napts, a Mutation affecting sodium channel activity in Drosophila, Is an allele of mle a regulator of X chromosome transcription

Cell, Vol. 66, 949-959, September 6, 1991, Copyright 0 1991 by Cell Press

napfS, a Mutation Affecting Sodium Channel Activity in Drosophila, Is an Allele of m/e, a Regulator of X Chromosome Transcription Maurice J. Kernan,‘? Mitzi I. Kuroda,*§ Robert Kreber,’ Bruce S. Baker,* and Barry Ganetzky’ *Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 *Department of Biological Sciences Stanford University Stanford, California 94306

tial. Trains of action potentials are the neuronal signals that govern locomotor activity in most animals. Paralysis of nap’* mutants is associated with the loss, at restrictive temperatures, of action potentials in many axons of the larval and adult nervous systems (Wu et al., 1978; Elkins and Ganetzky, 1990). Physiological and pharmacological evidence indicates that membrane excitability and sodium channel activity are reduced even at permissive temperatures in napfs mutant flies (Wu and Ganetzky, 1980) and in neurons cultured from nap” third

Summary

suggest that it is the number of sodium channels that is reduced in extracts from heads of adult nap’* homozygotes (Kauvar, 1982; Jackson et al., 1984). However, direct measurements of sodium current in cultured embryonic neurons show no difference in sodium current density between napIs and wild-type cells (O’Dowd and Aldrich, 1988). Genetic evidence also indicates a constitutive defect involving sodium channels. A decrease in sodium channel number would tend to decrease the electrical excitability of cell membranes. In fact, it has been shown that even at permissive temperature, nap’” suppresses the effects of a variety of mutations (Shaker, ether-a-go-go, Hyperkinetic) that affect potassium channels and cause increased membrane excitability (Ganetzky and Wu, 1982). Moreover, napIs interacts with paralytic (para), a gene now known to encode sodium channels (Loughney et al., 1989a). In a nap+ background, some hypomorphic alleles of para, such as para’s’, cause rapid temperature-sensitive paralysis (Suzuki et al., 1971); null alleles are homozygous lethal (Ganetzky, 1984). These mutations have more severe effects in a nap’” background: parats’ homozygotes are now lethal andpara null mutations are lethal even when heterozygous (Ganetzky, 1984). Conversely, increasing the dose of the wild-type para gene suppresses the temperaturesensitive paralysis of nap’” (Stern et al., 1990). These data show that the effect of nap” is consistent with a reduction in sodium channel activity and that this effect can be overcome by increased doses of a wild-type channel structural gene. The nature of the nap+ gene product is otherwise unspecified: it could also be asodium channel component, or could be involved in any stage of channel synthesis or operation. In this paper, we show that naprs is in fact an allele of a gene involved in X chromosome dosage compensation. In many heterogametic organisms, a dosage compensation mechanism is required to equalize the amount of X-linked gene product expressed by the single male X and the two female X chromosomes. In Drosophila, this is achieved by a male-specific enhancement of X chromosome transcription (reviewed in Baker and Belote, 1983; Jaffe and Laird, 1986; Lucchesi and Manning, 1987). In males, a group of transacting factors doubles the transcription rate of most X-linked genes; in females this process is negatively regulated by the action of the Sex-lethal locus such that hypertranscription of the X chromosome does not occur. The

instar larvae (Wu et al., 1983). Studies

nap” is a recessive mutation that affects the level of sodium channel activity and, at high temperature, causes paralysis associated with a loss of action potentials. We show, by genetic complementation tests, germline transformation, and analysis of mutations, that nap” is a gain-of-function mutation of m/e, a gene required for X chromosome dosage compensation and male viability. Molecular analyses of nap and m/e mutations indicate that m/e+, nap+, and nap” activities are encoded by the same open reading frame and suggest that nap’* is due to a single amino acid substitution. Although naprs is known to act via para+, an X-linked sodium channel structural gene, its effect is not due to a simple defect in para+ dosage compensation. Introduction As part of a genetic approach to understanding the molecular basis of nervous system function, many types of behavioral mutant have been isolated in Drosophila melanogaster (reviewed in Hall, 1982). napts (no action potential, femperature-sensitive) is one of a class of mutations that cause rapid temperature-sensitive paralysis (reviewed in Ganetzky and Wu, 1986). Homozygous nap” flies and larvae are paralyzed immediately upon transfer to temperatures above 35%; on return to room temperature, recovery is complete and equally rapid (Wu et al., 1978). Both males and females are affected. nap’* is recessive: a single copy of the wild-type gene is sufficient to overcome the effect of one or two copies of the mutated version. The paralytic phenotype of nap’” is believed to be the result of a reduction in the number or activity of voltagegated sodium channels.

These are proteins that span the

plasma membrane of neuronal cells, forming an aqueous pore that opens in response to depolarizing changes in the membrane electrical potential. The open pore is specifically permeable to sodium ions; the resulting inward sodium current can initiate and propagate an action poten-

t Present address: Howard Hughes Medical Institute, Cellular and Molecular Medicine M-049, University of California-San Diego, La Jolla, California 92093. 5 Present address: Department of Cell Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030.

of toxin binding

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Table 1. nap and m/e Alleles Used in Thus Work Class

Allele

Stock

Mutagen

nap”m/e+

nap”’ nap’s2

1 2

napcs3

3

EMS Hybrid dysgenesis Gamma ray

3

EMS

nap m/e+

nap’s’ nap’s5 nap6

4

EMS

nap m/e-

T(2;3)nap’

3

Gamma ray

115,649

nap’““’

5

Gamma ray

4,700

nap-m/e

# Screened 5,000 53,637 115,649 3,411

Selection

Reference

Comments

1s paralysis when homozygous Failure to complement nap”’ paralysis Farlure to complement nap”’ paralysis Failure to complement lethality of nap”’ rn para” background Serendipitous: isolated due to lethality of linked mutation j(2) 17-l Failure to complement nap’$’ paralysis Reduction in temperature sensitivity in combination with nap”’ and pafaa”’ Male lethality when homozygous Male lethality when homozygous Male lethality when homozygous

Wu et al. (1978) This work

A, C A, 6 C

This work

A.

This work

A, C

This work

C

This work

C

This work

C

m/e’ mleMAr m/eR”“” m/eD”‘2

Spontaneous Spontaneous Spontaneous EMS

mkF8

EMS

m/e” mle’38

EMS Gamma ray

2,600 23,126

Male lethality when homozygous Male lethality when homozygous

m/e*203

Gamma ray

23,126

Male lethality when homozygous

mle+2’

Gamma ray

23,126

Male lethality when homozygous

mle’235

Gamma ray

23,126

Male lethality when homozygous

m/e.z”

Gamma ray

23,126

Male lethality when homozygous

mlef286

Gamma ray

23,126

Male lethality when homozygous

mlefzq3

Gamma ray

23,126

Male lethality when homozygous

m/eRX

EMS

1,834

Male lethalrty when homozygous

C

Fukunaga et al. (1975) Golubovsky and lvanov (1972) Loverre and Cicchetti (1980) T. Schupbach (unpublished data) T. Schupbach (unpublished data) Belote and Lucchesi (1980b) M. Scott and J. Lucchesi (unpublished data) M. Scott and J. Lucchesi (unpublished data) M. Scott and J. Lucchesi (unpublished data) M. Scott and J. Lucchesi (unpublished data) M. Scott and J. Lucchesi (unpublished data) M. Scott and J. Lucchesi (unpublished data) M. Scott and J. Lucchesr (unpublished data) This work

Class: alleles are classified according to their behavior in complementation tests (see text, Figure 5). Stock: the parental stocks from which the chromosomes were derived. These are as follows: w, wild-caught chromosome; 1, a Canton S stock in the laboratory of Dr. S. Benzer; 2, a wild-caught P-type stock, from the laboratory of Dr. W. Engels; 3, a Canton S population: 4, an isogenic cn bw stock; 5, nap”’ cn; 6, a cn bw stock in the laboratory of Dr. T. Schupbach; 7, a cn bw stock in the laboratory of Dr. J. Lucchesi; 8, a bw stock in the laboratory of Dr. J. Lucchesi; 9, bw; st. Mutagens: EMS, ethyl methanesulfonate; #Screened: number of second chromosomes screened in each mutagenesis. Comments: A, as will be demonstrated the different nap’” alleles may not have arisen independently. B, although naptE2was isolated under hybrid dysgenic conditions, no P element DNA was detected at the nap locus by in situ hybridization, nor was this allele revertible in dysgenic crosses. C, as will be demonstrated, nap mutations are allelic to m/e: we suggest that these mutations be renamed in future as follows: m/enaprsr,m/enaprs2, m/e”ap’s3,m/e”BP*E’m/e”“Q’ , s5,m/enap6,T(2;3)m/eoBp7, and mle”“~““‘.

cis-acting regulatory sites appear to be dispersed throughout the X chromosome and, in at least some cases, are closely linked to individual genes. maleless (m/e) is one of four autosomal genes known to be required specifically for dosage compensation. Mutations in any one of these genes are male-specific lethals, probably because of insufficient X transcription (Belote and Lucchesi, 1980a). Females are apparently unaffected. m/e is also required for development of the male germline (Bachiller and Sanchez, 1986). In an accompanying paper (Kuroda et al., 1991) the isolation of the m/e+ locus and the molecular characterization of its wild-type product are reported. Here we show that the napr” mutation is a gain-of-function allele, and m/e mutations are loss-of-function alleles of the same gene. We

also present a molecular analysis of the different types of mutation and we interpret the effect of r@ on sodium channels in terms of the’ role of the m/e gene product in transcriptional regulation. Results nap’* Alleles, Though Recessive, Are Gain-of-Function Mutations The initial nap allele (nap” of Wu et al., 1978; this allele is here designated napfsl) was isolated as a recessive mutation that caused paralysis of flies at high temperature and had no other obvious phenotypic effects. Additional alleles with these properties were subsequently isolated as mutations that failed to complement either the paralytic pheno-

nap Is an Allele of m/e 951

30

O

Figure 1. Nonstandard ciencies

Complementation

of nap”, nap6, and nap Defi-

Flies from the stocks nap6 cnlSM66, nap”‘lSM66, Df(2R)naps//n(2LR)G/a, and Df(2R)bw”~2LC~2R//n(2LR)Gla were mated in five combinations to produce the progeny classes nap6 cnlnap”’ (lane l), Df(2R)bwy”zLcWnapfs’ (lane 2), nap6 cn/Df(2Rjbw”DB2Lc~zR(lane 3), Df(2R)napa/ nap’” (lane 4) and napecn/Df(2R)napS (lane 5). Flies of the genotypes nap6cn/nap”4 and Df/nap”‘are paralyzed at 39% and fall to the bottom of the chamber, but recover at 30%. napVDf flies show wild-type behavior-rapid, coordinated movement-at both temperatures. nap’ is closely linked to a lethal mutation in a separate complementation group, so that its behavioral phenotype when homozygous is not known.

type of napfs’ or its lethal interaction with para (Table 1). This class of allele is here referred to collectively as nap”. Alleles isolated due to their behavioral effects in combination with nap”, but which do not cause paralysis when homozygous, are referred to as nap. The first indication that nap” was not a simple loss-offunction mutation came from comparison of the relative contributions to the paralytic phenotype of nap’* alleles (Table 1) versus deletion mutations. nap’* alleles heterozygous with the allele nap6 cause paralysis at high temperatures (39°C) but deletions of nap+ such as M(2R)bt~“~~~~y~~ or Df(2t?)naps do not cause paralysis when heterozygous with nap6(Figure 1). This result can be interpreted as showing either negative complementation between two hypomorphic alleles in the heterozygote nap%ap”, or that nap’* mutations are gain-of-function mutations that by themselves have a more severe phenotypic effect than a deletion of the gene. Experiments comparing the severity of paralysis in nap%apts homozygotes and nap’*/Df(nap) heterozygotes confirmed the second interpretation. Over a range of temperatures, napfs’/napfs7 flies showed a more severe defect, i.e., showed more severe paralysis at a given temperature and tended to paralyze at lower temperatures, than napfs’/Df(2ff)nap77 heterozygous siblings (Figure 2). The same result was obtained (testing at a single temperature) for another deficiency chromosome, Df(2R)nap9, as compared with napts’, naptsn,and nap@‘(Figure 3). In each of these experiments, the sibling classes compared were identical with respect to chromosomes other than the second, on which nap is located; thus unlinked modifiers (nap suppressors in deletion stocks or enhancers in nap’” stocks) cannot account for these results.

Figure 2. nap”’ cnlnap” cn Homozygotes Are More Temperature Sensitive Than nap’s’ cnl Df(2R)nap” cn* Heterozygotes

A

34.5

37.5

36

temperature

B

33 O

36’

n

0

0

0

39

Flies from the stocks nap”’ chap’” cn and Df(2ff)nap” cn’h(2LR)Gfa were crossed to give nap”’ cnlDf(2R)nap” cn+ heterozygotes. Males of this genotype were backcrossed to nap”’ cnlnap”’ cn homozygotes. The F2 progeny were reared at 21 OC and sorted by sex and eye color (cn vs. cn’). Open symbols, napfs’ cnDf(2Ff)nap” cn’; closed symbols, napIs’ ml nap”’ cn. Squares, males; circles. females. (A) Quantitation of paralysis. Flies were aspirated, five at a time, into a vial held at a given temperature, and those unable to right themselves between 25 and 30 s after aspiration were counted as paralyzed. Although nap’“flies recover rapidly from, and appear to be unaffected by, brief periods of paralysis, flies were allowed to recover for at least 2 hr before retesting at another temperature. Between 30 and 50 flies of each progeny class were tested; each data point is the mean of groups of ten flies. Standard error bars are shown only for males, for clarity; those for females are equal or smaller. (B) Photographs of eight males and eight females of each type were taken in a separate experiment, up to 3 min after each temperature shift.

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Table 2. nap Loss-of-Function Allelic to m/e

Mutations Are Male Lethal and

T(2;3)nap7 m nap-hapnap-/m/e’ nap-lmPAK nap-lmle@2 nap-lmkPa nap-/m/e” nap-lmler2” nap-/m/eRK nap +I+msl- 1 nap-+l+msl-2 nap-l+; +/ms/-3

Figure 3. Df(2R)napQ Has a Weaker Effect on Temperature Than Any of Three nap” Alleles

Sensitivity

Df(2R)nap’ cnlln(2LR)Gla flies were crossed to each of three nap’” stocks: nap”’ cn+, napfS2cn+, and nap”’ cn+. napYDf(2R)nap9 cn progeny females were crossed to anap”’ cn tester stock and the F2 progeny sorted by sex and eye color. The Df(2R)nap9 deficiency uncovers both nap and cn, so that no recombination should occur between these two loci. Paralysis testing was carried out as for Figure 1, at 36% only. Each nap%ap” cn combination should be compared with its sibling Df(2R)napg chap’“’ cn class. Differences in the phenotype of Df(2R)nap* cnlnap”’ cn flies arising from different crosses may be the result of background variation in the different nap’” stocks, or of maternal or epigenetic effects.

A modifier closely linked to nap could explain any one of the results, but is unlikely to be segregating similarly in all of the crosses performed. These results show that napfs alleles, though recessive to nap+, behave as gain-of-function mutations. They cannot be hypermorphs (mutations that cause overexpression of a wild-type activity); were this the case, nap%ap+ would be more mutant in phenotype than napVDf(nap)-in fact, the converse is true. The characterization of napfs alleles as gain-of-function mutations raises the question: what is the phenotype of a loss-of-function mutation at this locus?

nap Loss-of-Function Mutations Are m/e Alleles Two approaches have been used to isolate putative lossof-function mutations of the gene identified by nap’“. The first was based on the finding that napVDf(nap) has a paralytic phenotype, whereas nap’*/+ is wild type. Thus, T(2; 3)nap’ was isolated in a screen for gamma ray-induced mutations that fail to complement the paralytic phenotype of naptS7.Cytological examination showed it to be a reciprocal translocation with breakpoints in 42A2-7-the region known to contain nap+ (Wu et al., 1978; Kuroda et al., 1991)-and in the centric heterochromatin of chromosome arm 3R. The second approach was based on the finding that napVDf(nap) is less severe in its paralytic phenotype than is napVnaptS. Thus, napfs7”was isolated as a gamma ray-induced derivative of naptS with an altered temperature sensitivity: nap fS”‘lnapr* flies are still paralyzed, but with a less severe phenotype than nap’* homozygotes. When homozygous, neither T(2;3)nap7 nor naptsfr’

nw”“’ f

0 0 0 0 0

34 95 35 57 72

0 0 41

70 00 34

m

f 88 49 153 89 67 49 45 24

46 26

31 29

Males(m) and females (9 from the stocks T(2;3)nap’/CyO and nap’““‘/ In(2LR)Gla were crossed to females and males from the same stocks, or from stocks carrying m/e and msl mutations together with the balancers CyO, In(2LR)Gla. SM68, or T&43. Progeny heterozygous for nap and m/e or msl mutations were distinguished by their lack of balancer markers (Cy, G/a, Ser) and sorted by sex. Blanks, not done. Both males and females of all other expected progeny classes were also recovered from all crosses. Reciprocal crosses were carried out for most combinations; the data from these were pooled. The single male of apparent genotype nap’““‘/m/e may in fact have been nap”“‘/CyO, as Cy was occasionally difficult to score in this cross.

causes paralysis at any temperature tested (up to 39%) in females, but surprisingly, both cause lethality of homozygous males (Table 2). Since the male-lethal mutation m/e was known to map nearby (Kuroda et al., 1991) we tested the loss-of-function mutations for failure to complement m/e. For all seven m/e mutations tested, T(2;3)nap’/ mle and napts’r’lmle heterozygotes were male lethal; females were viable and did not display a temperaturesensitive phenotype (Table 2). The failure to complement male-specific lethal mutations was specific to alleles of the m/e locus: males doubly heterozygous for nap’*“’ or T(2; 3)nap7and male-lethal mutations at three other loci, msl-7, msl-2, and msl-3, were viable. Thus, two probable loss-offunction mutations, isolated on the basis of their effect on the napLStemperature-sensitive phenotype, are also malelethal m/e alleles. Most m/e Alleles Fail to Complement napLs Because the nap” and m/e phenotypes are so dissimilar, and because both nap”“’ and T(2;3)nap7 were gamma ray induced, we considered-itjikely that these mutations were both affecting two closely linked or nested genes with different functions. To test this hypothesis, m/e mutations with no cytologically visible abnormality were tested for their ability to complement the paralytic phenotype of nap” mutations (Table 3, Figure 4). Surprisingly, 13 of 14 m/e alleles tested showed some degree of paralysis (i.e., failed to complement) in combination with naprS mutations; the 14th, rn!,‘+(, showed complete complementation. The observed failure of complementation was not the result of a dominant, nonspecific interaction between nap” and genes affecting dosage compensation, because double heterozygotes for nap’” and msl-2 or msl-3 mutations did not show paralysis. Furthermore, the paralytic phenotype

nap Is an Allele of m/e 953

Table 3. nap’*/m/e Complementation

Test

nap

ts1 ts ts ts ts 1s ts + ts ts ts ts ts ts ts + +

ts2 ts ts ts ts +

ts3

h-4

ts5

ts ts ts ts ts

ts ts ts ts ts ts +

ts ts ts ts

+ ts ts ts ts ts ts ts

+ +

35

O

2.5

O

ts +

+ +

Stocks containing different m/e alleles were crossed to nap’” stocks, and nap/m/e flies were sorted from the Fl progeny and tested for paralysis at 39%. +, wild-type behavior; rapid coordinated movement with no sign of paralysis. ts, any degree of paralysis, ranging from intermittent with a gradual accommodation to continuous and complete. Blank, not tested.

of napfs/m/e flies mapped specifically to the nap region: a Y-linked duplication of this region rescued the mutant phenotype (Figure 4). It is unlikely that 13 of 14 m/e mutations, isolated only on the basis of their male-lethal phenotype, affect two separate genes; we conclude instead that nap’* and m/e mutations are allelic, i.e., occur in the same gene or in genes that share at least some coding region. Although most m/e mutations fail to complement the paralysis of nap’“, nap9 m/e males are viable. Thus, nap’* mutations complement m/e male lethality and so must retain the activity required for dosage compensation. interpretation of the Genetic Data Two wild-type activities have been genetically defined for this locus (see Figure 5). These are m/e+, which is required for dosage compensation in males but has no apparent function in females, and nap+, which is at present defined only as an antagonist of (and dominant to) nap’“. The majority of mutations at the locus result in the reduction or loss of both activities; most m/e mutations, together with T(2; 3)nap7 and naprs”‘, are of this type (m/e-nap-). However, two mutations cause the loss of only one of the two activities and demonstrate that the nap+ and m/e+ activities are potentially independent. Thus, m/eRK lacks m/e+ function but retainsnap+ activity(m/e-nap+), whilenapsism/e+nap-. In nap’* mutants, the nap+ activity has been altered to produce a novel neomorphic or antimorphic activity that acts in both sexes; nap” mutants retain m/e+ activity (mle+napts). One further combination of activitiesm/e-nap”-is theoretically possible. The corresponding type of mutation has not been observed, and its phenotype cannot be predicted from the genetic data, as it is not known whether m/e+ function is required for nap” activity.

Figure 4. Paralysis of nap’Vmle Heterozygotes Males from the stock nap*‘/SMGB (males and females from this stock are shown in lane 1) were mated to m/eMAK//n(2LR)G/a females, and napsrlmleMAK heterozygotes from their progeny were sorted by sex (females in lane 2; males in lane 3). Lane 4 contains +/Ynap+; nap9 mleMAKmales, the product of mating +/Ynap+; nap’“‘lSM6B to m/e’9 In(2LR)Gla. Photographs were taken at 35% and then at 25%.

A 10.5 kb DNA Fragment Rescues Both napts Paralysis and m/e Male Lethality The nap-m/e region has been isolated as aseries of cloned inserts of genomic DNA in bacteriophage and cosmid vectors, and the position of the T(2;3)nap7 breakpoint has been located (Kuroda et al., 1991). A 10.5 kb fragment of cloned normal

mle activity+ + 8/“q6K +-

napts,\\ 0+ ts

Figure 5. Interpretation

- +

mle

? 0 ts

0_ -

of the Genetic Data

The locus is represented as a single unit with two independently mutable activities, The wild-type state is shown at the top; states corresponding to four observed types of mutation are below. The question mark labels a potential state that has not yet been observed. Arrows indicate the direction of known mutational events.

Cell 954

A

1

genomic DNA in a P element for the rescue tions (Kuroda

2

B

1

spanning the breakpoint has been shown, transformation experiment, to be sufficient of the male-lethal phenotype of m/e muta-

et al., 1991). When the same P element insertion was crossed into a nap” background, it also rescued the temperature-sensitive paralytic phenotype (Figure 6A). These results indicate that the same 10.5 kb of DNA contains sequences encoding both m/e+ and nap+but not necessarily nap’“-activities. Only one transcription unit located completely within this fragment has been identified in screens of cDNA libraries (Kuroda et al., 1991) lending support to the hypothesis that both wildtype activities are encoded by the same gene.

Transformation with flap’* Mutant DNA If napr’ is a gain-of-function mutation in m/e+, it should be possible to transform homozygous m/e flies (male lethal, females unaffected) to naprs (males and females viable and temperature sensitive), with a cloned nap’” gene. A bacteriophage library was constructed with nap’* genomic DNA, and the 10.5 kb fragment corresponding to that which in wild type encodes the nap+ and m/e+ activities was isolated and introduced into an m/e mutant background by P element-mediated transformation and genetic crosses. Two independent inserts of this fragment conferred male viability, and temperature-sensitive paralysis (the nap” phenotype) in both sexes, on m/e homozygotes (Figure 66). No inserts were found that affected only one of these phenotypes. This confirms that nap’” is a gain-of-function mutation and that the nap’* activity is encoded within the same 10.5 kb of DNA as the m/e+ activity. Molecular Analysis of Mutations: Deletions We have shown that two distinct types of activity are properties of a single gene and that both are encoded within a defined fragment that contains a single known transcription unit. What is the structural basis of the different activities? How can they be mutated independently? As an approach to these questions, we undertook a molecular analysis of the different types of mutation.

2

3

Figure 6. Transformation with Wild-Type and Mutant Genes (A) A cloned m/e+ gene rescues tfie nap& phenotype. Flies carrying an insert of a ry+m/e+ transposon on the third chromosome, nap”‘/ Cy; ry P[ry+]m/es.Iby, were crossed to nap”‘/ Cy; ry/ry. The Cy’ progeny were sorted by eye color, to give nap*‘/nap”‘; ry/ry (lane 1) and nap/nap’“‘; ry P[ry+]m/eG.l/ry (lane 2) and photographed at 39%. (8) The same gene cloned from a nap” stock confers both male viability and temperature sensitivity on m/e homozygotes. An insert of a ry+nap’” transposon on the second chromosome (P[ry+]naplJ2.3)was crossed into the stock m/erzo3cn/SM6B; ry/ry, to produce the following classes: m/e~203cn/m/e~zo3cn;g&y, females only (lane 1); m/eY203cn P[ry+]nap”2.3/ m/e’Tn; rylryfemales (lane 2) and males (lane 3). A similar result was obtained with an insert (P[ry+]nap”4) on the third chromosome.

One possibility is that the different activities arise from different transcript forms. Indeed, m/e cDNAs representing two alternatively spliced transcript forms have been found (Kuroda et al., 1991); they contain long (1293 amino acids) or short (226 amino acids) open reading frames (ORFs) and correspond to 4 kb transcripts seen in Northern blots. Could the alternatively spliced or different size transcripts correspond to the different activities? Examination of .lesions associated with m/e mutations suggests not. In a Southern blot analysis, 8 of 13 m/e mutations showed small deletions at sites throughout the gene; 7 of the 8 do not delete coding sequences from the shorter ORF (Figure 7A). As both m/e+ and nap+ activities are lost in all 12 mutations, both activities appear to be encoded by the longer type of ORF. It remains possible, however, that some unidentified splice variants that share most of the coding region might underlie the functional diversity of the locus, Molecular Analysis of Point Mutations In attempting to determine the structural bases of the different activities, those mutations affecting only one of them are of particular interest. This class includes nap’“, naps, and m/eRK.Southern blots showed no restriction fragment length changes associated with these mutations. To locate smaller alterations, a nuclease assay for mismatched base pairs (Myers et al., 1985) was performed on mutant genomic DNAs and wild-type controls. The sequence of any altered site was then determined and compared with that of the wild-type probe DNA. RNA probes used in the assay were transcribed from subcloned wildtype genomic DNA (originating from a Canton S genomic library; Maniatis et al., 1978) and covered all sequences known to be transcribed, but not the entire 10.5 kb used in transformation experiments (Figure 7A). The results of one nuclease assay experiment are shown and interpreted in Figures 78 and 7C. mleRKgenomic DNA showed a single alteration relative to the isogenic strain frdm which it was derived, in a 0.94 kb Hincll fragment. Comparison of the mleRKsequence to

nap Is an Allele of m/e 955

Figure 7. Molecular Analysis of Mutations (A) Location of mutations. A map of the XbalSpel fragment of genomic DNA is shown, with Hincll restriction sites marked below the line. Above are the two known transcript types, with long and short ORFs indicated by boxes. Below are alterations associated with each of the alleles indicated. The triangle indicates an insertion. Closed bars indicate deletions of the corresponding lengths. Bracketed bars show the region within which each lesion is located. These are the simplest interpretations of changes seen on Southern blots of Hincll- and BamHI- plus Xbal-cut genomic DNA. The small arrows indicate the position of the two point mutations found by RNAase assay. Hatched bars represent fragments used as probes in Southern blot experiments and transcribed to generate probes for RNAase assays. B C (6) Results of an RNAase assay, showing alter1 23 45 ations in nap4, naprs5, and m/eRK. Fragment a 0.94 phe . -. r in (A) was transcribed with T3 RNA polymerase 0.85 and the RNA probe hybridized to Hincll-cut geCanton s 1 *I nomic DNAfrom Canton S (lane I), napealnapar ts4, 0.61 0.34 ,SS 2,s "P nap (lane 2), napfsSIDf(2R)bwVa*LCvzR(lane 3) bwl bw, the stock from which mleRK was derived -0.61 (lane 4) and m/eRK bw/m/eRK bw (lane 5). The sizes of the altered fragment (0.94 kb) and of the fragments generated by RNAase cleavage at mismatches are indicated. (C) Interpretation of the results of RNAase assays, confirmed by DNA sequencing. Three polymorphismswere found in the 0.94 kb Hincll fragment. A net 6 bp insertion common to probe and napts DNA results in a 0.85 kb cleavage product in Canton S. bw, and mleRK bw. (The predicted 0.09 kb product is lost in this experiment.) A single base-pair substitution in nap” generates 0.63 and 0.34 kb cleavage products, and in ml@, the 0.85 kb fragment is further cleaved at a single base-pair substitution to give 0.43 and 0.41 kb products. (D) All nap’” alleles carry the same single base-pair alteration. A Southern blot of Mael-cut genomic DNA probed with fragment c (shown in [A] above) reveals a 0.87 kb fragment (bottom band) present in all nap” alleles, but absent from Canton S. The persistence of the wild-type 1.25 kb fragment (second from bottom) in nap’$ alleles and the presence of larger fragments are due to incomplete Mae1 cleavage. Lane 1, Canton S; lane 2, nap”’ cn; lane 3, nap”’ cn+; lane 4, nap”“’ cn; lane 5, nap@; lane 6, napfs3; lane 7, nap’““, lane 8. naprs5/Df(2R)bwY”“c~z~.

the probe DNA showed a C to T transition at the predicted location (C ‘W in the cDNA sequence; Kuroda et al., 1991), resulting in a deduced substitution of leucine for proline 385. All nap” alleles showed the same two alterations relative to the Canton S stock from which napts3,naprs4,and napts5 were thought to have been derived. One change, specific to the five nap’* alleles, is 90 bp downstream of the change found in m/eRK. A comparison of probe and napIs’ DNA sequences at this position, PU, showed a transversion to G in nap’“‘. This generates an Mael restriction site (CATGJ. Southern blot analysis of Mael-digested genomic DNA (Figure 7D) showed an identical novel fragment in all five nap’” alleles, confirming that all carry the same base-pair substitution. The alteration results in a missense change from the deduced wild-type amino acid sequence, replacing threonine 415 with serine. A second polymorphism was found 0.5 kb upstream of the missense mutation. Here, the probe sequence and all five nap” alleles appear identical to each other, but differ from all other genomic DNAs examined, including the Can-

ton S laboratory stock in which three of the napfs alleles were generated. DNA sequencing showed a net 6 bp insertion (replacement of 1 bp with a 7 bp insert) within an intron, in nap’*‘, and in probe DNA, relative to one of these other genomic DNAs (m/e”‘). As the probe was derived from the cloned DNA that was shown by transformation to be nap+ and m/e+, this change cannot alone be responsible for the napts phenotype. The presence of both polymorphisms in all five napfs alleles strongly suggests that they did not arise independently. Even if the observed amino acid substitution were the only change that could cause the nap” phenotype, this could not account for the reoccurrence of the identical base-pair change at the observed frequencies (Table 1): two other nucleotide substitutions can give rise to the same amino acid change. A nonindependent origin of these alleles is at odds with the history of their isolation; at present we cannot explain these events. The m/eRKand nap@ mutations each alter one of the two activitiesof the gene product, leaving theother unaffected, yet the amino acid substitutions associated with these mu-

Cell 956

tations are only 30 amino acids apart, and no known cDNA sequence shows them to be separated by mRNA splicing. If these are the causative changes in each mutant, the nap and m/e activities are most likely properties of a single type of polypeptide product. No alteration relative to a parental stock was found by nuclease assay in nap6 or napfslfi genomic DNA. Either these mutations lie outside the region examined or are undetectable by RNAase cleavage if they reside within it. As the nuclease assay for point mutations is not exhaustive, other changes may exist in any of the genomic DNAs, within the region examined or upstream of the gene. For this reason, and because of the probable nonindependent origin of the different napfs alleles and the consequent uncertainty regarding the identity of their parental stocks, we cannot be sure that it is the Thr to Ser change that causes the nap” phenotype. Experiments to test this are in progress. Is nap Activity Related to Dosage Compensation? Thus far, the molecular data imply that the nap and m/e activities arise from the same polypeptide, but the independent mutability of the different activities draws a functional distinction between them. To examine further the relation between nap activities and dosage compensation, we tested whether the msl genes, which are required for dosage compensation, are involved in nap’” or nap+ activity. nap’* mutations were introduced by genetic crosses into flies mutant at each of the three msl loci. nap; msl double-mutant females were temperature sensitive, while nap%ap’; msl females were not (data not shown). Although it is not known if themslgene products are present in females, this result argues that even if present, they are not required in females for either nap’* or nap+ activity. Discussion Allelism of nap and m/e Mutations The main conclusion of this paper is that nap”, a mutation isolated because of its paralytic phenotype and subsequently shown to affect voltage-gated sodium channel activity, is an allele of m/e, a gene previously known for its role in dosage compensation. Our results further indicate that the effects on sodium channels and the effects on dosage compensation are distinct functions of the m/e locus and that an m/e product may function in females. We have presented genetic and molecular evidence to show that nap’” is a gain-of-function mutation, that the wild-type nap+ and m/e+ activities are encoded by the same gene, that nap” activity is encoded by a mutant version of this same gene, and that the nap and m/e activities, though encoded by the same gene and most likely contained in the same polypeptide, are occasionally mutated independently and are therefore in some ways functionally distinct. That nap’* is a gain-of-function mutation is evident from the fact that it makes a stronger contribution to the paralytic mutant phenotype than does a deletion of the gene. This conclusion is borne out by the transformation of a null to a napfS mutant phenotype by introduction of a cloned

fragment of nap” DNA. Because nap’” is recessive to nap+, its gain-of-function activity was surprising. One explanation for the dominance of nap+ is that the nap” and nap+ products bind competitively to a substrate for which nap+ has a stronger affinity than nap’*: thus in a nap’*/nap’ heterozygote, the nap” product would be displaced from its site of action. The failure of most m/e alleles to complement the paralytic phenotype of nap” mutations shows that loss of nap+ activity is usually concurrent with mutation to m/e. Assuming that loss of both activities is in most cases a single event (a reasonable assumption for 13 independent alleles, isolated solely on the basis of their male lethality) it appears that mle+ and nap+ activities are encoded by the same gene. The ability of a wild-type DNA fragment, containing only one known transcription unit, to rescue both nap’” and m/e mutant phenotypes supports this hypothesis. That nap” activity is encoded by a mutant version of this same gene is indicated by the simultaneous loss of m/e+ and naptS activity in the mutation nap’““‘, the simultaneous restoration of these two activities to m/e homozygotes by transformation with napfS DNA, and the location in the same ORF of nucleotide changes associated with the nap” and m/eRKmutations. Given the allelism of nap and m/e mutations, and the original identification of the locus as m/e, we suggest that all mutations at the locus be henceforth referred to as m/e, with the nap desibnation, where present, being superscripted: m/e’, m/e’“, mlenaPtSf,mIena@,etc. (see legend to Table 1). The Cause of the nap’* Paralytic Phenotype The identification of naprs as an allele of a gene that acts to control transcription and the characterization of this gene’s product (MLE) as a nuclear, chromosome-binding protein with similarities to nucleic acid helicases (Kuroda et al., 1991) suggest that the defect underlying the paralytic phenotype is likely to be at the level of gene expression. Previous analysis of certain mutations of para, a sodium channel structural gene, has led to the idea that the temperature-sensitive phenotype of these mutants is the consequence of a temperature-independent decrease in sodium channel expression rather than the production of structurally altered sodium channels (Loughney et al., 1989a, 1989b). Experimental evidence in other organisms has shown that as temperature is elevated, an increasing fraction of the availablesodium channels is required to maintain propagation of action potentials; at a sufficiently high temperature, action potentials are blocked even in normal individuals (Hodgkin and Katz, 1949; Colquhoun and Ritchie, 1972). Thus, a constitutive reduction in expression ofpara could result in sodium channel levelssufficient to conduct action potentials at low temperature but not to meet the demands of elevated temperature. This interpretation can also readily explain the nap” phenotype if the consequence of this mutation is to cause a temperature-independent reduction in the production of functional para transcripts (Loughney et at., 1989b; Stern et al., 1990). This suaestion is supported by results of

nap Is an Allele of m/e 957

pharmacological experiments demonstrating a constitutive reduction in sodium channel numbers in nap” flies (Kauvar, 1982; Wu et al., 1983; Jackson et al., 1984) and genetic experiments revealing that the napr* paralytic phenotype can be rescued by an extra dose of par& (Stern et al., 1990). Because loss-of-function m/e alleles affect the rate of transcription of X-linked genes (Belote and Lucchesi, 1980a)nap’sallelesprobablyalsoaffect the initiation or elongation steps of transcription. However, we cannot exclude the possibility that nap@ acts by affecting para RNA splicing, particularly as the putative helicase sequences most similar to MLE are those of three splicing factors (Kuroda et al., 1991). The para gene contains a large number of introns (Loughney et al., 1989a) and would be sensitive to a reduction in splicing efficiency or accuracy. A direct interaction between the nap andpara gene products was proposed (Ganetzky, 1984) on the basis of allelespecific interactions between nap’*’ and a series of para mutations. It had been found that the relative severity of phenotype (reduction in viability) of the para mutations was different between a nap+ and a nap” background: for instance, the mutation parasT’os has a relatively strong effect in a nap+ background, but shows the weakest interaction with nap’*. If, however, nap+ encodes a transcription factor and para’ encodes a sodium channel, how can this allele specificity be explained? There may still be a direct interaction, not between both proteins, but between the nap” or the nap+ protein and the para gene, the site of this interaction being mutated in para alleles such asparaST’Og. How might the amino acid substitution associated with naprS alter the activity of the protein so as to bring about a reduction in gene expression? The mutated threonine (Thr’) is adjacent to a consensus nucleotide-binding sequence (Walker et al., 1982) Gly x x Gly x Gly Lys Thr Thr*-the first of seven conserved motifs identify the protein as a putative helicase (Kuroda et al., 1991). While not part of the strict consensus, threonine occurs at this position in 36 of 101 viral sequences of this type (Gorbalenya and Koonin, 1989) and in the three yeast proteins (PFtP2, -16, and -22) to which MLE is most similar. However, it is unlikely that the conservative substitution of serine eliminates nucleotide binding, as serine is found at this site in four of the viral sequences. It is difficult to imagine how a purely quantitative change in nucleotide binding or hydrolysis could give rise to the antimorphic properties of the nap’* mutation; it may be the coupling of these processes to an activity affecting transcription that is altered. A coupling of ATP hydrolysis to sequence recognition has been proposed to explain the gain-of-function properties of a suppressor mutation in PRP16 (Burgess et al., 1990): mutation of a conserved amino acid, five residues away from the position altered in nap”, from tyrosine to aspartate, enables the PRP16 protein to recognize a variant splice branchpoint. Thus it is possible that a mutation affecting nucleotide binding or hydrolysis could be the basis of the gain-of-function phenotype of nap’“. These interpretations are, of course, still contingent upon a definitive proof that the base change we have observed is indeed the cause of the nap’* mutation.

The Relation between nap” Activity and Dosage Compensation Is the proposed effect of nap on para expression related to the normal function of the gene in dosage compensation? para+ is on the X chromosome and, from comparison of the phenotypes ofpara’*’ hemizygous males and homozygous females (both are viable and temperature sensitive) with paratSi/paraOheterozygous females (lethal), we infer that it is dosage compensated in males. Can a perturbation of dosage compensation explain the paralytic phenotype of nap’s mutations? A complete failure of dosage compensation in males would be equivalent to a 50% reduction in X-linked gene dosage. However, in females, a 50% reduction in para+ dosage is not sufficient to cause paralysis: paraVpara+ heterozygotes are not temperature sensitive. Thus, the nap” paralytic phenotype is probably not due to a simple failure to dosage compensate para’. Conversely, nap” does not greatly affect dosage compensation: nap’” males are no less viable than females. Comparing the phenotypes of paraW; napfS males (viable and temperature sensitive) and paraVpara+; nap’” females (lethal) (Ganetzky, 1984) we infer that the para locus is still dosage compensated in napfS males, even though its expression is reduced in both sexes. Thus, dosage compensation of X-linked genes in general, and of para in particular, does not appear to be greatly perturbed by nap’“. The most obvious distinction between the nap” effect and dosage compensation is a lack of sex specificity: nap” affects both females and males. (In many experiments, males showed a slightly more severe phenotype than females of the same nap” genotype. This could indicate a partial effect of nap” on dosage compensation, but might also result from unrelated sex-limited traits such as size.) Paralysis of nap’* males and females is concordant with the observed expression of the wild-type gene in both sexes (Kuroda et al., 1991). Some other factor, perhaps one or more of the msl gene products, must be involved in the male-specific regulation of dosage compensation; this factor must neither be required for, nor interfere with, the activity of nap’“. In fact, we have shown that naprS acts independently of the msl gene products. Thus, by several functional criteria, nap*’ activity is distinct from the action of the protein in dosage compensation. Is it a reflection of a role played by the wild-type protein in some other, non-sex-specific regulatory mechanism? Is There a Normal nap+ Function? We have defined genetically an activity, nap+, as being antagonistic and dominant to the effect of nap’*. Like nap”, it is not sex specific, and it is independent of the msl gene products; it is also independent of m/e+ activity. Our proposed explanation for the dominance of nap+ -displacement of the mutant protein from its site of action in nap’“/ nap+ heterozygotes-suggests that the protein may be present at that site even in the absence of nap”. Does nap+ have a role in the transcriptional control of para’ or other genes, in wild-type flies? The presence of the MLE protein in both sexes suggests

Cdl 958

a function additional to dosage compensation of the male X chromosome. One possibility is that it acts to boost the expression at the level of either transcription or splicing, of para or of a set of genes including para, in both sexes. The normal action of nap+ at these loci could be subtle, perhaps a Pfold increase in transcription similar to (though independent of) the effect of dosage compensation. In this case, acomplete loss of nap+ activity would not necessarily be expected to result in a discernible phenotype: the situation would be equivalent to heterozygosity for a deficiency of several loci, which is usually without‘apparent phenotypic effect. In particular, as stated above, a 50% reduction in para dosage is known to have no overt effect. It is also possible that nap+ is a redundant activity. Although mle’ appears, in Southern blot analyses, to be a single-copy gene, two pieces of evidence suggest the existence in Drosophila of other proteins sufficiently similar to it in structure to share some of its activities: three proteins, all with substantial similarity to MLE, are found in yeast (Company et al., 1991; Kuroda et al., 1991), and anti-MLE antisera detect cross-reacting proteins in Western blots and on polytene chromosomes (Kuroda et al., 1991). However, in the absence of direct evidence-a phenotypic effect of m/e null mutations in females- the case for a normal function for nap+ remains unproven. What is the evolutionary relation between the different activities of the protein? The independence of nap+ activity from other known transacting factors, in contrast to the requirement for three other gene products in dosage compensation, suggests that the putative non-sex-specific function might be the more primitive (perhaps originally being a splice factor activity that came to influence transcription, in a system in which splicing and transcription were coupled) and might subsequently have come under sex-specific control. However, nap+ activity could be an escaped component of a preexisting dosage compensation system, acting on sites at which either sequence properties or transacting factors relieve it of the requirement for its sex-specific cofactors. The visibly different pattern of MLE binding between the sexes suggests that different binding modes or sites underlie the nap’ and m/e+ activities: an understanding of the actual binding substrate of the protein in each case may clarify both the relation between the different activities and their evolutionary origins. Experimental

Paralysis Testing All flies to be tested were reared at a constant 21 OC. If sorted under ether anesthesia, they were allowed to recover for at least 24 hr prior to testing. For testing, flies were aspirated into glass vials partially submerged in a water bath held at the required temperature. For photography, flies were confined to compartments in a chamber constructed from two 50 x 75 m m microscope slides separated by a 4 m m silicone rubber gasket, the chamber being partially submerged in a water bath. Isolation of nap”’ Derivatives Gamma ray-irradiated nap’*’ cn chromosomes (4700) were made heterozygous with nape, in apara”’ background, and screened for failure to paralyze at 31°C. Flies carrying an unmutated napB’ allele in this background were paralyzed at 28°C-300C; flies carrying instead a deficiency for nap were paralyzed only above 33OC. Two chromosomes, nap’““’ and napa”z, which consistently showed a reduction in temperature sensitivity, were recovered. napTQ behaved as a complete revertant to wild type; its analysis is still in progress and it is not discussed further in this paper. Cloning of nap” and m/en” Genomic DNA and Germline Transformation Genomic DNA was isolated from nap” and mleRX homozygotes, digested with Xbal, and fractionated on sucrose gradients. Fractions were assayed by Southern blot for the presence of a 13 kb Xbal fragment containing the nap gene, and DNA from positive fractions was cloned into the Xbal sites of the vector 12001 (Stratagene). The resulting libraries were screened with a cloned nap+ DNA probe. For transformation with nap” DNA, a 10.5 kb Xbal-Spel fragment was subcloned into the Xbal site of the P element transformation vector pDM23(D. MismerandG. M. Rubin, unpublisheddata), whichcontains the fy+ (rosy) eye color marker. The resulting 21 kb construct, P[ry+]nap”, was injected intorylryembryos, together with the helper plasmid p(A2,3), asourceof Pelementtransposase(Laskietal., 1986). Injected flies were mated to ry/ry homozygotes and their progeny screened for ry+ transformants. Molecular Analysis of Mutatlons For Southern blotting, genomic DNAs were electrophoresed on 1.2% (cut with Hincll), 1% (Mael), or 0.7% (other restriction enzymes) agarose gels, capillary blotted lo nitrocellulose, and probed with DNA labeled with 3ZP by nick translation. Blots were washed in 2 x SSC, 0.1% SDS at 68“C and exposed to film for 2-10 days. RNAase assays were carried out according to the method of Myers et al. (1985). To prepare bidirectional RNA probes, DNA was subcloned into a Bluescript vector (Stratagene), linearized by restriction, and transcribed with T3 or T7 RNA polymerase (Melton et al., 1984). Probes were hybridized to target genomic or cloned DNA overnight at 47°C; RNADNA hybrids were treated with lo-50 mglml RNAase A at 25OC for 30-90 min. Reactions were analyzed on 4% sequencing gels. DNA sequencing of putative mutant sites was carried out directlyon doublestranded plasmid subclones, by the dideoxy chain termination method (Sanger et al., 1977), using Sequenase (US Biochemical Corp.).

Procedures Acknowledgments

Mutant Strains The nap and m/e mutations used in this study are described in Table 1. Unlessotherwisereferenced, descriptionsof thefollowingmutations and chromosomes can be found in Lindsley and Grell (1966). Df(2R)bw”Bz‘c@n is a multiply rearranged chromosome that is deficient for the region 41 B to 42A2-7. Df(2R)nap is deleted for 41 D2-El to 42A2-7, Df(2R)nap’for 42Al-2 to 42E6-Fl , and Df(2R)nap” for 41 E2-4 to 42A710. The last three were gamma ray induced and were isolated due to their failure to complement nap”’ paralysis (R. K. and B. G., unpublished data). Map+ represents T(Y;2)L72, a Vanslocation of the region 41-43A inserted into BVy+ (Wu et al., 1978). SM68 and In(2LRjCyO are second chromosome balancers marked with Cy(Cur/y wings). ln(2LR)G/a is a second chromosome balancer marked with Glazed. msC7, msl-2, and msC3 (also known as m/e-3) are recessive male-lethal mutations at three different loci (Belote and Lucchesi, 1980b; Uchida et al., 1981; Lucchesi et al., 1982).

r

We are indebted lo C. Peterson for carrying out sequencing and transformation experiments. We thank A. Loverre, T. Schupbach, M. J. Palmer, M. Scott, and J. Lucchesi for generously supplying m/e mutants, A. Bejsovec for advice on RNA&e assays, M. Stern for enlightening discussions on several aspec& of this project, and members of the Ganetzky laboratory for advice and comments on the manuscript. This work was supported by predoctoral fellowship from the University of Wisconsin and a Lubrizol Industrial Fellowship to M. J. K., a postdoctoral fellowship from the American Cancer Society to M. I. K., NIH grant NS15396, a grant from the Lucille P. Markey Charitable Trust, a Klingenstein fellowship to B. G., and an NIH grant to B. S. B. This is paper number 3197 from the Laboratory of Genetics, University of Wisconsin-Madison. The costs of publication of thTS article were defrayed in part by the payment of page charges. This article must therefore be hereby

nap Is an Allele of m/e 959

marked “advertisement” in accordance solely to indicate this fact.

with 18 USC Section

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