INTRACISTRONIC MAPPING
O F
ELECTROPHORETICSITES
INDROSOPHILA MELANOGASTER: FIDELITY
O F
INFORMATIONTRANSFER
BY
GENE CONVERSION1MARGARET McCARRON, WILLIAM GELBART A N D ARTHUR CHOVNICK Genetics and Cell Biology Section, Biological Sciences Group, The University
of Connecticut, Storrs, Connecticut 06268
Manuscript received July 5, 1973 Revised copy received October 23, 1973
ABSTRACT
A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophih mezanogaster. A number of wild-type isoalleles of the rosy locus have been iso- lated which are associated with the production of electrophoretically distin- guishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a non- selective marker in the cross. Additionally, a large-scale recombination experi- ment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of cross- over and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic char- acter, this observation argues against a special mutagenesis hypothesis to ex- plain conversions resulting from allele recombination studies.
ALLELE recombination studies in Drosophila have been restricted heretofore to experiments utilizing mutants associated with visible phenotypic effects and/or enzyme inactivation. The present report describes a convenient method for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster which may be useful for several areas of investigation.
The use of electrophoretic variants as markers facilitates investigation of two kinds of questions bearing upon the fidelity of the conversion process. The first question relates specifically to the two sites of heterozygosity in a n individual heterozygous for two different mutant alleles. One point that has not been estab- lished questions whether, for those sites of heterozygosity, the conversion process in Drosophila is a conservative one, utilizing the preexisting genetic input, or whether conversion generates novel information at those sites. This question may be approached by searching for isoallelic differences among the wild-type excep- tions produced in large-scale intracistronic recombination experiments. If the mutants used in this experiment were induced on the same isoallelic background,
This investigation was supported by a research grant, GM-09886, from the Public Health Service
290 M. MCCARRON, W. GELBART A N D A. CHOVNICK
wild-type exceptions of new isoallelic classes would indicate a lack of fidelity in the conversion process. A search of this nature simultaneously questions fidelity of information transfer involving a segment of DNA far greater than the two sites of heterozygosity discussed above, possibly involving most of the DNA
sequence that codes for the polypeptide under investigation.
With the development of suitable genetic systems for detailed analysis of con- version in Drosophila, a compelling case can be made that relates conversioll to the process of linked exchange (CHOVNICK, BALLANTYNE and
HOLM
1971). An alternative interpretation relates conversion to the enigmatic case of the “mut- able” reddish-alpha allele in DrosophiZa uirilis (DEMEREC 1928). This viewpoint suggests that conversion, as seen in Drosophila, really is a special mutagenesis that only occurs in heterozygous females (HEXTER 1963). By attempting to uncover differences in the wild-type alleles generated by conversion as compared to crossover products recovered from females heterozygous for two mutant alleles of a given cistron, some limit may be placed on the special mutagenesis hypothe- sis. Traditionally, the crossovers are inferred to represent exchanges of separable parts of the cistron, thereby restoring the original wild-type allele. If convertants arise by a mutation-like event, then one might anticipate that this class would include a n array of wild-type isoalleles which would be distinguished from the original wild-type allele through a n examination and comparison of the function of their respective gene products. Just such investigations have failed repeatedly to uncover differences between crossover and convertant products involving the garnet ( CHOVNICK 1958),
maroon-like (FINNERTY, DUCK and CHOVNICK 1970) and rosy (CHOVNICK et aZl970) loci. However, in all of these studies, the power of the methods used to distinguish between slight differences in wild-type iso- alleles remained as a serious question. The present report describes an experiment which explores this point through electrophoretic characterization of crossover and convertant products.MATERIALS A N D METHODS
The genetic system: The msy cistron in Drosophila melanogaster (ry:3-52.00) is a solitary unit concerned with the enzyme xanthine dehydrogenase (XDH), located within an intensely mapped short region of the right arm of chromosome 3 (salivary section 87D). Strong genetic and biochemical evidence argues that rosy is a structural gene for XDH (GRELL 1962; YEN and GLASSMAN 1965). Mutations restricted t o the rosy cistron are homozygous viable, and fall into two groups: (1) a class of “wild-type isoalleles” which produce electrophoretic variants of the enzyme (YEN and GLASSMAN 1965) ; and (2) a large group of mutants which are enzymatically inactive and exhibit a brownish mutant eye color phenotype resulting from a reduction in the red (drosopterin) pigments (CHOVNICK et al. 1964). Study of the latter class of mutants failed to find any evidence of allele complementation (SCHALET, KERNAGHAN and CHOVNICK 1964), and investigation of allele recombination heretofore has been restricted to this class of mutants. Figure 1 presents a map of the centromere-proximal region of the right arm of chromosome 3 of Drosophila melunogaster indicating the location of rosy, the centromere, and other markers used
in the study (LINDSLEY and GRELL 1968). In addition, Figure 1 presents a summary map of
CONVERSION IN DROSOPHILA 29 1
M(3 )s34 r i Cfd cu k a r ry & ( 3 ) 2 6 Sb UbX
A
-
hk.3 47.0 47.5
1
9.0 10-3 Map U n i t sI
FIGURE 1.-A genetic map of the rosy region of chromosome 3. The map of various mutants used in this study is indicated, and the genetic fine structure of the rosy cistron is summarized.
for the amino acid sequence of the XDH polypeptide (GELBART, MCCARRON, PANDEY and CHOV-
NICK, manuscript in preparation).
Selectiue system matings: The experiments described in the present report involve two series of matings of rosy mutant heterozygotes females to tester males as indicated in Figure 2. Progeny were reared on purine-supplemented medium which effectively kills all zygotes lacking XDH activity. The experimental protocol is identical to that reported earlier (CHOVNICK 1973). Unless otherwise noted, all mutants and rearrangements used i n the study are described in LINDSLEY and GRELL (1968). Tp(3)MKRS is a complex multiple break arrangement which is a n effec- tive balancer for the middle of the third chromosome including the region from M(3)S34 to Sb (Figure 1) (LEFEVRE, personal communication, 1970).
Genetic tests of exceptional progeny: Surviving individuals of the selective system crosses (Figure 2), invariably ry+Sb or ry+Sb+ in phenotype, are mated t o flies of the genotype, Tp(3)MKRS, M(3)S34 kar rys Sb / karz, Df(3)ry75. Eye color phenotypes of the progeny of each cross permit confirmation of the original diagnosis of the genetic composition of each ex- ceptional chromosome with respect to kar and ry. Additionally, two further tests of each excep-
Male Parent Ixperiment Female Parent,
I In(3L3):-269, k a r ry" L26
+ ry5 i
kar', D f ( 3 ) r ~ ~ ~ X
In(3LR)MKRS. M(3)S34 kar ry2 Sb
8
I1 tu l c a r r y 126
+ i r$ +
292 M. MCCARRON, W. GELBART A N D A. C H O V N I C K
tion are carried out: (A) The ry+Sb F, progeny carry the exceptional r y + bearing chromosome over Tp(3)MKRS. Such flies are mated to ry5 E26 / In(3LR), cu kar UbxA, and their prDgeny permits classification of the exceptional r y + chromosome with respect to 126 and cu (where ap- plicable). (B) The r y + S b + F, progeny carry the exceptional r y + allele on one chromosome, and are heterozygous for Df(3)ry75 which is missing the region of chromoosme 3 from salivary band 87D2 to 87D14, which includes the rosy cistron at 87D8-12 (LEFEVRE personal communica- tion and 1971). These flies, each with a single exceptional r y f allele, serve as a source of XDH
for electrophoretic characterization.
Mutagenesis experiments: New rosy mutants, ryloz, rylo9, ry106 and ryllo were recovered from X-ray mutagenesis experiments involving treatment (3,000 rads from a Cs-137 source) of aged virgin males whose third chromosomes were homozygous for a ry+ isoallele designated as r y + l (Table 1). Such males were mated to a n excess of aged virgin females of the genotype,
h r 2 , Df(3)ry75 / Tp(3)MKRS, M(3)S34 kar rys Sb. After three days, the treated males were provided with a fresh group of virgin females for a second three-day brood. New rosy mutants were recovered as rare phenotypically rosy F, individuals carrying the newly induced mutant over either karl, Df(3)ry75 or the MKRS chromosome. Subsequent crosses: (1) provided con- firmation of each new mutant; (2) tested that each new mutant was not associated with a de- ficiency extending into the rosy cistron; (3) established each mutant in a homozygous stock.
Enzyme preparation and electrophoresis: With two minor modifications, the procedures used are identical to those described by YEN and GLASSMAN (1956). Principal differences are: the gel consists of 3.8% acrylamide, 0.2% N,N'-methylenebisacrylamide, 10-4 M fl-diphospho- pyridine nucleotide (NAD) and 10-4 M dithiothreitol; the assay mixture contains M
phenazine methosulfate, 2.5
x
10-4 M p-nitroblue tetrazolium, 3.0x
10-4 NAD and 4.0x
M hypoxanthine in a pH 8.5 buffer of 0.2 M tris (hydroxymethyl) aminomethane-HCl and 0.001M disodium ethylenediaminetetraacetate. With t h i s solution, gels are allowed to develop in the dark at room temperature for a t least one hour. Electrophoretic variants of XDH are illustrated i n Figure 3A, and the results of testing unknown ry+ alleles against standards is illustrated in Figure 3B.
RESULTS
As previously noted (YEN and GLASSMAN 1965; CHARLE~WORTH and CHARLES-
WORTH 1973), electrophoretic variants of
XDH,
which map to the rosy cistron, are readily isolated from laboratory stocks and natural populations of DrosophilaTABLE 1
Wild-type isoalleles of the rosy cistron of Drosophila melanogaster, and mutant derivatives
Wild-type
ls~allele Source Mutants
5 8 41
r y
,
1-9 , r yw
+Z Oregon-R Stock r y Z o o s e r i e srY
+z
Oregon-R-C S t r a i n zyZoo s e r i e sry +3 South Amherst wild type q 3 0 0 s e r i e s
+4 Pacific Stock w4O0 s e r i e s
ry c5 Z ; ~ y + laboratory stock ry5O0 s e r i e s
+6 Hikone Stock r y 6 O 0 s e r i e s
and others
+
cu kar ry
f O
YY
collected by P. T. Ives
ry
CONVERSION IN DROSOPHILA 293
FIGURE 3.-(A) Acrylamide gel electrophoretic variants of xanthine dehydrogenase. (a) r y + 5 (b) r y + 4 (c) ry+* (d) r y + O (e) y+J. (B) Acrylamide test gel of three ry+ exceptions from experiment 11. (a)
e
convertant (b) r y + o standard (c) r y 1 O J convertant (d) r y + l standard (e) crossover between f l and r y 1 O J .melanogaster. Stable lines exhibiting single bands of
XDH
of uniform character upon electrophoresis may be established by selection of a single third chromo- some overTp(3)MKRS,
and then, in most cases, establishing a homozygous line.A number of such lines have been isolated, and those presently under investi- gation are listed in Table 1. Figure 3A illustrates the range of electrophoretic variation. The r y + O allele produces our slowest migrating
XDH,
ry+’ and ry+*produce distinct but intermediate XDHs, while produces a fast
XDH.
Thery+‘ allele produces an XDH which migrates at a rate indistinguishable from ry+l, produces a fast XDH indistinguishable from r ~ + ~ , and ry+8 produces a slow
XDH
indistinguishable from r y + O . The ry+‘, ry+6 and ry+6 designations reflect their isolation from sources quite distinct from ry+I, ry+s and ry+O respectively. We are able to confirm the observation ofYEN
andGLASSMAN
(1965) that mixtures of the various combinations of slow, intermediate and fast isozymes are separable upon electrophoresis. Moreover, isoallele heterozygotes of the slowest and fastest types yield enzyme preparations which, upon electro- phoresis, exhibit three clear bands (slow, fast, and an intermediate “hybrid” band), suggesting that the active XDH molecule is a dimer. The present report describes experiments which utilize the ry+O and ry+’ alleles and null enzyme rosy mutants recovered from X-ray treatment of these wild-type isoalleles.
294 M. M C C A R R O N , W. G E L B A R T A N D A. C H O V N I C K
same mutagenesis yielded a third chromosome multiple break rearrangement,
c u kar, UbxA (LINDSLEY and GRELL 1968), which involves some 10 breaks
including a transposition of a piece of chromosome 4 (E. B. LEWIS, personal communication, 1964), and which has served as a very useful balancer chromo- some for the rosy region. The ry+ allele present on this rearrangement chromo- some produces the slowest moving XDH (Table 1) in our collection, and this allele has been designated as ry+O.
Experiment I: Large scale recombination test matings of ry41/ry5 mutant
heterozygous females (Figure 2) were carried out, and their progeny reared on purine enriched selective medium.
It
will be noted that the markers kar ry4I 126are carried within the inverted region of a large pericentric inversion In(3LR)C-
269, and the cross is heterozygous for this rearrangement. From a n estimated
1.25 X lo6 zygotes sampled, a total of 62 ry+ progeny were recovered as single flies randomly distributed among the matings. Analysis of their third chromo- somes (MATERIALS A N D METHODS) revealed that each of the ry+ bearing chromo-
somes was of maternal origin, and, as in all past experiments with this system, the ry+ exceptional chromosmes fell into three categories: (1) a single class of 16 were associated with crossing over for the flanking markers kar and 126 con- sistent with the prior localization of ry5 to the left of ry41 on a standard chromo- some order map (Figure 1 ) ; (2) a class of 35 carrying the parental recessive markers kar and 126, and thus classified as conversions of ry41; ( 3 ) a class of 11 carrying the parental markers kar+ and 126+, and thus classified as ry5 con- vertants. All of the 62 r y + exceptions were characterized by electrophoretic
analysis of their XDHs produced in hemizygotes carrying Df(3)ryT5 (MATERIALS
AND METHODS). Experiments were repeated at least once in all cases, and all of
the ry+ exceptions behaved as ry+O alleles.
Experiment II: Several large-scale purine selection system crosses were carried
out in standard order chromosome homozygotes (Figure 2, Experiment
11)
involving recombination tests between the mutant, rys, which is located in a relatively central position on the existing rosy cistron map (Figure 1 ),
and newly induced rosy mutants of the 100 series (Table 1).Exceptional ry+ progeny were recovered from all of the matings, and the analysis of their third chromosomes revealed that all of the r y f bearing chromo- somes arose as random meiotic products of maternal origin (Table 2). In our past experience, now numbering some 103 ry+ exceptions analyzed, mutant heteroallele tests such as those described herein yield a single class of flanking marker crossover exceptions, and these provide f o r consistent site mapping. Thus, despite the small numbers of ry+ exceptions recovered from each of the crosses described in Table 2, we are confident in the relative positions of the rosy mutant sites indicated by the flanking marker crossover classes, and summarized at the bottom of Table 2.
Localization of electrophoretic site: I n addition to the lesions which gave rise
CONVERSION I N DROSOPHILA
295
TABLE 2Analysis of ry+ exceptional chromosomes recouered from crosses of the indicated females to males of the genotype, karz, Df ( 3 ) ry75/In (3LR) MKRS, M(3) S34 kar r y z Sb
Exceptional v + chrOmDsOmeS
T o t a l praqeny
~ ti
Female parent PI + U 9 s e i io 7 d " n %+ ' 5
cu k a r r y 8 226
+ + ry102 + 2
cu ka r r y 8 226
+ + ry103 +
8
cu kar r y 226
+ + r3'O6 +
cu k a r r y 8 126
+ + ,yllO +
1
4
1 2 0.90 x l o 6
2 1 0.87 x l o 6
0.60 x l o 6
2 1 2 0.55 x 10'
106
Y4
110
'"Y
,y103 8 102
TY rY '
X-ray treatment of ry+l bearing third chromosomes carried out within the past year. (2) However, the rys mutation was, no doubt, induced on a ry+O back- ground as evidenced by, (a) rys was recovered from a large scale X-my mutation experiment which involved treatment of males of a cu kar ry+ homozygous stock whose ry+ alleles were of unknown electrophoretic character, and indeed, might have been heterogeneous; (b) the Ubx" rearrangement (see earlier dis- cussion) was recovered from this mutagenesis, and it carries a ry+O allele; (c) the
ry5 and ry41 mutants also were recovered from this mutagenesis, and the results
of experiment I are consistent with the notion that both mutants were induced on ry+O bearing chromsomes; (d) the population of ry+ exceptionals recovered in experiment I1 (Table 2) includes both r y f o and ry+l alleles. Additional sup- port for the argument that ry8 was induced on an ry+O allele emerges from an examination of Table 2. All of the ry+ exceptions which carry parental flanking markers indicating their origin as conversions of rys are, in fact, ry+O exceptions, while conversions of the rosy-I00 series mutants are ry+l exceptions.
If one accepts the inference that each of the crosses of Table 2 is heterozygous for at least one site concerned with the electrophoretic difference between ry+O
and ry+I, as well as the sites which lead to loss of XDH activity, then examina-
tion of the electrophoretic character of the ry+ crossover classes provides relative localization of that electrophoretic site. The dotted line of Figure 4A represents the crossover event that gave rise to the ry+ crossover chromosomes recovered from the rys/ry102 test (Table 2, first row)
,
and is consistent with locating ry102296 M. MCCARRON, W. GELBART A N D A. CHOVNICK
cistron
'1
I I
'102
;
+ + + rY
r o s y c i s t r o n
(I
;k1;
I r r1
2; rylo' +_ _ _ _ _ _ _ _ _ - -
_ -
I _ _ _ _ _ _ _ _ _ _ _ _ _ - - -
( D )
FIGURE 4.-(A) Crossover between rys and an rylou series mutant located to the right of zy8. (B) Crossover between ry8 and an ry100 series mutant located to the left of ry*.
in electrophoretic character argues that the electrophoretic site must be to the right of ry8. This conclusion is supported by the electrophoretic character of the
ry+ crossovers recovered from the ry8/ry103 test (Table 2, second row; Figure 4B)
,
as well as the remaining crossover data of Table 2.DISCUSSION
Experiment I was conceived to ascertain two facts. In a recent report (CHOV-
NICK 1973)
,
recombination between rosy mutant alleles was examined in a smallparacentric inversion heterozygote, In(3R)P1,, in which the rosy region is centrally located. In that experiment, classical single crossovers between the rosy mutants were dramatically suppressed, but conversions occurred for all rosy mutant alleles in all heterozygous combinations in the inversion heterozygote. Moreover, the order of magnitude of conversion frequencies seen in the in- version heterozygote did not change from that seen in a prior standard chro- mosome homozygote study (CHOVNICK, BALLANTYNE and HOLM 1971). In view
of the significance of these observations for notions concerning the extent of infor- mation transfer in the presence of rearrangement barriers to crossing over (see
CONVERSION I N DROSOPHILA 297
The second purpose of experiment
I
was to screen for evidence of two kinds of infidelity in the conversion and crossing over processes. Wild-type convertants or recombinants whose XDH moieties migrate at a different rate than does the XDH characteristic of ry+O would constitute such evidence. Since all of the 62 excep- tions were electrophoretically indistinguishable from ry+O, no evidence indicat- ing a lack of fidelity in the conversion and crossing over processes exists. With respect to the two specific sites of heterozygosity in a heterozygote for two mutant alleles, if the conversion process were to generate codons leading to the insertion of novel amino acids, the testing procedure would have identified those conver- sions which created electrophoretically distinct wild-type isoalleles.Consider next the hypothesis that conversion involves a special mutagenesis which takes place as a non-reciprocal event in certain heterozygous females. One might envisage that such special mutation would involve the production of an array of alterations imposed upon the rosy cistron, and only those which restore some enzyme activity would survive the purine selective system. Among these alterations, one might expect to find a series of “second-site” lesions as well as reversions of the original rosy mutation. Those mutational events that restored the original wild-type DNA sequence would produce XDH molecules that con- tained the original parental amino-acid sequence, and would thus have a net surface charge that would identify an ry+O allele. A more likely expectation of the special mutagenesis hypothesis would have the ry+ convertant products exhibit an array of diff went electrophoretic types reflecting the individuality of each mutational event (HUNT, SOCHARD and DAYHOFF 1972). However, all 46 conversions were identical to the 16 recovered crossovers, and all of the 62 r y +
exceptions classified as identical to the original ry+ wild-type allele from which the mutants were derived originally. Taken together with the fact, reported earlier ( CHOVNICK et al. 1970), that the
XDH
activity associated with ry+ alleles produced by conversion events were uniform and identical to activity levels associated with crossover events as well as the original parental wild-type allele, these observations argue very strongly against a special mutagenesis hypothesis for the origin of convertants.Given (a) the stock stability of the XDH electrophoretic character associated with each of the ry+ isoalleles thus for isolated, and (b) that the electrophoretic character of the XDH molecule is a stable non-selective marker in otherwise mutant heteroallele crosses (documented in experiment I), then the genetic sites responsible for differences in XDH electrophoretic character may be iden- tified from mutant heteroallelic crosses involving rosy mutants induced on electrophoretically distinct r y + isoalleles. Experiment
I1
represents a first effort at intracistronic mapping of an electrophoretic site in Drosophila, and constitutes a demonstration experiment illustrating consistency of this procedure to identify the position of such a site relative to the position of other known markers within the cistron.298 M. MCCARRON, W. GELBART A N D A. CHOVNICK
one infers that there exists in a population an array of wild-type isoalleles, each providing the structural information for a molecule with a fixed net molecular surface charge under a specified set of experimental conditions of electrophoresis. Electrophoretic isoallele frequency studies have been important in comparative studies of patterns of variability in different kinds of populations (AYALA, POWELL and DOBZHANSKY 1971;
PRAKASH,
LEWONTIN and HUBBY 1969), in studies of linkage disequilibrium involving alleles at various loci ( CHARLESWORTH and CHARLESWORTH 1973; KOJIMA, GILLESPIE and TOBARI 1970), and in studies of linkage disequilibrium related to inversion polymorphism ( CHARLESWORTH and CHARLESWORTH 1973;PRAKASH
and LEWONTIN 1968, 1971;PRAKASH
and MERRITT 1972). In all of this work, electrophoretic identity of a given enzyme molecule from different individuals, whether from the same population sample or from quite distinct populations, is taken to reflect allele identity. Clearly, it is impossible to determine the amino acid sequence for each sample of all enzymes studies. However, the assumption of allelic identity of electrophoretically identi- cal mdecules of the same enzyme has received a general acceptance which is dis- quieting in view of the important inferences drawn from population studies of molecular polymorphism. Moreover, the fact that amino acid sequence analysis f o r hemoglobin has established the existence in human populations of nonidenti- cal but electrophoretically indistinguishable forms of a molecule (GIBLETT 1969; HUNT, SOCHARD and DAYHOFF 1972) suggests that a limited examination of this Question is warranted for those genetic systems which have been used to investi- p t e the dynamics of population variation. Indeed, WRIGHT and MACINTYRE( 1965) have noted differences in heat stability between electrophoretically indis- thguishable products of the esterase4 gene in Drosophila melanogaster. The pre-ent investigation eventually will question the genetic identity of electro- phoretically indistinguishable isoalleles.
In addition, the present experimental system is useful i n the traditional genetic approach to the study of genetic organization through recombination analysis. Since electrophoretic sites represent sites of variation within the structural infor- mation of a genetic unit (i.e., that part of the DNA which is transcribed to pro- duce an mRNA which, in turn, is translated to produce a specific polypeptide chain)
,
fine structure maps which include such sites along with sites which pro- duce inactive molecules and thermolabile molecules clearly set spatial limits to a structural gene. Localization of sites which give rise to variants which alter the tissue distribution, quantity and/or rate of synthesis ultimately may provide insight into the functional organization of a genetic element in eukaryotes.LITERATURE CITED
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