Molec. gem Genet. 153, 191-198 (1977) © by Springer-Verlag 1977
Investigations on the Organization of Genetic Loci in Drosophila melanogaster" Letha| Mutations Affecting 6-phosphogluconate Dehydrogenase and Their Suppression V.A. Gvozdev, T.I. Gerasimova, G.L. Kogan, and J.M. Rosovsky Kurchatov Institute of Atomic Energy, Moscow, USSR
Summary. The molecular nature of lethal and semilethal mutations in the Pgd locus of D. melanogaster coding for 6-phosphogluconate dehydrogenase (6PGD) was studied. All the 11 mutations affect the structural gene of the Pgd locus: 3 semilethal mutations resulted in altered 6PGD molecules with decreased catalytic activities; the rest 8 lethals were " n u l l " alleles characterized by mutant polypeptides capable of reacting with antisera against highly purified 6PGD. " N u l l " or low activity alleles for glucose-6-phosphate dehydrogenase induced by ethyl methanesulfonate were shown to be suppressors for the lethal mutations in the Pgd locus. A monocistronic type of organization of the Pgd locus is suggested taking into account the biochemical mechanism of suppression of the Pgd-lethals and their location in the structural gene coding for 6PGD.
and biochemical characteristics of the mutant phenotype permit to interpret the obtained data in terms of genetic loci structure (Gelbart et al., 1974; Rawls and Fristrom, 1975). In the present work mutations in the Pgd and Zw loci of D. melanogaster coding for 6-phosphogluconate-dehydrogenase and glucose-6phosphate dehydrogenase respectively were studied. The relationship between these two types of mutations was also investigated. The data were interpreted as evidence for a monocistronic type of organization of the Pgd locus.
Materials and Methods Methods of induction and screening of lethal or semilethal mutations affecting 6-phosphogluconate dehydrogenase were described elsewhere (Gvozdev et al., 1975). 6PGD and G6PD isozymes were assayed essentially as described earlier (Gvozdev et al., 1970). The detailed descriptions of mutations and special chromosomes are given in the catalogue of Lindsley and Grell (1968). In Table 1 is a synopsis of the most frequently used gene symbols.
Introduction The principles of organization of eukaryotic gene units remain obscure up to date. A 20-30 fold excess of DNA exists per one chromomere or genetic unit as compared to the DNA content necessary to code for a polypeptide with an average molecular weight (Beermann, 1973; Lefevre, 1974; Brenner, 1974). The functional role of the "excess" DNA is not elucidated. Organization of eukaryotic genetic loci in operon-like polycistronic structure or alternatively in single cistrons with the adj acent extended control nucleotide sequences is widely discussed (Georgiev, 1969; Davidson and Britten, 1973). Approaches to study the organization of eukaryotic genes using classic genetic methods appear now to be effective if the gene product (RNA or enzyme) may be well characterized. In these cases molecular
Table 1. Synopsis of the sex-linked gene symbols
Gene symbol
Phenotype
Pgcl
Electrophoretic variants of 6-phosphogluconate dehydrogenase (6PGD): Fast form Slow form Alleles resulting in decreased or zero level of 6PGD Brownish eyes
Pgd A Pgd ~ Pgdpn Zw Zw A Zw B
Zw-(suPgd)
Electrophoretic variants of glucose-& phosphate dehydrogenase (G6PD): Fast form Slow form Alleles resulting in decreased or zero level of G6PD, suppressors of the Pgd- alleles.
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
192
/
Pgd + "'F--.........~ '"v~ ~
Attctched X-chromosomes
Pgd + , Y-chromosome
I
I
Pgd-
pn
Pgd +
pn+
I
I
X
EMS •
×-chromosome w+Y-chromosome
f
F1 Pgd + Pgd- pn
Pgd+[• Pgd +
??
pn +
Y- chromosome
r
jd
Fig. 1. Crosses for suppressor mutation selection
Results
Three mutations, 13, 50 and 109, were semilethals resulting in a 3-8-fold decrease of viability. When the relatively large quantities of protein extract were applied to gel tubes, unstable 6PGD isozymes with diminished catalytic activity were revealed. Electrophoretic mobility of the fast form of 6PGD was not appreciably changed as a result of mutation 13 (Fig. 2, B, b, c, d) or mutation 109. Specific activity of 6PGD in crude extracts of flies carrying mutation 13 was decreased on the average by 60% as compared to normal flies. When the normal quantities of protein extract were applied to gel tubes, no fast electrophoretic variant of 6PGD was detected in extracts of heterozygous 13/Pg~ females, although an active hybrid form with intermediate mobility can been vi-
1. Biochernical and Biological Characteristics of Mutations Affecting the Activity of 6PGD. Sodium dodecyl sulfate gel electrophoresis reveals that Drosophila 6PGD consists of two identical subunits of 50,000 daltons (Kogan et al., in press). This result is in accord with earlier observations obtained by different methods (Kazazian, 1966). A hybrid AB isozyme and two parental forms of 6PGD (AA and BB) are detected in heterozygous females carrying the PgdA and P g ~ alleles (Fig. 2A, a). 11 mutations in the Pgda or Pgd~ loci affecting the activity of 6PGD were obtained (Gvozdev et al., 1975). A single one, Nr. 111, was induced by 7-irradiation and the rest by EMS treatment. The bulk of mutations induced (35, 39, 45, 71, 93, 94, 100, 111) were practically lethals resulting in drastic (30-1000fold) decreases of the number of surviving hemi or homozygous individuals (Table 2). In these cases the hybrid 6PGD and a slow or fast electrophoretic variant were absent in heterozygous females (l/PgdA or 1/Pgd B) corresponding to the type of the Pgd allele treated by the mutagen.
Fig. 2. 6PGD izozymes in wild type and mutant flies when homoor heterozygous. A-heterozygous females : a-PgdA/Pgd B, b- 13/Pgda; B, a-males carrying mutation 50, 10-fold excess of protein extract; mixtures of extracts of homozygous individuals: b-semilethals 13 and 50, 10-fold excess of protein extract, c-semilethals 13, 50 and wild type P g ~ flies, d-Pgdk and PgdB, e-semilethal 50 and Pg~, f-semilethal 13 and Pg~; g-heterozygous 50/30 females
Revertants carrying suppressor mutations for the lethal Pgdmutations were induced by ethyl methanesulfonate as described earlier (Gvozdev et al., 1975). Males carrying the lethal Pgd and recessive pn mutations in the X-chromosome and w+ Y duplication of the X-chromosome including Pgd + and pn + loci were treated with EMS. The viable pn males (revertants) were selected in the offspring of the cross of the Pgd-/w+Y males to females with attached X-chromosomes (Fig. 1). Purification of 6PGD, the immunization schedule and the characterization of antisera directed to the highly purified sample of 6PGD are described elsewhere (Kogan et al., in press). Immunochemical analysis was performed using double Ouchterlony diffusion in agar plates (Ouchterlony, 1953). A micromodification of the complement fixation tests (Wasserman and Levine, 1961) was used in order to estimate the mutant forms of 6PGD quantitatively.
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
193
Table 2. Characteristics of mutations in the Pgd locus NN of mutations
Mutagen treated Pgd allele
Phenotypic characteristics
% of individuals survived
6PGD activity in % to the normal level Pgd- males
Females Pgd-/Pgd-
Pgd+/Pgd -
Semilethals: 13
Pgd A
males fertile, females sterile
13
40-70
50
Pgd A
males sterile
30
22-40
109
Pgd A
females sterile," males inviable
30
35
Pgd A ]
3
39 45 71 93 94 100 111
Pgd Pgd A A I Pgd A Pgd B Pgd B Pgd B Pgd A
41.5 1 0.5 0.1 1
lethals
10-24 < 3 (35/35) b < 1.5 (39/Df) < 2 (45/45) < 2 (71/71) < 5 (39/93) < 5 (94/45) -
52
65 50 55 57
a The Pgd-/Df females carrying the deficiency uncovered the Pgd locus were examined; b lethals carrying by homozygous or heterozygous female excapers are indicated in parenthesis; - 6 P G D activity not detected
sualized easily (Fig. 2, A, b). This result was possibly due to the rapid inactivation of enzyme molecules containing two identical mutant polypeptide chains. Drastic increase in thermolability of 6PGD and a two-fold increase of the Km value for 6-phosphogluconate shows the alterations of the properties of 6PGD as a result of mutation 13. Semilethal mutation 50 induces increase in the electrophoretic mobility of 6PGD (Fig. 2, B, a, d). Association of the mutant polypeptide coded by this allele with the wild type "slow" subunit of 6PGD resulted in an active dimer of 6PGD whose electrophoretic activity was approximately the same as that of the fast form of enzyme (Fig. 2, B, d, e, g). The intensively stained band representing the heterologous dimer shows its relatively high stability and activity as compared to the mutant enzyme containing two identically altered subunits. Alteration of 6PGD due to semilethal mutation 50 is followed by a 2.5-5.0 fold decrease of enzyme activity in fly extracts. The data show that semilethals 13 and 50 affect the structural gene of the Pgd locus which codes for the polypeptide of 6PGD. As a result of mutation 109 the specific activity of 6PGD was reduced to a level of 5-10% of the wild type activity. Data will be present below showing that this mutation is also localized in the structural gene coding for 6PGD. Semilethal mutations in the Pgd locus are pleiotropic: they affected male or female fertility (Table 2) and lengthened the rate of development. Female and male escapers carrying lethals 35, 45, 71 are also
sterile and in several cases display an abnormal wing phenotype. Analogous effects of mutations in the Pgd locus were reported earlier (Bewley and Luechesi, 1975). Changes in 6PGD activity due to lethal mutations in the Pgd locus were estimated indirectly: the specific activity of 6PGD was reduced in heterozygous l/Pgd+ females to a level of 55-65% of the wild type activity (Table 2). It was shown earlier that the heterozygous deficiency in the X-chromosome including the Pgd locus resulted in a similar decrease of the activity of 6PGD (Gvozdev et al., 1975). The level of 6PGD activity in female escapers carrying lethals 35, 45 or 71 (Table 2) was shown to be no more than 2-5% of the normal activity. Taking into account these results we suggested that the obtained lethals are the " n u l l " alleles of the Pgd locus. Interallelic complementation between the lethals may indicate the presence of inactive polypeptide in the mutant extracts as a result of a lesion in the structural gene. However all the mutations studied fail to complement although 6PGD is a dimer containing two identical polypeptide subunits. Female escapers carrying different lethal mutations in the heterozygous state showed no enhancement of the enzyme activity and the number of these individuals did not exceed the number of homozygous escapers. The absence of interallelic complementation in a series of mutations that affect the structural genes for proteins containing two identical subunits has also been reported by others (Hartman et al., 1971). Thus
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
194
Table 3. Frequency of suppression of the Pgd-lethals and characteristics of suppressors Genotype of the EMS-treated males
Number of chromosomes screened
Number of revertants (suppressors)
Frequency of suppression
Characteristic of G6PD
139pnZwA/w + Y
49,250
3 (su6, suT, suB)
2 x 10 _5
< l 5% of the normal activity
171pnZwB/w+Y
140,000
3 (sul, su2, sus)
6 x 10 - s
increase of the Km values (Gvozdev et al.0 1976) KmG6P-30 60 times Kn~NADP-2--7times
the complementation data throw no light on the molecular nature of induced lethal mutations in the Pgd locus.
2. Suppression of the Lethal Mutations in the Pgd Locus. As a result of EMS treatment of the 139pn/ w+Y or 171pn/w+Y males, six revertants carrying 139pn or 171 pn X-chromosomes without the w+ Yduplication (see Methods) were obtained with a frequency of 2-6 x 10- 5 (Table 3). However the restoration of viability was riot followed by increase in the activity of 6PGD. Revertants were characterized by " z e r o " level of 6PGD peculiar to flies carrying the lethal 39 or 71 mutations. Thus the viability was restored without reversion of the normal function of the Pgd locus. It was very surprizing that decreased or null activity of G6PD, another enzyme of the pentose phosphate pathway, was demonstrated in all revertants. The corresponding data are presented in Table 3 ; in part, they have been reported earlier (Gvozdev et al., 1976). It was concluded that the increase in viability was due to suppressor mutations (SUl, su2, sus, su6, su7, sus) defective in G6PD. Biochemical studies of the sul, su2 and su5 mutations showed the drastic drop in the G6PD activity and increase in the Km values for substrate and coenzyme (Gvozdev et al., 1976). Mutations su6, suT, sub resulted in zero level of G6PD activity, which has been near the sensitivity limit of enzyme assays (Table 3). Alterations of the electrophoretic mobility of G6PD as a result of the su~, su2 and su5 mutations (Gvozdev et al., 1976) indicate that the mutational lesions affect the corresponding structural gene in the X-linked Zw locus coding for G6PD. The high frequency of crossing over in the X-chromosome between the Pgd and the su2 or sus mutations is in accord with the well known data showing the location of the Pgd and Zw loci in distal and proximal parts of the X-chromosome respectively (Lindsley, 1968). Inviability of the Pgd- Zw-/Pgd- Zw + females showed the recessive nature of induced sex-linked suppressors. The su2 and sus mutations were transferred by crossingover to the X-chromosomes carrying other Pgd-lethals. The viability of males carrying different Pgd-lethals and
suppressor mutations showed that suppressors are allele-nonspecific. Suppression of the Pgd-mutations provided sufficient quantity of flies needed for the further biochemical and immunochemical studies of the Pgd-mutations. The biochemical mechanism of suppression has been considered by us earlier (Gvozdev et al., 1976), but some aspects of suppression related to the problem of the organization of the Pgd locus will be presented in the Discussion.
3. 6PGD Activity in Flies Carrying Suppressors. The presence of the lethal Pgd-mutations in the suppressed state premitted a more precise estimation of the levels of 6PGD activity due to the Pgd- mutations, using a sufficient number of flies for analysis. No detectable 6PGD activity was revealed in flies in which the level of.enzyme activity did not exceed 0.5% (lethal 35), 0.2% (lethal 39), 0.1% (lethal 45), 0.15% (lethal 94), 3% (lethal 100), 2% (lethal 111) of the normal 6PGD activity. The absence of complementation between the Pgdmutations was confirmed in experiments showing that the level of 6PGD activity in females carrying different suppressed Pgd-lethals did not exceed 1% of the normal activity. In strains carrying semilethal Pgd mutations and suppressors the activities of 6PGD were approximately those presented in Table 2.6PGD activity attained a level of 5-25% (semilethal 13), 2-15% (semilethal 50), and 0-22% (semilethal 109) of the normal activity. These variations of 6PGD activities may be explained by the instability of the mutant forms of 6PGD. 4. Immunochemical Studies of the Pgd Mutations. In order to detect the product of the mutant Pgd locus, an antiserum against the highly purified 6PGD coded by the Pgd B allele was obtained (Kogan et al., in press). In Oucherlony immunodiffusion tests on agar plates the highly purified slow or fast variants of 6PGD demonstrate a single continuous precipitation line without detectable antigenic differences (Fig. 3). In most experiments two precipitation lines were de-
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster 1
O
0 Fig. 3. Ouchterlony double diffusion precipitin analysis of the wild type and mutant extracts. The center well contained antisera directed to the purified slow form of 6PGD; to the outer wells were added: 1-purified slow enzyme, 2-purified fast enzyme, 3 and 6-wild type extract, 4-extract from the first group of mutants, 5-extract from the second group of mutants
monstrated in fly extracts prepared from the wild type strains, the internal line being identical to that formed by purified enzyme. Extracts of flies carrying suppressed Pgd-lethals were checked by immunodiffusion tests. As a result the examined Pgd-mutations were divided into two groups. The first group included semilethals 13, 50 and 109 and two lethals, 35 and 45, with complete immunological similarity to normal fly extracts. No differences in position and intensity of the precipitation lines were observed between wild type and mutant extracts containing equal concentration of protein. Thus there was no appreciable change of antigen content as a result of mutations, taking into account the fact that a two-fold dilution of fly extracts resulted in a drastic weakening of the precipitation line and its shift it to the outer antigen containing well. The same result was obtained for the lethal 109 which, as shown above, results in a 5 10-fold drop of 6PGD activity. These results suggest that the semilethal 109, like the two other semilethals, 13 and 50, is located in the structural gene coding for 6PGD. These mutations affect the properties of the gene product but have no influence on the quantity of the enzyme molecules. In other words, immunochemical analysis showed that mutations of the first group (13, 50, 109, 35, 45) are located in the corresponding structural gene. In these cases alterations in primary polypeptide structure of the mutant subunits of 6PGD exerted no effect on the properties of antigenic determinants as revealed by immunodiffusion tests. No internal precipitation line peculiar to the normal purified enzyme was demonstrated in fly extracts carrying the second group mutants (39, 71, 93, 94, 100 and 111) although an external precipitation line ("external antigen") has been detected in all cases. This line was also often seen in wild-type extracts. The nature of the "external antigen" as a product of the Pgd locus was clarified in experiments (Kogan
195
et al., in press) in which treatment of extracts with 2% sodium dodecyl sulfate in the presence of 0,05% 2-mercaptoethanol resulted in elimination of the external precipitation line with simultaneous appearance of the internal line. We suggest that the production of two precipitation lines by an enzyme molecule reflects two different discrete conformations of polypeptide chains without common precipitative antigenic determinants. A conformation similar but not identical to the native form is completely excluded in a cell as a result of the second group mutations. The appearance of the "external antigen" in the wild type extracts remains unexplained. Observation of a gene dosage effect for the "external antigen" supplied additional evidence in favour of the suggestion that this protein is a product of the Pgd locus. The antigen quantity was assayed by complement fixation tests. Aliquots of protein extract were mixed with the serum in order to detect an antigen-antibody ratio by which 50% utilization of the added complement is achieved. In the first place it was shown that 6 P G D quantity evaluated by this method well corresponds to the level of the enzyme activity in flies carrying variable doses of the normal Pgd locus. It is well known that a single Pgd locus in males is two-fold as active as in females and that this results in dosage compensation (Kazazian et al., 1965; Gvozdev et al., 1970). Yet the level of 6PGD activity is dosage dependent in both sexes: females carrying a heterozygous X-chromosome deficiency that uncovers the Pgd gene have a 40% reduced level of 6PGD specific activity, and the enzyme activity in males carrying two Pgd doses was 1.5-2.0 times as high as in normal males. In agreement with these
Table 4. Dosage effects of the Pgd genes assayed by antisera directed to 6PGD. In parenthesis an antigen quantity is indicated in arbitrary units Genotypes
Number of the Pgd genes
Females DJ/ Pgd +
1
Males Pgd+/Pgd +w+Y
2
Females Dr, su5/194 su5
1
Females, 194sus/194su 5
2
Females, Df, sus/193sus
1
Females, 193sus/193su 5
2
Quantity of protein extract (p,g) needed for 50% complement fixation Exp. 1
Exp. 2
12.2 (33)
31.2 (29)
4.0 (100)
15.2 (48) 7.5 (100) 15.8 (51) 8.1 (100)
9.1 (100)
39.0 (51) 20.0 (100) 40.0 (48) 19.2 (100)
196
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophilamelanogaster
data, complement fixation tests demonstrated a 3-3.5 fold excess of 6PGD quantity in males carrying two Pgd doses as compared to the females with a single Pgdlocus (Table 4). Quantities of the "external antigen" were estimated in females carrying one or two doses of the mutant Pgd-locus (lethals 93 and 94). The data revealed a two-fold drop in the "external antigen" quantity as a result of the twofold decrease in the number of Pgd loci, thus showing striking dosage dependence. Results presented in Table 4 indicate also that the mutations studied have no effect on the absolute gene product quantity as compared to the normal Pgd ÷ alleles. qmmunochemical analysis revealed that all the semilethal and lethal mutations were due to alterations in the corresponding structural gene and most likely represent missence mutations. Evidence for the missence type of the induced mutations was obtained by polyacrylamide gel elecrophoresis, which demonstrated no changes in molecular weights of mutant antigens purified by means of cellulose-immobilized antibodies (Kogan et al., in press).
Discussion In many cases, the number of genetic loci determined for a given chromosome region in saturation experiments for induced lethals corresponds well to the number of bands in this region (Lefevre, 1974). The 20 30-fold excess of D N A per chromomere of a polytene chromosome of D. melanogaster over that necessary to code for a polypeptide with an average molecular weight raises a major question concerning the functional role of this "excess D N A " . A polycistronic operon-like structure of a chromomere was proposed by several authors in order to explain the functions of the "excess" D N A (Davidson and Britten, 1973; Olenov, 1974). It is possible to suppose that in saturation experiments lethal mutations attributed to a single locus may affect the different genes of a polycistronic structure in a polar way and thus appear to be non-complementary, whereas the lethal effect would be due to the inactivation of several genes coding for a corresponding enzyme complex. According to this hypothesis a lethal mutation may be localised in a non-essential gene and result in the switching-off of adjacent vital genes (Davidson and Britten, 1973). This possibility cannot be neglected in cases in which nothing in known about the gene products of the loci studied and only lethal effects indicate alterations of gene function. We will consider our data concerning the Pgd locus in connection with the surmised polycistronic structure of a chromomere. The Pgd locus coding
for 6PGD is situated in the 2D3-5 region of the Xchromosome where the relatively faint bands are located. Saturation studies revealed 3-4 genetic units in this region (Gvozdev et al., 1975). Feulgen densitometry of D N A content in the 2D region permits to suggest the presence of a 5-8-fold excess of D N A per chromomere over the quantity needed to code for a polypeptide subunit of 6PGD of 50,000 mol. weight (Ananiev and Barsky, 1975). This result shows a D N A abundance also in these faint bands. Bewley and Lucchesi (1976) described a lethal in the Pgd locus but could not rule out a possibility that the lethal effect was due to mutation in a locus adjacent to the Pgd. The present paper analyses the nature of mutations in the Pgd locus; with a single exception all were induced by EMS. Three semilethal mutations affected 6PGD enzymatic characteristics, the rest 8 mutations resulted in zero activity although an immunologically defective product of the Pgd gene with a tool. weight identical to that of the normal 6PGD subunit (Kogan et al., in press) has been demonstrated in all cases. Thus in our experiments only the missence type mutations were discovered, although it is known that ethylmethanesulfonate is able to induce chromosomal aberrations (Auerbach and Kilbey, 1971 ; Bishop and Lee, 1973). The major conclusion is that all the mutations are located in a structural gene coding for 6 P G D and the alteration of 6PGD production is not due to mutational lesions in adjacent genes. In other words there are no grounds for suggesting a polycistronic structure of a chromomere inclusing the Pgd gene and for attributing the lethal effects to inactivation of several genes. An alternative interpretation, assuming an operon-like polycistronic structure with a single vital Pgd gene as a proximal one followed by distal non-vital genes, seems very doubtful. Our data concerning the suppression of the lethal mutations in the Pgd locus also may be considered in relation to chromomere organization. Suppression is attained by mutations in the Zw locus and results in drastic decreases of G6PD activity. This allelenonspecific suppression as well as a previously reported suppression of rudimentary mutations (Bahn, 1973) seem to be the rare cases where a suppression mechanism in animals may be explained in biochemical terms. Consideration of the biochemical nature of suppression is especially attractive because of the evidence in favour of a monocistronic structure of the Pgd locus. The Pgd and Zw loci control two oxidative reactions in t h e beginning of the pentose phosphate pathway. The pentose phosphate route in animal cells is considered usually as a major source of reduced nicotinamide-adenine dinucleotide phosphate and
V.A. Gvozdev et al. : Organization of Genetic Loci in
Drosophila melanogaster
Glucose-5-phosphate ~_____.___ ~
.•" fructose-6-phosphate 1 ~
fructose -1.5-diphosphate ~ n t d O ; ~" gtycercddehyde-3- phosphate
Gtycolysis
icise
197
Zw [OCUS ~- NADPH
6- phosphogluconote LPgd I°cus ~ NADPH ~ - 5 - phosphate r]bulose [ pentoses)
trcmsketoLose nucleic acid biosynthesis
Pentose phosphate pctthwcty
pentoses needed for nucleic acid biosynthesis (Lehninger, 1972). Pentose synthesis may also be accomplished by the use of metabolites of glycolysis; fructose-6-phosphate and glyceraldehyde-3-phosphate (Fig. 4). A switch-off the second oxidative reaction of the pentose phosphate pathway by a Pgd mutation has a lethal effect on Drosophila. The viability of the flies is restored by suppressor mutations in the Zw locus, which result in the decrease of G6PD activity, although the oxidative step of the pentose phosphate pathway now is blocked completely. These oxidative reactions seem to play no essential and indispensable role in metabolism and the deficit of NADPH is readily compensated for (Gvozdev et al., 1976). Pentose biosynthesis in flies carrying the Pgd and Zw negative mutations is possibly accomplished via the transaldolase and transketolase reactions using glycolytic metabolites (Fig. 4). The lethal effect of the Pgd mutation may be explained by accumulation of 6-phosphogluconate which, as has been shown in mammals, inhibits some enzymatic step of glycolysis (Parr, 1956). We suppose that as a result of the Pgd mutation both metabolic routes for pentose biosynthesis (Fig. 4) are interrupted. Suppressor mutations' in the Zw locus provide a flux of glucose-6-phosphate in glycolysis, preventing at the same time the deleterious accumulation of 6-phosphogluconate. Thus the suppressor mutations open a possibility of pentose biosynthesis via glycolytic and reversible non-oxidative reactions of the pentose phosphate pathway. Similar cases of suppression as a result of elimination of deleterious accumulation of toxic sugar phosphate esters were reported in yeasts and bacteria (Hartman et al., 1973). The supposed mechanism of suppression of the Pgd lethals is in good accord with our observations that the development of Drosophila larvae carrying the " Z e r o " Pgd mutations stops at the end of the first instar larval stage, at which time a severalfold increase in the activity of G6PD is attained (Wright and Shaw, 1970).
Fig. 4. Metabolic relations between the pentose phosphate pathway and glycolysis
Consideration of the suppression data suggests that the lethal effect of the Pgd mutations is due only to mutations in a single gene coding for 6PGD and provides no basis for speculations on alterations of another vital gene in a corresponding chromomere. Defects induced by the independently obtained mutations in the Pgd locus are repaired to the same extent by mutations in the Zw locus. In other words, consideration of the biochemical mechanism of suppression also demonstrates that the Pgd-mutations affect only 6PGD production and exert no effect on the other essential enzymes. It is necessary to underline that these data do not imply that the DNA of each chromomere codes for but one polypeptide. It was shown recently that the rudimentary locus coded for three enzymes for successive steps of pyrimidine biosynthesis (Rawls and Fristrom, 1975). On the other band repetitive DNA sequences were revealed in the Balbiani ring or ribosomal genes. It would be more likely to suppose that the different principles of genetic organization of chromomeres may reflect their functional divergence. An extended regulatory region in eukaryotic loci is often proposed in explanation of the relatively large quantity of DNA in a chromomere (Georgiev, 1969; Judd et al., 1972; Beermann, 1973). In this case a substantial part of induced mutations would be expected to affect a controlling region of a gene. We have obtained the opposite result--all the 11 lethal mutations were located in the structural gene coding for 6PGD. This result agrees with the conclusion (Schalet a. Sankaranarayanan, 1976) that mutational intralocus changes in Drosophila occur predominantly in DNA which codes for the structural portion of the gene. Most of the spontaneous mutations affecting human G6PD production were considered to be point mutations inside the structural element (Luzzatto, 1974). At least 40% of the 105 nitrosoguanidine induced mutations affecting hypoxanthine-phosphoribosyl transferase in mouse L-cells
198
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
were also shown to be structural gene alterations (Wahl et al., 1974). Similar results were recently obtained for the locus coding for alcohol dehydrogenase in D. melanogaster (Schwartz and Sofer, 1976). No extended regulatory zone was demonstrated for the rosy locus in D. rnelanogaster (Gelbart et al., 1974). These data may be explained by the mutational resistance of eukaryotic DNA sequences containing controlling elements. For example deficiencies of nucleotide sequences or simultaneous alterations in multiple sites may be necessary to affect the functioning of gene-controlling regions. However different point of view is possible if it is supposed that the size of regulatory zones of eukaryotic genes at least does not exceed the length of the adjacent structural gene (or genes). It is difficult to reconcile the our data and those cited with the suggestion that mutational rates in genetic loci increase in eukaryotes proportionally to DNA content in a wliole genome and possibly in a genetic locus (Abrahamson et al., 1973). The functions of the bulk DNA are difficult to determine, but hypotheses that this DNA may be involved for example in chromosome mechanics or evolution of eukaryotic DNA sequences cannot now be neglected. Acknowledgements. The authors are greatly indebted to Dr. L.A. Zamchuck for guidance in the complement fixation experiments, Prof. Yu.M. Olenov and Prof. R.B. Khesin for discussion and to Mrs. G.T. Khromeshina, O.Yu. Braslavskaya, M.I. Volkova and V.P. Kopaeva for generous help.
References Abrahamson, S., Bender, M.A., Conger, A.D., Wolf, C.: Uniformity of radiation induced mutation rates among different species. Nature (Lond.) 245, 460-462 (1973) Ananiev, E.V., Barsky, V.E. : DNA content, replication and transcription rate in different sites of the X-chromosome of D. melanogaster. Molec. Biologia (Russ.) 9, 752 760 (1975) Auerbach, C., Kilbey, B.J.: Mutations in eukaryotes. Ann. Rev. Genet. 5, 163-218 (1971) Bahn, E. : A suppressor locus for the pyrimidine requiring mutant: rudimentary. Dros. Inform. Service 49, 98 (1972) Beermann, W. : Chromomeres and genes. In: Results and problems in cell differentiation, Vol. 4, pp. 1-33. Berlin-Heidelberg-New York: Springer 1973 Bewley, G.C., Lucchesi, J.C.: Lethal effects of low and " n u l l " activity alleles of 6-phosphogluconate dehydrogenase in D. melanogaster. Genetics 79, 451-457 (1975) Bishop, J.B., Lee° W.R. : Chromosome breakage in D. melanogaster induced by a monofunctional alkylating agent (EMS). Mutation Res. 21, 327-333 (I973) Brenner, S. : The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974) Davidson, E.H., Britten, R.J. : Organization, transription and regulation in the animal genome. Quart. Rev. Biol. 48, 565 613 (1973) Gelbart, W.M., McCarron, M., Pandey, J., Chovnick, A. : Genetic limits of the xanthine dehydrogenase structural elements within the rosy locus in D. melanogaster. Genetics 78, 869-886 (1974) Georgiev, G.P.: On the structural organization of operon and the regulation of RNA synthesis in animal cells. J. theor. Biol. 25, 473-490 (1969)
Gvozdev, V.A., Birstein, V.J., Faizullin, L.Z.: Gene dependent regulation of 6-phosphogluconate dehydrogenase activity of Drosophila melanogaster. Molec. Biologia (Russ.) 6, 876-887 (1970) Gvozdev, V.A,, Gerasimova, T.I., Kogan, G.L., Braslavskaya, O.Yu. : Role of the pentose phosphate pathway in metabolism of Drosophila melanogaster elucidated by mutations affecting glucose-6-phosphate and 6-phosphogluconate dehydrogenases. FEBS Letters 64, 85-88 (1976) Gvozdev, V.A., Gostimsky, S.A., Gerasimova, T.I., Dubrovskaya, E.S., Braslavskaya, O.Yu. : Fine genetic structure of the 2D32F5 region of the X-chromosome of D.melanogaster. Mol. gen. Genet. 141, 269-275 (1975) Hartman, P.E., Hartman, Z., Stahl, R.C., Ames, B. : Classification and mapping of spontaneous and induced mutations in the histidiu operon of Salmonella. Advanc. Genet. 16, 1-34 (1971) Hartman, P.E., Roth, J.R.: Mechanisms of suppression. Adv. in Genet. 17, 1-106 (1973) Judd, B.H., Shen, M.W., Kaufman, T.C. : The anatomy and function of a segment of the X-chromosome of D. melanogaster. Genetics 71, 139-156 (1972) Kazazian, H.H., Young, W.J., Childs, B. : X-linked 6-phosphogluconate dehydrogenase in Drosophila: Subunit association. Science 150, 1601-1602 (1965) Kazazian, H.H. : Molecular size studies in 6-phosphogluconate dehydrogenase. Nature (Lond.), 212, 197-198 (1966) Kogan, G.V., Rosovsky, Ya.M., Gvozdev, V.A.: Purification and immunological characteristics of the wild type and mutant forms of 6PGD. Molec. Biologia (Russ.). in press Lefevre, G. : The relationship between genes and polytene chromosome bands. Ann. Rev. Genet. 8, 51-62 (1974) Lehninger, A.L. : Biochemistry. New York: Worth publishers 1972 Lindsley, D.L., Grell, E.H. : Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627 (1968) Luzzatto, L.: Genetic heterogeneity and pathophysiology of glucose-6-phosphate dehydrogenase deficiency. Brit. J. Haematol. 28, 151-155 (1974) Olenov, Yu.M. : On the genome organization in Metazoa. Cytologia (Russ.) 16, 403-418 (1974) Ouchterlony, O.: Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta. Path. Microbiol. Scand. 32, 231-240 (1953) Parr, C.W.: Inhibition of phosphoglucose isomerase. Nature (Lond.) 178, 1401 (1956) Rawls, J.M., Fristrom, J.W. : A complex genetic locus that controls the first three steps of pyrimidine biosynthesis in Drosophila. Nature (Lond.) 255, 738-740 (1975) Schalet, A.P., Sankaranarayanan, K. : Evaluation and re-evaluation of genetic radiation hazards in man. I. Interspecific comparison of estimates of mutation rates. Mutation Res. 35, 341 370 (1976) Schwartz, M., Sorer, W. : Alcohol dehydrogenase-negative mutants in Drosophila: Defects at the structural locus? Genetics 83, 125-136 (1976) Wahl, G.M., Hughes, S.H., Capecchi, M.D. : Immunological characterization of hypoxanthine-guanine phosphorybosyl transferase mutants of mouse L cells : evidence for mutations at different loci in the HGPRT gene. J. Cell Physiol. 85, 307-320 (1975) Wasserman, E., Levine, L.: Quantitative micro-complement fixation and its use to study of antigenic structure by specific antigen-antibody inhibition. J. Immunol. 87, 290595 (1961) Wright, D.A., Shaw, C.R. : Time of expression of genes controlling specific enzymes in Drosophila embryos. Biochem. Genet. 4, 385-394 (1970)
Communicated by Ch. Auerbach Received September 10 / November 6, 1976
Investigations on the Organization of Genetic Loci in Drosophila melanogaster" Letha| Mutations Affecting 6-phosphogluconate Dehydrogenase and Their Suppression V.A. Gvozdev, T.I. Gerasimova, G.L. Kogan, and J.M. Rosovsky Kurchatov Institute of Atomic Energy, Moscow, USSR
Summary. The molecular nature of lethal and semilethal mutations in the Pgd locus of D. melanogaster coding for 6-phosphogluconate dehydrogenase (6PGD) was studied. All the 11 mutations affect the structural gene of the Pgd locus: 3 semilethal mutations resulted in altered 6PGD molecules with decreased catalytic activities; the rest 8 lethals were " n u l l " alleles characterized by mutant polypeptides capable of reacting with antisera against highly purified 6PGD. " N u l l " or low activity alleles for glucose-6-phosphate dehydrogenase induced by ethyl methanesulfonate were shown to be suppressors for the lethal mutations in the Pgd locus. A monocistronic type of organization of the Pgd locus is suggested taking into account the biochemical mechanism of suppression of the Pgd-lethals and their location in the structural gene coding for 6PGD.
and biochemical characteristics of the mutant phenotype permit to interpret the obtained data in terms of genetic loci structure (Gelbart et al., 1974; Rawls and Fristrom, 1975). In the present work mutations in the Pgd and Zw loci of D. melanogaster coding for 6-phosphogluconate-dehydrogenase and glucose-6phosphate dehydrogenase respectively were studied. The relationship between these two types of mutations was also investigated. The data were interpreted as evidence for a monocistronic type of organization of the Pgd locus.
Materials and Methods Methods of induction and screening of lethal or semilethal mutations affecting 6-phosphogluconate dehydrogenase were described elsewhere (Gvozdev et al., 1975). 6PGD and G6PD isozymes were assayed essentially as described earlier (Gvozdev et al., 1970). The detailed descriptions of mutations and special chromosomes are given in the catalogue of Lindsley and Grell (1968). In Table 1 is a synopsis of the most frequently used gene symbols.
Introduction The principles of organization of eukaryotic gene units remain obscure up to date. A 20-30 fold excess of DNA exists per one chromomere or genetic unit as compared to the DNA content necessary to code for a polypeptide with an average molecular weight (Beermann, 1973; Lefevre, 1974; Brenner, 1974). The functional role of the "excess" DNA is not elucidated. Organization of eukaryotic genetic loci in operon-like polycistronic structure or alternatively in single cistrons with the adj acent extended control nucleotide sequences is widely discussed (Georgiev, 1969; Davidson and Britten, 1973). Approaches to study the organization of eukaryotic genes using classic genetic methods appear now to be effective if the gene product (RNA or enzyme) may be well characterized. In these cases molecular
Table 1. Synopsis of the sex-linked gene symbols
Gene symbol
Phenotype
Pgcl
Electrophoretic variants of 6-phosphogluconate dehydrogenase (6PGD): Fast form Slow form Alleles resulting in decreased or zero level of 6PGD Brownish eyes
Pgd A Pgd ~ Pgdpn Zw Zw A Zw B
Zw-(suPgd)
Electrophoretic variants of glucose-& phosphate dehydrogenase (G6PD): Fast form Slow form Alleles resulting in decreased or zero level of G6PD, suppressors of the Pgd- alleles.
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
192
/
Pgd + "'F--.........~ '"v~ ~
Attctched X-chromosomes
Pgd + , Y-chromosome
I
I
Pgd-
pn
Pgd +
pn+
I
I
X
EMS •
×-chromosome w+Y-chromosome
f
F1 Pgd + Pgd- pn
Pgd+[• Pgd +
??
pn +
Y- chromosome
r
jd
Fig. 1. Crosses for suppressor mutation selection
Results
Three mutations, 13, 50 and 109, were semilethals resulting in a 3-8-fold decrease of viability. When the relatively large quantities of protein extract were applied to gel tubes, unstable 6PGD isozymes with diminished catalytic activity were revealed. Electrophoretic mobility of the fast form of 6PGD was not appreciably changed as a result of mutation 13 (Fig. 2, B, b, c, d) or mutation 109. Specific activity of 6PGD in crude extracts of flies carrying mutation 13 was decreased on the average by 60% as compared to normal flies. When the normal quantities of protein extract were applied to gel tubes, no fast electrophoretic variant of 6PGD was detected in extracts of heterozygous 13/Pg~ females, although an active hybrid form with intermediate mobility can been vi-
1. Biochernical and Biological Characteristics of Mutations Affecting the Activity of 6PGD. Sodium dodecyl sulfate gel electrophoresis reveals that Drosophila 6PGD consists of two identical subunits of 50,000 daltons (Kogan et al., in press). This result is in accord with earlier observations obtained by different methods (Kazazian, 1966). A hybrid AB isozyme and two parental forms of 6PGD (AA and BB) are detected in heterozygous females carrying the PgdA and P g ~ alleles (Fig. 2A, a). 11 mutations in the Pgda or Pgd~ loci affecting the activity of 6PGD were obtained (Gvozdev et al., 1975). A single one, Nr. 111, was induced by 7-irradiation and the rest by EMS treatment. The bulk of mutations induced (35, 39, 45, 71, 93, 94, 100, 111) were practically lethals resulting in drastic (30-1000fold) decreases of the number of surviving hemi or homozygous individuals (Table 2). In these cases the hybrid 6PGD and a slow or fast electrophoretic variant were absent in heterozygous females (l/PgdA or 1/Pgd B) corresponding to the type of the Pgd allele treated by the mutagen.
Fig. 2. 6PGD izozymes in wild type and mutant flies when homoor heterozygous. A-heterozygous females : a-PgdA/Pgd B, b- 13/Pgda; B, a-males carrying mutation 50, 10-fold excess of protein extract; mixtures of extracts of homozygous individuals: b-semilethals 13 and 50, 10-fold excess of protein extract, c-semilethals 13, 50 and wild type P g ~ flies, d-Pgdk and PgdB, e-semilethal 50 and Pg~, f-semilethal 13 and Pg~; g-heterozygous 50/30 females
Revertants carrying suppressor mutations for the lethal Pgdmutations were induced by ethyl methanesulfonate as described earlier (Gvozdev et al., 1975). Males carrying the lethal Pgd and recessive pn mutations in the X-chromosome and w+ Y duplication of the X-chromosome including Pgd + and pn + loci were treated with EMS. The viable pn males (revertants) were selected in the offspring of the cross of the Pgd-/w+Y males to females with attached X-chromosomes (Fig. 1). Purification of 6PGD, the immunization schedule and the characterization of antisera directed to the highly purified sample of 6PGD are described elsewhere (Kogan et al., in press). Immunochemical analysis was performed using double Ouchterlony diffusion in agar plates (Ouchterlony, 1953). A micromodification of the complement fixation tests (Wasserman and Levine, 1961) was used in order to estimate the mutant forms of 6PGD quantitatively.
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
193
Table 2. Characteristics of mutations in the Pgd locus NN of mutations
Mutagen treated Pgd allele
Phenotypic characteristics
% of individuals survived
6PGD activity in % to the normal level Pgd- males
Females Pgd-/Pgd-
Pgd+/Pgd -
Semilethals: 13
Pgd A
males fertile, females sterile
13
40-70
50
Pgd A
males sterile
30
22-40
109
Pgd A
females sterile," males inviable
30
35
Pgd A ]
3
39 45 71 93 94 100 111
Pgd Pgd A A I Pgd A Pgd B Pgd B Pgd B Pgd A
41.5 1 0.5 0.1 1
lethals
10-24 < 3 (35/35) b < 1.5 (39/Df) < 2 (45/45) < 2 (71/71) < 5 (39/93) < 5 (94/45) -
52
65 50 55 57
a The Pgd-/Df females carrying the deficiency uncovered the Pgd locus were examined; b lethals carrying by homozygous or heterozygous female excapers are indicated in parenthesis; - 6 P G D activity not detected
sualized easily (Fig. 2, A, b). This result was possibly due to the rapid inactivation of enzyme molecules containing two identical mutant polypeptide chains. Drastic increase in thermolability of 6PGD and a two-fold increase of the Km value for 6-phosphogluconate shows the alterations of the properties of 6PGD as a result of mutation 13. Semilethal mutation 50 induces increase in the electrophoretic mobility of 6PGD (Fig. 2, B, a, d). Association of the mutant polypeptide coded by this allele with the wild type "slow" subunit of 6PGD resulted in an active dimer of 6PGD whose electrophoretic activity was approximately the same as that of the fast form of enzyme (Fig. 2, B, d, e, g). The intensively stained band representing the heterologous dimer shows its relatively high stability and activity as compared to the mutant enzyme containing two identically altered subunits. Alteration of 6PGD due to semilethal mutation 50 is followed by a 2.5-5.0 fold decrease of enzyme activity in fly extracts. The data show that semilethals 13 and 50 affect the structural gene of the Pgd locus which codes for the polypeptide of 6PGD. As a result of mutation 109 the specific activity of 6PGD was reduced to a level of 5-10% of the wild type activity. Data will be present below showing that this mutation is also localized in the structural gene coding for 6PGD. Semilethal mutations in the Pgd locus are pleiotropic: they affected male or female fertility (Table 2) and lengthened the rate of development. Female and male escapers carrying lethals 35, 45, 71 are also
sterile and in several cases display an abnormal wing phenotype. Analogous effects of mutations in the Pgd locus were reported earlier (Bewley and Luechesi, 1975). Changes in 6PGD activity due to lethal mutations in the Pgd locus were estimated indirectly: the specific activity of 6PGD was reduced in heterozygous l/Pgd+ females to a level of 55-65% of the wild type activity (Table 2). It was shown earlier that the heterozygous deficiency in the X-chromosome including the Pgd locus resulted in a similar decrease of the activity of 6PGD (Gvozdev et al., 1975). The level of 6PGD activity in female escapers carrying lethals 35, 45 or 71 (Table 2) was shown to be no more than 2-5% of the normal activity. Taking into account these results we suggested that the obtained lethals are the " n u l l " alleles of the Pgd locus. Interallelic complementation between the lethals may indicate the presence of inactive polypeptide in the mutant extracts as a result of a lesion in the structural gene. However all the mutations studied fail to complement although 6PGD is a dimer containing two identical polypeptide subunits. Female escapers carrying different lethal mutations in the heterozygous state showed no enhancement of the enzyme activity and the number of these individuals did not exceed the number of homozygous escapers. The absence of interallelic complementation in a series of mutations that affect the structural genes for proteins containing two identical subunits has also been reported by others (Hartman et al., 1971). Thus
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
194
Table 3. Frequency of suppression of the Pgd-lethals and characteristics of suppressors Genotype of the EMS-treated males
Number of chromosomes screened
Number of revertants (suppressors)
Frequency of suppression
Characteristic of G6PD
139pnZwA/w + Y
49,250
3 (su6, suT, suB)
2 x 10 _5
< l 5% of the normal activity
171pnZwB/w+Y
140,000
3 (sul, su2, sus)
6 x 10 - s
increase of the Km values (Gvozdev et al.0 1976) KmG6P-30 60 times Kn~NADP-2--7times
the complementation data throw no light on the molecular nature of induced lethal mutations in the Pgd locus.
2. Suppression of the Lethal Mutations in the Pgd Locus. As a result of EMS treatment of the 139pn/ w+Y or 171pn/w+Y males, six revertants carrying 139pn or 171 pn X-chromosomes without the w+ Yduplication (see Methods) were obtained with a frequency of 2-6 x 10- 5 (Table 3). However the restoration of viability was riot followed by increase in the activity of 6PGD. Revertants were characterized by " z e r o " level of 6PGD peculiar to flies carrying the lethal 39 or 71 mutations. Thus the viability was restored without reversion of the normal function of the Pgd locus. It was very surprizing that decreased or null activity of G6PD, another enzyme of the pentose phosphate pathway, was demonstrated in all revertants. The corresponding data are presented in Table 3 ; in part, they have been reported earlier (Gvozdev et al., 1976). It was concluded that the increase in viability was due to suppressor mutations (SUl, su2, sus, su6, su7, sus) defective in G6PD. Biochemical studies of the sul, su2 and su5 mutations showed the drastic drop in the G6PD activity and increase in the Km values for substrate and coenzyme (Gvozdev et al., 1976). Mutations su6, suT, sub resulted in zero level of G6PD activity, which has been near the sensitivity limit of enzyme assays (Table 3). Alterations of the electrophoretic mobility of G6PD as a result of the su~, su2 and su5 mutations (Gvozdev et al., 1976) indicate that the mutational lesions affect the corresponding structural gene in the X-linked Zw locus coding for G6PD. The high frequency of crossing over in the X-chromosome between the Pgd and the su2 or sus mutations is in accord with the well known data showing the location of the Pgd and Zw loci in distal and proximal parts of the X-chromosome respectively (Lindsley, 1968). Inviability of the Pgd- Zw-/Pgd- Zw + females showed the recessive nature of induced sex-linked suppressors. The su2 and sus mutations were transferred by crossingover to the X-chromosomes carrying other Pgd-lethals. The viability of males carrying different Pgd-lethals and
suppressor mutations showed that suppressors are allele-nonspecific. Suppression of the Pgd-mutations provided sufficient quantity of flies needed for the further biochemical and immunochemical studies of the Pgd-mutations. The biochemical mechanism of suppression has been considered by us earlier (Gvozdev et al., 1976), but some aspects of suppression related to the problem of the organization of the Pgd locus will be presented in the Discussion.
3. 6PGD Activity in Flies Carrying Suppressors. The presence of the lethal Pgd-mutations in the suppressed state premitted a more precise estimation of the levels of 6PGD activity due to the Pgd- mutations, using a sufficient number of flies for analysis. No detectable 6PGD activity was revealed in flies in which the level of.enzyme activity did not exceed 0.5% (lethal 35), 0.2% (lethal 39), 0.1% (lethal 45), 0.15% (lethal 94), 3% (lethal 100), 2% (lethal 111) of the normal 6PGD activity. The absence of complementation between the Pgdmutations was confirmed in experiments showing that the level of 6PGD activity in females carrying different suppressed Pgd-lethals did not exceed 1% of the normal activity. In strains carrying semilethal Pgd mutations and suppressors the activities of 6PGD were approximately those presented in Table 2.6PGD activity attained a level of 5-25% (semilethal 13), 2-15% (semilethal 50), and 0-22% (semilethal 109) of the normal activity. These variations of 6PGD activities may be explained by the instability of the mutant forms of 6PGD. 4. Immunochemical Studies of the Pgd Mutations. In order to detect the product of the mutant Pgd locus, an antiserum against the highly purified 6PGD coded by the Pgd B allele was obtained (Kogan et al., in press). In Oucherlony immunodiffusion tests on agar plates the highly purified slow or fast variants of 6PGD demonstrate a single continuous precipitation line without detectable antigenic differences (Fig. 3). In most experiments two precipitation lines were de-
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster 1
O
0 Fig. 3. Ouchterlony double diffusion precipitin analysis of the wild type and mutant extracts. The center well contained antisera directed to the purified slow form of 6PGD; to the outer wells were added: 1-purified slow enzyme, 2-purified fast enzyme, 3 and 6-wild type extract, 4-extract from the first group of mutants, 5-extract from the second group of mutants
monstrated in fly extracts prepared from the wild type strains, the internal line being identical to that formed by purified enzyme. Extracts of flies carrying suppressed Pgd-lethals were checked by immunodiffusion tests. As a result the examined Pgd-mutations were divided into two groups. The first group included semilethals 13, 50 and 109 and two lethals, 35 and 45, with complete immunological similarity to normal fly extracts. No differences in position and intensity of the precipitation lines were observed between wild type and mutant extracts containing equal concentration of protein. Thus there was no appreciable change of antigen content as a result of mutations, taking into account the fact that a two-fold dilution of fly extracts resulted in a drastic weakening of the precipitation line and its shift it to the outer antigen containing well. The same result was obtained for the lethal 109 which, as shown above, results in a 5 10-fold drop of 6PGD activity. These results suggest that the semilethal 109, like the two other semilethals, 13 and 50, is located in the structural gene coding for 6PGD. These mutations affect the properties of the gene product but have no influence on the quantity of the enzyme molecules. In other words, immunochemical analysis showed that mutations of the first group (13, 50, 109, 35, 45) are located in the corresponding structural gene. In these cases alterations in primary polypeptide structure of the mutant subunits of 6PGD exerted no effect on the properties of antigenic determinants as revealed by immunodiffusion tests. No internal precipitation line peculiar to the normal purified enzyme was demonstrated in fly extracts carrying the second group mutants (39, 71, 93, 94, 100 and 111) although an external precipitation line ("external antigen") has been detected in all cases. This line was also often seen in wild-type extracts. The nature of the "external antigen" as a product of the Pgd locus was clarified in experiments (Kogan
195
et al., in press) in which treatment of extracts with 2% sodium dodecyl sulfate in the presence of 0,05% 2-mercaptoethanol resulted in elimination of the external precipitation line with simultaneous appearance of the internal line. We suggest that the production of two precipitation lines by an enzyme molecule reflects two different discrete conformations of polypeptide chains without common precipitative antigenic determinants. A conformation similar but not identical to the native form is completely excluded in a cell as a result of the second group mutations. The appearance of the "external antigen" in the wild type extracts remains unexplained. Observation of a gene dosage effect for the "external antigen" supplied additional evidence in favour of the suggestion that this protein is a product of the Pgd locus. The antigen quantity was assayed by complement fixation tests. Aliquots of protein extract were mixed with the serum in order to detect an antigen-antibody ratio by which 50% utilization of the added complement is achieved. In the first place it was shown that 6 P G D quantity evaluated by this method well corresponds to the level of the enzyme activity in flies carrying variable doses of the normal Pgd locus. It is well known that a single Pgd locus in males is two-fold as active as in females and that this results in dosage compensation (Kazazian et al., 1965; Gvozdev et al., 1970). Yet the level of 6PGD activity is dosage dependent in both sexes: females carrying a heterozygous X-chromosome deficiency that uncovers the Pgd gene have a 40% reduced level of 6PGD specific activity, and the enzyme activity in males carrying two Pgd doses was 1.5-2.0 times as high as in normal males. In agreement with these
Table 4. Dosage effects of the Pgd genes assayed by antisera directed to 6PGD. In parenthesis an antigen quantity is indicated in arbitrary units Genotypes
Number of the Pgd genes
Females DJ/ Pgd +
1
Males Pgd+/Pgd +w+Y
2
Females Dr, su5/194 su5
1
Females, 194sus/194su 5
2
Females, Df, sus/193sus
1
Females, 193sus/193su 5
2
Quantity of protein extract (p,g) needed for 50% complement fixation Exp. 1
Exp. 2
12.2 (33)
31.2 (29)
4.0 (100)
15.2 (48) 7.5 (100) 15.8 (51) 8.1 (100)
9.1 (100)
39.0 (51) 20.0 (100) 40.0 (48) 19.2 (100)
196
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophilamelanogaster
data, complement fixation tests demonstrated a 3-3.5 fold excess of 6PGD quantity in males carrying two Pgd doses as compared to the females with a single Pgdlocus (Table 4). Quantities of the "external antigen" were estimated in females carrying one or two doses of the mutant Pgd-locus (lethals 93 and 94). The data revealed a two-fold drop in the "external antigen" quantity as a result of the twofold decrease in the number of Pgd loci, thus showing striking dosage dependence. Results presented in Table 4 indicate also that the mutations studied have no effect on the absolute gene product quantity as compared to the normal Pgd ÷ alleles. qmmunochemical analysis revealed that all the semilethal and lethal mutations were due to alterations in the corresponding structural gene and most likely represent missence mutations. Evidence for the missence type of the induced mutations was obtained by polyacrylamide gel elecrophoresis, which demonstrated no changes in molecular weights of mutant antigens purified by means of cellulose-immobilized antibodies (Kogan et al., in press).
Discussion In many cases, the number of genetic loci determined for a given chromosome region in saturation experiments for induced lethals corresponds well to the number of bands in this region (Lefevre, 1974). The 20 30-fold excess of D N A per chromomere of a polytene chromosome of D. melanogaster over that necessary to code for a polypeptide with an average molecular weight raises a major question concerning the functional role of this "excess D N A " . A polycistronic operon-like structure of a chromomere was proposed by several authors in order to explain the functions of the "excess" D N A (Davidson and Britten, 1973; Olenov, 1974). It is possible to suppose that in saturation experiments lethal mutations attributed to a single locus may affect the different genes of a polycistronic structure in a polar way and thus appear to be non-complementary, whereas the lethal effect would be due to the inactivation of several genes coding for a corresponding enzyme complex. According to this hypothesis a lethal mutation may be localised in a non-essential gene and result in the switching-off of adjacent vital genes (Davidson and Britten, 1973). This possibility cannot be neglected in cases in which nothing in known about the gene products of the loci studied and only lethal effects indicate alterations of gene function. We will consider our data concerning the Pgd locus in connection with the surmised polycistronic structure of a chromomere. The Pgd locus coding
for 6PGD is situated in the 2D3-5 region of the Xchromosome where the relatively faint bands are located. Saturation studies revealed 3-4 genetic units in this region (Gvozdev et al., 1975). Feulgen densitometry of D N A content in the 2D region permits to suggest the presence of a 5-8-fold excess of D N A per chromomere over the quantity needed to code for a polypeptide subunit of 6PGD of 50,000 mol. weight (Ananiev and Barsky, 1975). This result shows a D N A abundance also in these faint bands. Bewley and Lucchesi (1976) described a lethal in the Pgd locus but could not rule out a possibility that the lethal effect was due to mutation in a locus adjacent to the Pgd. The present paper analyses the nature of mutations in the Pgd locus; with a single exception all were induced by EMS. Three semilethal mutations affected 6PGD enzymatic characteristics, the rest 8 mutations resulted in zero activity although an immunologically defective product of the Pgd gene with a tool. weight identical to that of the normal 6PGD subunit (Kogan et al., in press) has been demonstrated in all cases. Thus in our experiments only the missence type mutations were discovered, although it is known that ethylmethanesulfonate is able to induce chromosomal aberrations (Auerbach and Kilbey, 1971 ; Bishop and Lee, 1973). The major conclusion is that all the mutations are located in a structural gene coding for 6 P G D and the alteration of 6PGD production is not due to mutational lesions in adjacent genes. In other words there are no grounds for suggesting a polycistronic structure of a chromomere inclusing the Pgd gene and for attributing the lethal effects to inactivation of several genes. An alternative interpretation, assuming an operon-like polycistronic structure with a single vital Pgd gene as a proximal one followed by distal non-vital genes, seems very doubtful. Our data concerning the suppression of the lethal mutations in the Pgd locus also may be considered in relation to chromomere organization. Suppression is attained by mutations in the Zw locus and results in drastic decreases of G6PD activity. This allelenonspecific suppression as well as a previously reported suppression of rudimentary mutations (Bahn, 1973) seem to be the rare cases where a suppression mechanism in animals may be explained in biochemical terms. Consideration of the biochemical nature of suppression is especially attractive because of the evidence in favour of a monocistronic structure of the Pgd locus. The Pgd and Zw loci control two oxidative reactions in t h e beginning of the pentose phosphate pathway. The pentose phosphate route in animal cells is considered usually as a major source of reduced nicotinamide-adenine dinucleotide phosphate and
V.A. Gvozdev et al. : Organization of Genetic Loci in
Drosophila melanogaster
Glucose-5-phosphate ~_____.___ ~
.•" fructose-6-phosphate 1 ~
fructose -1.5-diphosphate ~ n t d O ; ~" gtycercddehyde-3- phosphate
Gtycolysis
icise
197
Zw [OCUS ~- NADPH
6- phosphogluconote LPgd I°cus ~ NADPH ~ - 5 - phosphate r]bulose [ pentoses)
trcmsketoLose nucleic acid biosynthesis
Pentose phosphate pctthwcty
pentoses needed for nucleic acid biosynthesis (Lehninger, 1972). Pentose synthesis may also be accomplished by the use of metabolites of glycolysis; fructose-6-phosphate and glyceraldehyde-3-phosphate (Fig. 4). A switch-off the second oxidative reaction of the pentose phosphate pathway by a Pgd mutation has a lethal effect on Drosophila. The viability of the flies is restored by suppressor mutations in the Zw locus, which result in the decrease of G6PD activity, although the oxidative step of the pentose phosphate pathway now is blocked completely. These oxidative reactions seem to play no essential and indispensable role in metabolism and the deficit of NADPH is readily compensated for (Gvozdev et al., 1976). Pentose biosynthesis in flies carrying the Pgd and Zw negative mutations is possibly accomplished via the transaldolase and transketolase reactions using glycolytic metabolites (Fig. 4). The lethal effect of the Pgd mutation may be explained by accumulation of 6-phosphogluconate which, as has been shown in mammals, inhibits some enzymatic step of glycolysis (Parr, 1956). We suppose that as a result of the Pgd mutation both metabolic routes for pentose biosynthesis (Fig. 4) are interrupted. Suppressor mutations' in the Zw locus provide a flux of glucose-6-phosphate in glycolysis, preventing at the same time the deleterious accumulation of 6-phosphogluconate. Thus the suppressor mutations open a possibility of pentose biosynthesis via glycolytic and reversible non-oxidative reactions of the pentose phosphate pathway. Similar cases of suppression as a result of elimination of deleterious accumulation of toxic sugar phosphate esters were reported in yeasts and bacteria (Hartman et al., 1973). The supposed mechanism of suppression of the Pgd lethals is in good accord with our observations that the development of Drosophila larvae carrying the " Z e r o " Pgd mutations stops at the end of the first instar larval stage, at which time a severalfold increase in the activity of G6PD is attained (Wright and Shaw, 1970).
Fig. 4. Metabolic relations between the pentose phosphate pathway and glycolysis
Consideration of the suppression data suggests that the lethal effect of the Pgd mutations is due only to mutations in a single gene coding for 6PGD and provides no basis for speculations on alterations of another vital gene in a corresponding chromomere. Defects induced by the independently obtained mutations in the Pgd locus are repaired to the same extent by mutations in the Zw locus. In other words, consideration of the biochemical mechanism of suppression also demonstrates that the Pgd-mutations affect only 6PGD production and exert no effect on the other essential enzymes. It is necessary to underline that these data do not imply that the DNA of each chromomere codes for but one polypeptide. It was shown recently that the rudimentary locus coded for three enzymes for successive steps of pyrimidine biosynthesis (Rawls and Fristrom, 1975). On the other band repetitive DNA sequences were revealed in the Balbiani ring or ribosomal genes. It would be more likely to suppose that the different principles of genetic organization of chromomeres may reflect their functional divergence. An extended regulatory region in eukaryotic loci is often proposed in explanation of the relatively large quantity of DNA in a chromomere (Georgiev, 1969; Judd et al., 1972; Beermann, 1973). In this case a substantial part of induced mutations would be expected to affect a controlling region of a gene. We have obtained the opposite result--all the 11 lethal mutations were located in the structural gene coding for 6PGD. This result agrees with the conclusion (Schalet a. Sankaranarayanan, 1976) that mutational intralocus changes in Drosophila occur predominantly in DNA which codes for the structural portion of the gene. Most of the spontaneous mutations affecting human G6PD production were considered to be point mutations inside the structural element (Luzzatto, 1974). At least 40% of the 105 nitrosoguanidine induced mutations affecting hypoxanthine-phosphoribosyl transferase in mouse L-cells
198
V.A. Gvozdev et al. : Organization of Genetic Loci in Drosophila melanogaster
were also shown to be structural gene alterations (Wahl et al., 1974). Similar results were recently obtained for the locus coding for alcohol dehydrogenase in D. melanogaster (Schwartz and Sofer, 1976). No extended regulatory zone was demonstrated for the rosy locus in D. rnelanogaster (Gelbart et al., 1974). These data may be explained by the mutational resistance of eukaryotic DNA sequences containing controlling elements. For example deficiencies of nucleotide sequences or simultaneous alterations in multiple sites may be necessary to affect the functioning of gene-controlling regions. However different point of view is possible if it is supposed that the size of regulatory zones of eukaryotic genes at least does not exceed the length of the adjacent structural gene (or genes). It is difficult to reconcile the our data and those cited with the suggestion that mutational rates in genetic loci increase in eukaryotes proportionally to DNA content in a wliole genome and possibly in a genetic locus (Abrahamson et al., 1973). The functions of the bulk DNA are difficult to determine, but hypotheses that this DNA may be involved for example in chromosome mechanics or evolution of eukaryotic DNA sequences cannot now be neglected. Acknowledgements. The authors are greatly indebted to Dr. L.A. Zamchuck for guidance in the complement fixation experiments, Prof. Yu.M. Olenov and Prof. R.B. Khesin for discussion and to Mrs. G.T. Khromeshina, O.Yu. Braslavskaya, M.I. Volkova and V.P. Kopaeva for generous help.
References Abrahamson, S., Bender, M.A., Conger, A.D., Wolf, C.: Uniformity of radiation induced mutation rates among different species. Nature (Lond.) 245, 460-462 (1973) Ananiev, E.V., Barsky, V.E. : DNA content, replication and transcription rate in different sites of the X-chromosome of D. melanogaster. Molec. Biologia (Russ.) 9, 752 760 (1975) Auerbach, C., Kilbey, B.J.: Mutations in eukaryotes. Ann. Rev. Genet. 5, 163-218 (1971) Bahn, E. : A suppressor locus for the pyrimidine requiring mutant: rudimentary. Dros. Inform. Service 49, 98 (1972) Beermann, W. : Chromomeres and genes. In: Results and problems in cell differentiation, Vol. 4, pp. 1-33. Berlin-Heidelberg-New York: Springer 1973 Bewley, G.C., Lucchesi, J.C.: Lethal effects of low and " n u l l " activity alleles of 6-phosphogluconate dehydrogenase in D. melanogaster. Genetics 79, 451-457 (1975) Bishop, J.B., Lee° W.R. : Chromosome breakage in D. melanogaster induced by a monofunctional alkylating agent (EMS). Mutation Res. 21, 327-333 (I973) Brenner, S. : The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974) Davidson, E.H., Britten, R.J. : Organization, transription and regulation in the animal genome. Quart. Rev. Biol. 48, 565 613 (1973) Gelbart, W.M., McCarron, M., Pandey, J., Chovnick, A. : Genetic limits of the xanthine dehydrogenase structural elements within the rosy locus in D. melanogaster. Genetics 78, 869-886 (1974) Georgiev, G.P.: On the structural organization of operon and the regulation of RNA synthesis in animal cells. J. theor. Biol. 25, 473-490 (1969)
Gvozdev, V.A., Birstein, V.J., Faizullin, L.Z.: Gene dependent regulation of 6-phosphogluconate dehydrogenase activity of Drosophila melanogaster. Molec. Biologia (Russ.) 6, 876-887 (1970) Gvozdev, V.A,, Gerasimova, T.I., Kogan, G.L., Braslavskaya, O.Yu. : Role of the pentose phosphate pathway in metabolism of Drosophila melanogaster elucidated by mutations affecting glucose-6-phosphate and 6-phosphogluconate dehydrogenases. FEBS Letters 64, 85-88 (1976) Gvozdev, V.A., Gostimsky, S.A., Gerasimova, T.I., Dubrovskaya, E.S., Braslavskaya, O.Yu. : Fine genetic structure of the 2D32F5 region of the X-chromosome of D.melanogaster. Mol. gen. Genet. 141, 269-275 (1975) Hartman, P.E., Hartman, Z., Stahl, R.C., Ames, B. : Classification and mapping of spontaneous and induced mutations in the histidiu operon of Salmonella. Advanc. Genet. 16, 1-34 (1971) Hartman, P.E., Roth, J.R.: Mechanisms of suppression. Adv. in Genet. 17, 1-106 (1973) Judd, B.H., Shen, M.W., Kaufman, T.C. : The anatomy and function of a segment of the X-chromosome of D. melanogaster. Genetics 71, 139-156 (1972) Kazazian, H.H., Young, W.J., Childs, B. : X-linked 6-phosphogluconate dehydrogenase in Drosophila: Subunit association. Science 150, 1601-1602 (1965) Kazazian, H.H. : Molecular size studies in 6-phosphogluconate dehydrogenase. Nature (Lond.), 212, 197-198 (1966) Kogan, G.V., Rosovsky, Ya.M., Gvozdev, V.A.: Purification and immunological characteristics of the wild type and mutant forms of 6PGD. Molec. Biologia (Russ.). in press Lefevre, G. : The relationship between genes and polytene chromosome bands. Ann. Rev. Genet. 8, 51-62 (1974) Lehninger, A.L. : Biochemistry. New York: Worth publishers 1972 Lindsley, D.L., Grell, E.H. : Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627 (1968) Luzzatto, L.: Genetic heterogeneity and pathophysiology of glucose-6-phosphate dehydrogenase deficiency. Brit. J. Haematol. 28, 151-155 (1974) Olenov, Yu.M. : On the genome organization in Metazoa. Cytologia (Russ.) 16, 403-418 (1974) Ouchterlony, O.: Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta. Path. Microbiol. Scand. 32, 231-240 (1953) Parr, C.W.: Inhibition of phosphoglucose isomerase. Nature (Lond.) 178, 1401 (1956) Rawls, J.M., Fristrom, J.W. : A complex genetic locus that controls the first three steps of pyrimidine biosynthesis in Drosophila. Nature (Lond.) 255, 738-740 (1975) Schalet, A.P., Sankaranarayanan, K. : Evaluation and re-evaluation of genetic radiation hazards in man. I. Interspecific comparison of estimates of mutation rates. Mutation Res. 35, 341 370 (1976) Schwartz, M., Sorer, W. : Alcohol dehydrogenase-negative mutants in Drosophila: Defects at the structural locus? Genetics 83, 125-136 (1976) Wahl, G.M., Hughes, S.H., Capecchi, M.D. : Immunological characterization of hypoxanthine-guanine phosphorybosyl transferase mutants of mouse L cells : evidence for mutations at different loci in the HGPRT gene. J. Cell Physiol. 85, 307-320 (1975) Wasserman, E., Levine, L.: Quantitative micro-complement fixation and its use to study of antigenic structure by specific antigen-antibody inhibition. J. Immunol. 87, 290595 (1961) Wright, D.A., Shaw, C.R. : Time of expression of genes controlling specific enzymes in Drosophila embryos. Biochem. Genet. 4, 385-394 (1970)
Communicated by Ch. Auerbach Received September 10 / November 6, 1976
