleles laboratory). SINGH,LEWONTIN and FELTON of the XDH structural gene from 146 randomly isolated lines of Drosophila pseudoobscura. Taken together, these observations bespeak the considerable likelihood that any two isolated lines (i.e., ry+4 and any other ry+ isoallele) will possess structural element differences. However, these site differences may or may riot be responsible for the difference in level of X D H activity. Obviously, the extensive polymorphism exhibited by the X D H structural element may be observed as well in the adjacent control element; the difference in level of XDH activity might reflect such control element variation. Characterization of the ry+4 gene product. We may well begin by examining the possibility that there are structural differences between the X D H molecules produced by r ~ and + those ~ produced by other ry+ isoalleles which are responsible for their differencesin level of activity. Three sets of observations have failed to provide evidence in favor of a structural basis for the activity difference. 1. There is no systematic relationship between level of XDH actiuity and ebctrophoretic site diflerences. Consider ry+O, ry+I, ry+# and r y + f l ,which exhibit normal levels of XDH activity. VVe know that ry+O differs from ry+l in one identified electrophoretic site (see earlier discussion), and ry+* differs from ry+4 in two known sites, e217 and e408 (Figure 2C). On the other hand, ry+’ and ry+” produce XDH molecules of the same mobility as ry+4 (Table 1). 2. There is no systematic relationship between XDH thermolability and level of activity. One might suspect that the increased level of ry+4 enzyme activity is a reflection of a greater molecular stability resulting in increased numbers of
242
A. CHOVNICK
et al.
HEAT INACTIVATION of XDH
TIME i m , n ) AT 60"
FIGURE 5.-Heat
inactivation of XDH in extracts of r y f "
(N) and r y f 4 (H) homozygotes.
XDH molecules. Such differences may be exposed by examination of XDH thermolability associated with the several r y f isoalleles. Heat inactivation experiments over a range oi temperatures have been carried out on extracts from most of our r y f lines. While there do exist differences in thermolability of XDH molecules produced by the several ry+ isoalleles, no association with level of activity is apparent. Figure 5 summarizes heat inactivation experiments carried out to compare the thermolability of the r y f 4 and i y f J JX D H s at 60°, and indicates that there is no recognizable difference between the two1 samples. The isoalleles ry+O and ryf2 show similar thermolability curves. Clearly, thermolability differences cannot account for activity level differences seen in these lines.
60
M
v'
A0D650 12 min
4c
M
20
10
2 00
loo
b]
=
500
46)
[HYPOXANTHINE].~M
FIGURE 6.-XDH activity as a function of substrate concentration in matched extracts of ry+'l ( N ) and r y f 4 (H) homozygotes.
ROSY LOCUS CONTROL ELEMENT
243
3. We may next consider the possibility that differences in level of XDH activity reflect r y f structural element variants that alter the catalytic ability of the enzyme. Figures 6 and 7 summarize the results of one experiment that demonstrates, at least for ry+4 and ryf'I, that the differencein leuel of X D H activity between these two lines cannot be attributed to differences in affinity of the X D H molecules for substrate. Reaction velocities of matched, partially purified preparations of XDH from ry+4 (El) and ry+I1 (N) are plotted against substrate concentration in Figure 6. At each substrate concentration the activity of the ry+O enzyme is approximately 4~ that of the r y f " preparation With both preparations approaching a plateau at a concentration of 200 pM of hypoxanthine. Figure 7 presents a double reciprocal plot of the data of Figure 6. The abscissa intercept estimates 1 /Vma, for each preparation, while the ordinate intercept estimates the K, of each enzyme. The reader will note that the k, of the ry+4 enzyme estimated from this experiment is slightly greater than that of the ry+ll enzyme. I n repeat experiments, the K, estimates of these two enzymes differ negligibly. If the observed difference in V,,, between these preparations were due to substrate affinity variation, then we would have expected the r y f 4 enzyme to have a K,, that is $4 that of the ry+I1 enzyme. That the K,s are identical argues that the difference in level of activity is associated with variation in number of molecules of XDH/preparation.
Euidence from immunological experiments. From a series of immunological studies, further evidence emerges in support of the view that the difference in levels of activity reflects a difference in number
FIGURE 7.-Double reciprocal plot of the data of Figure 6. Ordinate intercept = l/Vmax: Abscissa intercept = Q.
244
et al.
A. CHOVNICK
of X D H molecules. Rabbits were immunized following a protocol (MATERIALS designed to produce an anti-XDH reagent that would react broadly with Drosophila melanogaster-XDH and would minimize known structural dif ferences. Ouchterlony plate experiments were carried out with crude extracts of adult wild-type flies as antigen. Over a broad range of antigen-antiserum concentrations, such experiments reveal a single major component which represents the X D H band as identified by staining for XDH activity. In addition, several very faint precipitin lines are observed which fail to stain for XDH. For some exper:ments, unabsorbed serum is used, while for other experiments (as indicated) this serum is pre-absorbed with extracts of the genotype Dfd Df(3)kars1 ryG0JkargDf ( 3 ) ~ yThis ~ ~ extract . possesses little or no X D H cross-reacting material, but is able to remove the extraneous reactivity of the serum. The Ouchterlony experiments also demonstrate that this serum is a competent anti-Drosophila X D H reagent. In addition to precipitating wild-type X D H , it is ( 1) capable of precipitating an enzymatically inactive counterpart to wild-type X D H from maroon-like mutant extracts, (2) capable of prccipitating weakly reactive X D H molecules from lxd extracts, and (3) unable to precipitate material from ry2mutant extracts (See review by FINNERTY 1976). Utilizing this antiserum as a reagent for the quantitative assay of X D H , we carried out experiments whose results confirm our inference that homozygous r y f 4 flies possess more XDI-I molecules than do flies homozygous for our other ry+ isoalleles. Figure 8 summarizes an antiserum titration experiment in which
AND METHODS)
% XDH
ACTIVITY REMAINING
164 132
116
I8
14 r
CONCENTRATION,ANTI- XDH SERUM
FIGURE8.-Titration of unabsorbed anti-XDH serum. % of XDH activity remaining in ry+e (N) and 4
- 4
(H) extracts following incubation in the indicated dilutions of serum.
245
ROSY LOCUS CONTROL ELEMENT
I
15
20
pI of MTRACT
FIGURE 9.-Quantitative analysis of “rocket” electrophoresis experiment. Height of rocket measured from top of well to apex of rocket. Matched extracts of homozygotes run against preabsorbed, anti-XDH serum. Volume of extract in each well is adjusted to 20 pl with buffer.
a set of dilutions of the unabsorbed anti-XDH serum is tested for ability to remove XDH activity from matched extracts of ry+4 and ry+*.The titration curves are very different for the two preparations with the ry+2extract being inactivated at an antiserum dilution that is incapable of removing more than 50% of the XDH activity from r y f 4 . Figure 9 summarizes the results of an immunoelectrophoresis experiment utilizing the method of quantitative “rocket electrophoresis’’ (LAURELL 1966; WEEKE 1973) to compare the relative number of molecules of XDH in matched extracts of r y f 4 ,ry+jj and ry+$homozygotes. In this experiment, several concentrations of ry+4 extract are run, and their resultant rocket heights describe a straight line relationship. Relative number of molecules of XDH in ry+I1 and ry+* preparations are extrapolated as indicated in the Figure. Clearly, the ry+4 extract possesses more molecules of XDH than do the other tested lines. Genetic fine structure localization. I n a previous section, experiments were described which localized the genetic basis for the high level of XDH activity associated with the ryt4-bearingchromosome to the rosy locus itself. Let us now turn to two groups of large-scale fine-structure experiments which further define this localization. Table 2 presents the results of experiments involving null enzyme mutants of the ry+4 isoallele tested against a standard series of mutants of the ry+O allele. The reader will note that from just these experiments (GELBART et al. 1974; GELBART, MCCARRON and CHOVNICK1976), recombination data provided for the localization of the null enzyme mutant sites ry4OZand ry406, as well as for the localization of the electrophoretic difference between the ry+O and ry+4 isoalleles. These results are summarized in the maps of Figure 2, with
A . CHOVNICK
et al.
TABLE 2 Number and classes of r y + chromosomes recovered from progeny of crosses of the indicated females to tester males of the genotype Dfd Df (3)kar31 rycO/kar* Df (3)ryiS
Zygotes were reared on purine-supplemented medium, permitting only rare r y f progeny to survive. Crossovers
+ + +
kar ry+ 126+
Feinale parent
+ + +
13[I102N]
,Y402 __-____ kar ryhl 126
,Y402 karsry5 126
0
,Y405 kar2ry5 126
0
kart rp+ 126
0
0
15[1.00 HI
Conv. ryhoo Xar+ ry+ 126+
8[1.02 HI
0
3[1.02 HI
0'
+
ry406 picG281
kar ry23
+
+ 126
0
2[l.OOH]
2[1.02H]'
Conv. rys
---
Zygotes sampled
kar rp+ 126
( X 10-e)
6[1.00N] 2 [ 1.02 NI
1.11
1[1.00N]
1.34
4[1.00N]
0.8
2[1.00N]
0.57
7[1.00N] I [1.00 HI
1.21
* At the time of these experiments, the ry406 bearing chromosome was lethal when homozygous, presumably due to one or more unknown recessive lethal mutations resulting from the same mutagenesis that yielded ry506. Subsequent analysis revealed the presence of a lethal allele of piccolo, picQasl,which is located immediately to the right of rosy (see SCHALET, KERNACHAN and CHOVNICK1964, and Figure 1). The indicated exceptional classes carry picG231, and must be reduced in number by virtue of the fact that one of the paternal tester chromosomes, kar2 Df(3)ryTS is deficient for the pic locus as well.
the electrophoretic site, e408, located at the right end of the map. I n the earlier presectations of these data, attention was focused upon the boundaries of the XDH structural element, and all consideration of variation in level of activity was omitted to simplify the presentation. Here, we offer the total data. Consider the first row of Table 2, which summarizes test of ryAoaagainst ryA1, a site located near the right end of the XDH structural element. With respect to the flanking markers, kar and 126, three classes of ryf recombinant chromosomes were recovered. The mobility and level of activity (H = High; N = Normal) associated with each ry+ recombinant allele was determined in subsequent electrophoresis and fluorescence assay experiments. Thirteen flanking marker crossovers were recovered, all kar l y + 126+, indicating that ry402 lies well to the left of ryA1.Moreover, these were recombinant for electrophoretic mobility and level of XDH activity. All showed the mobility of the r y + : parental allele (1.02) with the level of activity of the ry+O allele (N) . From this observation, we are able to infer that the genetic basis for the difference in mobility between r y f o and ry+4 (e408) lies to the right of all crossover points between ry402 and ryhl, while the basis for the difference in activity level falls to the left of all crossover points. Note that in all experiments described in Tables 2 and 3, no intermediate or nonparental classes with respect to XDH activity level were recovered. Additionally,
24 7
ROSY LOCUS CONTROL E L E M E N T
TABLE 3 Number and classes of ry+ chromosomes recovered from progeny of crosses of the indicated females to tester males of the genotype, Tp ( 3 )MKRS, M ( 3 )S34 kar ry2 %/In (3R)PI,,kar r y 4 l Ubx e4 Zygotes were reared on purine-supplemented medium, permitting only rare r y + progeny to survive. Conv. ry4OO
Conv. ry5
kar ry+ pic+
Crossovers kar+ ry+ pic
kar ryf pic
kart ryt pic+
1[1.03 HI
0
2[1.05 H] 1[1.02 HI
1C1.03 NI
1.99
4r1.00 HI
0
1cl.02 HI
5[1.ON] 1 [1.OO H]
0.98
--.___--
Female parent
kar2 ry4ofi picGz31
+
ryps214
+
kart ry406 pic0231
___ ______
+
.PO6
+
Zygotes sampled (X
ry+ recombinants exhibiting parental flanking markers (i.e., conversions) were recovered. Thus, eight conversions of the ryhoZallele were recovered, and all exhibited parental mobility and level of XDH activity. I n contrast, the conversions of the ry4’ allele exhibit some co-conversion with e408 reflecting proximity of these markers (Figure 2). However, each conversion of ry4’ exhibits the parental (Normal) level of activity. Obviously, the site giving rise to the increased XDH activity is different and separable from e408. Let us now designate this carrying i409H,and ry+O carrying i409N. site as i409,with The data of Row 2, Table 2 summarize the results of recombination test of ry402 against ry5,an XDH- mutant site located near the left end of the structural element. These data complement the results presented in Row 1 and serve only to locate the mutant ry402 quite close to ry5. Turning to the third row of Table 2, we see a pattern of results which reinforces the inferences drawn from the first cross. The mutant ry5, located near the left end of the XDH structural element, is tested against ry405. Fifteen flanking marker crossovers serve to locate ry405 to the right of ry5. All of these crossovers were recombinant for electrophoretic mobility and level of XDH activity. All exhibited the mobility of the ry+O parental allele (1.OO) with level of activity of the ry+4 allele (H). Again, we infer that the genetic basis for the difference in mobility between ry+O and ry+4 (e408)lies to the right of all crossover points between ry5 and ry405,while the basis for the difference in activity level (209) fall to the left of all crossover points. The experiments of rows 4 and 5 focus upon the left end of the rosy structural element by selecting for ry+ recombinants involving mutant sites located on that side of the map (Figure 2). Following the argument used in analysis of the data of rows 1 and 3, the crossover classes emphatically point to the left end of the rosy locus as the location of i409, the genetic basis for level of XDH activity, while e408, the mobility difference between the ry+Qand r y + 4alleles is located at the right end of the map. ?‘he remaining two experiments of this series are summarized in Table 3. Again, we concentrate upon the left end of the XDH structural element with the mutant ry4O6, a member of the ry4O0 series of mutants (Figure 2), tested
248
A . CHOVNICK
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against iyPSzi4and ry606.As with Table 2, part of these data are presented elsewhere (GELBART, MCCARRON and CHOVNICK 1976). The first cross of Table 3 describes the results of recombination in heterozygotes, ry4°6JryPsg14. The reader will recall ( GELBART et al. 1974) that the electrophoretic difference between iy+" and ry+O is resident in a site, e217, located in the left portion of the XDH structural element, while the electrophoretic difference between ry+4 and ry+O relates to a site, e408, h a t e d at the right end of the map. Crosses between derivatives of the iy+4 and r y f 2isoalleles differ at both sites. Thus, the cross of Row 1, Table 3 involves assay of recombination in heterozygous females of the genotype kaP 4- 406 e217S e408F picGgss1 - , where the relative positions of all rosy -I- ps214 -I- e217F e408S f locus sites are seen from Figure 2. The single flanking marker crossover recovered in this cross (Table 3, Row 1) carried i409H, and exhibited the mobility of the ry+" parental isoallele (XDH1.03),and thus must carry e217F and e408S. Of interest is the proximity of ry406 to the e217 site (Figure 2). This fact is reflected in the observation that two of the three conversions of ry4O6 exhibit co-conversion f o r the adjacent electrophoretic site as well. This results in a ry+ allele carrying i409H, e217F, e408F, which produces an XDH1.05with the level of activity of the iy+4 parent allele. The second cross of Table 3 involves the XDH-, allele complementing mutant, ry606,which is the leftmost known unambiguous structural mutant (Figure 2). Data presented elsewhere (GELBART 1976) argues that there is a single electrophoretic site difference responsible for the mobility difference between ry+4 and r ~ +This ~ . site is located on the right side of the structural element, and may = e408S). Thus, in the ry4°6/iy606 cross well be e408 (ry+4 = e408F: (Table 3, Row 2), the single electrophoretic site difference between these alleles serves merely as a marker €or the right side of the XDH structural element. Following the previous logic, all of the i y + recombinants associated with flanking marker exchange exhibited recombination for the i409 site and e408 (Table 3, Row 2). All exhibited XDH1.OOwith the high level of activity associated with i409H. All of the crossovers of Table 3 place the genetic basis for the observed difference in level of XDH activity to the left of all crossover points. Pooling the data of Tables 2 ar?d 3, the crossovers place i409 to the left of all observed exchange points within the XDH structural element. An additional feature of these data emerges from examination of the results involving i y Z s (Table 2) and rySo6(Table 3 ) . These mutants serve to mark the present left border of the XDH structural element. The crossover data would place 2 0 9 either to the left of both these mutants, or very close to the right, presumably just inside the structural element (Figure 2). If the latter position were correct, conversions of i y Z s ,iy606,iy4O2, i y 4 0 6 and ry5 would often be accompanied by co-conversion for the i409 site (CHOVNICK et al. 1974). However, both ryZ3and ry606show a low frequency of co-conversion with i409, while iy402, ry406 and ry5 have failed to exhibit co-conversion with i409. Taken together, interpretation of both crossover and co-conversion data would place i409 well to the left of both i y Z 3and ry606,and hence, beyond the left border of the XDH structural element.
249
ROSY LOCUS CONTROL E L E M E N T
XDH STRUCTURAL ELEMENT A06
!(3)S12
i409
r
402
FIGURE 10.-Map location of i409 site relative to 2(3)S12 and the XDH structural element.
Confirmation of the localization of the i409 site. Figure 10 summarizes the previous section’s localization of 2 0 9 relative to the left end of the XDH structural element. The mutants ry4OSand ry406 are represented as distinctive sites just inside that border. Although no effort has been made to separate those rosy mutants from each other, a pattern of comparative recombination data consistently places 402 just to the left of 406 (Figure 2). Note also, the relative position of 1(3)S12 (Figure 1). This heretolfore unreported mutant was recovered from an X-ray treatment of a karPry+ll chromosome. It was positioned between mes (SCHALET, KERNAGHANand CHOVICK1964) and ry by complementation and recombination tests (SCHALET, Personal Communication). Preliminary experiments revealed that the ka? 1(3)S12 ry+ll chromosome is associated with normal levels of XDH1.02 activity, ar?d is thus designated kar3 1(3)S12 i409N ~ y + ” . Despite its proximity to the rosy locus (< 0.01 map units in preliminary experiments), there is no evidence to consider 1(3)S12 as an alteration within the control element of the rosy locus. Rather, we believe it to be a site mutant in the very next genetic unit to the left of rosy. Figure 11 presents the genotypes of two classes of females which were crossed
+
M(3)S34 kar2 1 ( 3 ) S 1 2 i409N
(A)
+
+
+
i409H ry406 p i c
+
M(3)S34 kar2 1 ( 3 ) S 1 2 i409N (B)
+
+
+
+
i409H r y 4 0 2
Sb
females
G2 3 1
+
Sb
+
+
females
X
c u kar Df ( 3 ) r ~ [1(3)S12~ ~
r y - pic-]Sb+
Ubx e 4
males Tp
( 3 ) MKRS, M(3)S34
kar
ry2
Sb
FIGURE11.-Parents of large-scale selective recombination experiments. Resultant ry+ crossover chromosomes are described in Table 4.
250
A . CHOVNICK
et al.
to the indicated males in large scale matings carried out on purine-supplemented medium. I n the absence of crossing over in the region from M ( 3 ) S 3 4 S b , all zygote classes will die due to the homozygous or hemizygous lethality of M(3)S34,1(3)S12, ry, pic, and Sb either singly o r in combination. Of particular interest are single crossovers between 1(3)S12 and r y which produce a kar+ lS12+ r y + pic+ Sb recombination product. Zygotes receiving this product, as well as a cu kar Df(3)ryS7Sb+ Ubx e5 paternal chromosome, survive and will be kar+ + y + Sb Ubx in phenotype. What about the 209 site associated with such crossovers? I n the absence of the data of the previous section, three possibilities are apparent: (1) If i409 were inside the XDH structural element and located either to the right of ry402 and/or ry406 or just to the left of one o r both rosy mutants, then all (or almost all) of these crossovers would be i409N. (2) If i409 were located to the left of the structural element border, and somewhere between 1(3)S12 and both ryGoz and ry606, then the exchange classes should include ry+ chromosomes with i409H and i409N in proportions relative to the location of i409. (3) Finally, if i409 were, in fact, located to the left of 1(3)S12 or possibly just to the right, then all (or almost all) of this exchange class should be i409H. The pertinent results of the crosses outlined in Figure 11 are summarized in Table 4. A total of 16 crossover progeny were recovered, which upon further test were shown to fall into two classes with respect to the maternally derived exchange chromosome. These chromosomes were cytologically normal. The results are entirely consistent with the second alternative described above, and thus permit the conclusion that i409 is located well to the left of the structural element border. The relative proportions of the i409H and i409N classes permit location of i409 somewhat closer to 1(3)S12 than to the left border of the XDH structural element (Figure 10). The results of these crosses, therefore, provide a very strong confirmation of the localization of i409 inferred from the recombination data presented in the previous section. TABLE 4 Number and classes of ry f crossover chromosomes recovered from progeny of crosses described in Figure 11 Zygotes were reared on purine-supplementedmedium, permitting only rare r y + progeny to survive. Crossoversf
Cross
kar+ 1(3)S12+ i409H ry+ pic+ Sb
A B'
3
ZYgOteS
kar+ l ( 3 JS12-i i409N ry+ pic+ Sb
7
4 2
sampled x 10.0)
(
0.36 0.82
* 1 kar 2(3)5'12+ i409N r y + pic+ Sb chromosome was recovered and classified as a conversion, l(3)SlZ + 1(3)S12+. 16(4) x IO2 f Map distance, l(3)SlZ-ry = -___ = 0.0054 1.18 x 106 -f Map distance, i409-ry
lO(4)
x x
102
= ___--1.18
IO6
- 0.0034 -
ROSY I.OCUS CONTIWI. ICI.IXMI.NT
25 1
Of course. several additional distinguishable classes of survivors were recovered. These include double crossovers, a variety of triploids. aneuploids. and rare tandem duplicr\tions resulting from unequal exchanges in the rosv region. All of these classes survive by virtue of the lethals being "covered" in the duplicated region or rcmoved by Crossovers. .Might i409H rrprcscnt n tnndem duplication of tlic XDH structural clcment ? Ample precedence for such consideration exists in the several well-known cases W GREEN1959. 1963). On this notion, in Drosophila (Sec, for example. ~ ~ . A F I 1967; the r y f ' isoallclc would be considered to possess two functional XDH structural elements in tandem, presumably resulting from a n unequal exchange event. Such a model is precluded on several counts: ( I ) Cytological examination of polytene chromosomes revcals no such tandem duplication. (2) Tanclem duplications arc characterized by instability in homozygotes due to increased incidence of unequal exchange events. The ry+z stock has been quite stable. (3) Fine structure recombination expcrimcnts involving tests of ry:')''series mutants against other XDH- mutants hare been characterized by regular exchange events. and the complete absence of unequal crossing over. ( 4 ) The r y f ' allcle is associated with a single XDH electrophoretic band (Figure 3) of the mobility class, XDH'."' (Table 1). XDH is a homodimcr. and the presence of two electrophoretically distinct structural elements will produce individuals possessing three XDH moieties (Figure 12). The tandem duplication model then requires that the r y + J allele possess two XDH structural elements whose peptide products are indistinguishable, and of the mobility class, XDH'.O?.
A
B
FIGURE 12.-XDH rlectropherogram indicating the rrlatiw amounts of XDH"."", XDH"."G n n d XDH'."?prrsrnt in mntchrd rxtrncts of flies of the genotypes (A) i40YN ry+'/i409N ry+" ancl (R) i409H r y + J / i 4 0 9 N r y + l O .
252
A. CHOVNICK
et al.
Thus, i409H should be associated with an XDH1.02. On this point, the model fails. In all experiments which recombine i409H with other electrophoretically distinct structural elements, there is no evidence of the production of an XDH1.02 moiety. We see from all of the arguments presented above that i409H may not be considered to mark a duplication of the XDH structural element. Functional relationship between i409 and the XDH structural element. It is apppropriate at this point to consider that i409 marks a genetic element that serves, in one way or another, to regulate XDH. On the basis of evidence thus far presented, a broad array of regulatory roles are possible, and further specification would be premature. However, by a simple experiment, we are able to describe a key feature of this regulatory function. Under one class of regulatory roles, dominance-recessiveness, or incomplete dominance in heterozygotes of i409 may obtain. In still another class of roles, the regulatory function of a specific i409 element would be restricted to the specific XDH structural element adjoining it on the chromosome. We shall refer to this as a “cis-acting” reaplator. Consider the heterozygote i409N ry+’/i409N ry+lz. Such individuals should approximately equal quantities of the ry+I and ry+I2peptides. Assume further that random union of monomers takes place to yield active XDH dimers. Electrophoresis of extracts of such heterozygotes should reveal the presence of three XDH moieties, XDH0.Oo(ry+ldenzyme) , XDHo.QG (hybrid dimer) and XDH1.02 (ry+’ enzyme) in a ratio of 1:2: 1. Figure 12 is a photograph of an electropherogram which is printed as a negative in order to enhance the contrast between XDH banded regions and the acrylamide slab gel. Figure 12A presents the XDH molecular composition of extracts of i409N ry+’/i409N ry+Iz, and is entirely consistent with the expectation and its underlying assumptions. Consider next the XDH composition of an extract of the genotype i409H r ~ + ~ / i409N ry+’%(Figure 12B). The XDH structural element of the ry+4 allele is associated with an XDH possessing a mobility identical to that of ry+’ (XDH1.02). Thus, in terms of XDH moieties, this genotype should possess the same three mobility classes as seen in Figure l2A. However, this genotype differs from the former in that it is heterozygous, i409H/i409N. Under one class of regulatory models, dominance of one or the other form of these alternatives, or even incomplete dominance, may be expected. In such event, we should find the same 1:2: 1 ratio of the three mobility classes. However, all would be produced in high, standard or intermediate quantities depending upon the specific dominance relationship that obtains. Alternatively. each i409 element may operate to regulate only the specific XDH structural element (or product thereof) located adjacent to it on the same chromosome (i.e., a “cis-acting” regulator). Thus, in the heterozygote, i409H ry+4/i409N ry+‘z, this model would predict that greater quantities of the ry+4 polypeptide than ry+lapeptide would be produced. Random union of monomers would then result in increased quantities of XDH1.02 (ry+4 enzyme) and XDHo.96(hybrid dimer) at the expense of XDHo.90(ry+’$ enzyme). Examination of Figure 12B reveals that just such result obtains, and we are led to conclude that i409 operates as a “cis-acting” regulator.
ROSY LOCUS CONTROL ELEMENT
253
DISCUSSION
We have shown that i409H and i409N represent alternative forms of a genetic element associated with variation in number of molecules of XDH/fly. We have been unable to relate this variation to alteration in the structure of the XDH polypeptide or to a tandem duplication of the XDH structural element. Finestructure recombination studies locate this element very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Moreover, these experiments have produced exchange products (Tables 2 and 3) that carry new combinations of the 209 element with electrophoretically distinct structural elements. While previously (Table 1) our array of wild-type stocks carried i409H only in combination with an XDH structural element whose product belonged to the mobility class, XDHl 02, we now possess i409H with structural elements producing XDH1.OO,XDH1 O3 and XDH1.05molieties as well. Indeed, we believe that 2 0 9 is completely separable from the XDH structural element, and that the construction of every possible combination of XDH structural alleles and 2 0 9 alternatives would be a simple genetic exercise. Taken together, these observations provide a compelling argument in favor of a regulatory role for i409.Moreover, evidence is presented that limits our consideration of the specific function of the 209 segment to that of a “&-acting” regulator. We may eliminate several broad categories of regulatory roles which are characterized by “trans-acting” regulation and which should exhibit dominancerecessiveness or intermediate phenotypes in heterozygotes. Thus, we may eliminate all regulatory models which associate i409 with the synthesis of a negative or positive acting diffusible regulatory molecule (GILBERTand MULLER-HILL 1970; DICKSON et al. 1975). GOLDBERGER (1974) has recently reviewed the evidence for autogenous regulation, largely from prokaryote studies, and has suggested that similar cases may be found in higher organisms. The possibility that the XDH peptide serves a second function as an autogenous regulatory macromolecule has not been explored. Nonetheless, we note that the “cis-acting” nature of the i409 element also precludes the possibility that i409H and i409N are structure variants in a region of the XDH peptide that serves as an autogenous regulator. Rather, we are drawn to the possibility that i409 marks the 5t controll element of the rosy locus. Variants of this element would possess alterations in DNA sequences concerned with functions generally ascribed to such elements (see Introduction). The size of the control element may be estimated from the recombination data of Table 4. We believe that these data to produce underestimates by virtue of the fact that all of the relevant crossover individuals are Ubz in phenotype. Such individuals suffer an increased likelihood of “drowning” in the food shortly after eclosion, and thus not being scored. Ignoring this problem, we take the map distance estimated from the crossovers in the interval 209-ry (Table 4) as a minimap units), mal estimate of the size of the XDH control element (3.4 x while the crossovers in the interval Z(3)SIZ-ry provide a maximum estimate (5.4 x map units). In the accompanying report (GELBART, MCCARRON and CHOVNICK 1976), we have estimated the size of the XDH structural element at
254
A. CHOVNICK
et al.
5.0 X map units and related this to a DNA length of 3,000 base pairs from the XDH peptide size (160,000 daltons) . From this, we may estimate the size of the i409 segment to range from a minimum of 2,040 base pairs [3.4 ( 3 x IO3)/ 5.01 to a maximum of 3,240 base pairs C5.4 (3 X 103)/5.0]. At a maximum, the 2 0 9 control element is no larger than the XDH structural element. We recognize that the possibility of control sequences at the other end of the rosy locus remains to be explored. Nevertheless, we note that the present results lend little support to solutions of the chromomere paradox that postulate control elements an order of magnitude larger than their structural components (See review, BEERMAN 1972). We wish to acknowledge the skilled technical assistance of FLORA GRABOWSKA, HARRY LEVINE and FLORENCE JOHNSTON. LITERATURE CITED
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