X Chromosome Duplications Affect a Region of the Chromosome They Do

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1 Copyright by the Genetics Society of America X Chromosome Duplications Affect a Region of the Chromosome They Do Not Duplicate in Caenorhabditis elegans Philip M. Meneely and Kim D. Nordstrom Fred Hutchimon Cancer Research Center, Seattle, Washington Manuscript received December 18, 1987 Accepted February 25, 1988 ABSTRACT X chromosome duplications have been used previously to vary the dose of specific regions of the X chromosome to study dosage compensation and sex determination in Caenorhabditis elegans. We show here that duplications suppress an X-linked hypomorphic mutation and elevate the level of activity of an X-linked enzyme, although these two genes are located in a region of the X chromosome that is not duplicated. The effects do not depend on the region of the X chromosome duplicated and is stronger in strains with two doses of a duplication than in strains with one dose. This is evidence for a general elevation of X-linked gene expression in strains carrying X-chromosome duplications, consistent with the hypothesis that the duplications titrate a repressor acting on many " X-&ked genes. OSAGE compensation is the process by which D the level of X chromosome expression is equalized between animals with one X chromosome and those withtwo. It is an unusual example of gene regulation since the genes regulated coordinately can be separated by large physical and genetic distances. In the three systems where dosage compensation has been studied the best, coordinate regulation is apparently brought about by fundamentally contrasting mechanisms. In eutherian mammals, dosage compensation is accomplished by inactivationofall X chromosomes but one, resulting in heterochromatic sex chromatin bodies that are not expressed (GARTLER and RIGGS 1983). This system of chromosome-based coordinate regulation gives rise to the familiar mosaicism for X-linked genes in female mammals. No such natural mosaicism is seen in either of the other well-studied organisms, Drosophila and Caenorhabdztis elegans (BAKER and BELOTE 1983; HERMAN 1984). In each case, the level of X chromosome expression is apparently controlled by autosomal genes acting in trans on individual X-linked genes or groups of genes. In flies, the autosomal regulatory genes are thought to be positive regulators in males, partly because male-specific lethal mutations with abnormally low levels of X chromosome expression have been found (BELOTE and LUCCHESI 1980). Additional evidence for positive regulation in males comes from experiments involving X chromosome duplications. Most strikingly, MARONI and LUCCHESI (1980) have found that the total amount of transcription (measured by tritiated uridine incorporation in polytene chromosomes) is the same in males regardless of the amount of X chromosome material present. In other words, a duplication of the X chromosome in males decreases the expression of X-linked genes that are not duplicated. This leads to the general model for coordinate control of X chromosome expression in Drosophila in which certain autosomal genes produce an activator in limited amounts that is titrated by the X chromosome in some unknown fashion, possiblyby binding upstream ofx-linked genes. Because males have only a single X, they have twice the level of activator per gene (or per dosage compensation hit) that females have, and thus have a higher level of expression per gene than a 2X female does (reviewed in JAFFE and LAIRD 1986); The situation in C. elegans seems different yet, although the evidence is more limited. It is clear that 2X animals (hermaphrodites) heterozygous for X- linked mutations are not mosaics; otherwise, HERMAN (1984) wouldhave seen a high levelofmosaicism among his control hermaphrodites. This argues against a mechanism for dosage compensation based on X chromosome inactivation. Furthermore, X- linked genes translocated to autosomes via large duplications appear to be regulated as if still X-linked (MENEELY and HERMAN 1979; P. M. MENEELY and K. D. NORDSTROM, unpublished data), suggesting that X chromosome expression is regulated on a regional or perhaps even a gene-by-gene basis. Is the regulation of X chromosome expression occurring by positive control in males (as in Drosophila), by negative control in hermaphrodites, or by some combi- nation of these processes? Autosomal genes that affect the levelof X chromosome expression are known (WOOD et al. 1985; MEYER and CASSON 1986; MENEELY and WOOD 1987). Mutations in these genes affect 2X animals preferentially (HODGKIN 1983), although not exclusively in several cases (WOOD et al. Genetics 119: (June, 1988).

2 366 P. M. Meneely and K. D. Nordstrom 1985; MEYER and CASSON 1986; MENEELY and WOOD 1987). This is opposite what is seen in Drosophila, where the autosomal regulatory mutations are specific to 1X animals. Furthermore, the mutations in C. elegans appear to define negative regulators of X expression since recessive mutations in them result in abnormally high levels of expression in 2X animals (MEYER and CASSON 1986; MENEELY and WOOD 1987). In addition to the autosomal genes with apparent negative effects, there mayalsobex-linked genes withpositiveeffects on X chromosome expression (WOOD et al. 1985; MENEELY and WOOD 1987), but the evidence is not so strong. There are two X-linked genes, db-22 and db-23, each defined by a single mutant allele. The canonical phenotype for each mutant is that 2X animals are sick Dumpys (Dpy) and 1X animals are inviable or of low viability. However, the phenotype in both 2X and 1X animals is widely variable (MENEELY and WOOD 1987). When the effects of these two mutants on X-linked gene expression were tested, the dpy-22 mutant appeared to have lower levels of expression than wild-type in both 2X and 1X animals, whereas the dpy-23 mutant appeared to have lower expression of some but not all X-linked genes in 2X animals. In addition, dpy-23 may affect autosomal gene expression, while db-22 did not affect the expression of the autosomal genes tested (MENEELY and WOOD 1987). Other mutants with abnormal levels of X-linked gene expression in 1X or 2X animals or both are also known (VILLENEUVE and MEYER 1987; WOOD et al. 1987; B. MEYER, personal communication; J. MANSER and W. B. WOOD, personal communication; P. MENEELY, unpublished data). A competition or titration model similar in principle to that in Drosophila has been suggested (WOOD et al., 1987). This model attempts to unite dosage compensation and sex determination, processes whichmay be related since many genes appear to affect both. A component of the model is that X chromosome duplications elevate expression of the X globally in 2X animals. This is exactly oposite the results of MARONI and LUCCHESI (1980) in Drosophila because the fundamental mechanism appears to be opposite; that is, a major feature of dosage compensation in C. elegans may be negative regulation in 2X animals rather than positive regulation in 1X animals. The prediction that X chromosome duplications elevate overall X chromosome expression can be tested genetically by using hypomorphic mutations ofx-linkedgenes (MENEELY and WOOD 1987) and enzymatically by examining the level of activity of the X-linked enzyme ace-1 (JOHNSON et al. 1981). An elevated level of X chromosome expression will be seen genetically by suppression of an X-linked hypomorph or by a decrease in its penetrance. We report here that X chromosome duplications do de- crease the penetrance of an X-linked hypomorph. The degree of suppression depends on the dose of the duplication, consistent with a titration model. In confirmation of the genetic results, we also show that acetylcholinesterase activity encoded by the X-linked gene ace-l is elevated in hermaphrodites with X chromosome duplications that do not include the ace- 1 gene. MATERIALS AND METHODS Genes, alleles, and strains of C. ekguns: Handling and culture were by standard methods. All strains are derived from the Bristol strain of C. eleguns, which has the wildtype designation N2 (BRENNER 1974). The hypomorphic mutation Zin-I5(n765) has been previously described by MENEELY and WOOD (1987) and FERCUSON and HORVITZ (1985). Determination of the penetrance of the Muv mutant phenotype of lin-15 was done as previously described (MENEELY and WOOD 1987). Strain constructions were done by standard methods (MENEELY and WOOD 1987). The X chromosome duplications, mndp25, mndp33, and stdp2, have been described previously (MENEELY and WOOD 1984). mndp57 is a duplication isolated and characterized by M. SCHUYLER in the laboratory of R. HERMAN (personal communication), and sent to us by them. It is similar to the others in that it is stably attached to an autosome, in this case linkage group I, and is like mndp25 in that it is viable and fertile as a homozygote. Consistent with the observations of M. SCHUYLER and R. HERMAN (personal communication), we find that mndp57lmndp57 homozygotes are somewhat dumpy and slightly uncoordinated. Allelesof other genes and markers used are givenin the text; in most cases, the reference allele was used. Males were made by mating wild-type N2 males to hermaphrodites with a duplication. In some cases, selfprogeny males resulting from the non-disjunction mutation him.-5(e1490) (HODCKIN, HORVITZ and BRENNER 1979) were used, in a strain of general genotype Dp! + ; him-5; unc lin- 15(n765), or Opt+; him-5 dpy-21; unc lin-ii(n765). These cases are indicated in the tables, which give the complete genotype. About 35% of the self-progeny of such him-5 hermaphrodites are X0 males. Animals with the duplication were distinguished from animals lacking the duplication by the unc recessive marker on the X chromosome. We have not found any difference between self-progeny him-5 males and cross-progeny males in any of the assays we have used. The him-5 mutation was also necessary to examine males homozygous for the duplications mndp25 and mndp57. In these cases, we used the male self-progeny of mndp251 mndp25; him-5; unc-3(e151) lin-l5(n765) and mndp571 mndp57; him-5; unc-2(e55) lin-l5(n765). We infer that these males are homozygous for the duplication because neither duplication is readily lost in homozygous duplication strains, unlike the duplication mndplo (HERMAN, MADL and KARI 1979). We attempted to prove that these males were duplication homozygotes by mating them to marked hermaphrodites; the males are virtually sterile, and fewer than ten total cross-progeny were ever found from repeated matings. All of the cross-progeny found carried the duplication, consistent with the parental males being duplication homozygotes; however, the number of cross-progeny was too low to be sure of this. We found previously that some Dp! + ; dpy-21 X0 animals are sexually abnormal (MENEELY and WOOD 1984; also P. M. MENEELY, unpublished data). These abnormal males are typically less than 10% of the X0 progeny of the strains

3 Dosage Compensation in C. elegans 367 using mndp25 or mndp57, and a much lower percentage in smp2 strains; the exact fraction varies both within and between strains. Since the phenotype of lin-15 (n765) is sex-influenced (MENEELY and WOOD 1987), only morphologically normal males were counted in determining the penetrance of the Muv phenotype in the duplication strains. This interaction between dpy-21 and the duplication may cause us to underestimate the impact of the combination on xo animals, since some males we counted as morphologically normal may have had subtle sexual abnormalities or hermaphrodite characteristics; lzn-l5(n765) has a higher penetrance in hermaphrodites than in males (MENEELY and WOOD 1987). Assays for acetylcholinesterase activity: There are three genes encoding acetylcholinesterase in C. elegans, one of which is the X-linked gene ace-i. Thus, an additional assay for X-linked gene expression is to use ace-i-encoded acetylcholinesterase activity (what wewill call for convenience ace-i activity). Acetylcholinesterase activity can be determined by a radiometric assay of one or a few worms in a single vial (JOHNSON et al. 1981; C. JOHNSON and J. RAND, personal communication). The X-linked form of the enzyme, encoded by ace-i, can be assayed independently of the two autosomal forms since the ace-1 activity is resistant to the detergent deoxycholate (DOC); the autosomally encoded activities are sensitive to DOC (CULOTTI et al. 1981). We assayed total acetylcholinesterase activity by water soluble activity and ace-1 activity by DOC-resistant activity, using the published assay procedures with slight modifications (C. JOHNSON and J. RAND, personal communication). Tritiated acetylcholine chloride (Amersham) was purified as recommended by CULOTTI et al. (1981) by using a Bio-Rad AG 1-X8 ion exchange column, and the purified substrate pooled and stored frozen. Healthy adult worms were picked into 10 pl of buffer (100 mm HEPES, ph 7.0; 5 mg/ml bovine serum albumin; 1 mm sodium azide; 1 mm EDTA) in an Eppendorf vial, two worms per vial. The vials were put through eight to ten cycles of freezing in liquid nitrogen and thawing in cool water, and 5 ~1 of detergent (1% sodium deoxycholate) or distilled water added; the vials sat at room temperature for 1 hr. To each vial, 10 p,l of the thawed labeled substrate (2 X ~O-'M) were added and the reaction was allowed to incubate for 3 hr at room temperature. The reaction was stopped by the addition of 30 pl of stop mix (1 M chloroacetic acid, 0.5 M NaOH, 2 M NaCl, and 1.2 ml of scintillation fluid (0.5% PPO, 0.03% POPOP in toluene, with 10% butanol). The use of this scintillation fluid allowed us to count the product and not the substrate (JOHNSON and RUSSELL 1975). For each set of assays, two controls were done: a blank vial without any worms added (twovials, if both watersoluble and DOC-resistant activity were to be determined); and a vialwith 5 p1 NaOH to measure total hydrolysis. The reactions were run so that the blanks were less than 4% and th experimentals about 30% of the total hydrolysis. For convenience, we will refer to this protocol using a given thawed aliquot of the substrate as an experimental run. In contrast to published data, which used a slightly different assay, we find the DOC-resistant activity to be typically less than 25% of the water-soluble activity; this percentage varied severalfold with storage of the substrate. As with JOHNSON et al. (1981) and CULOTTI et al. (198 I), the activity as measured by total counts varied greatly (more than tenfold) from run to run, but less than 10% within a given run of animals. The reason for the between-run variation is not known, but could be due to different sensitivity of the acetylcholinesterase forms to changes in the substrate during storage. The level of enzyme activity in a given run depended linearly on the number of animals and on time for the reaction (not shown). Statistical analysis: Statistical differences in the penetrance of the Muv phenotype of lin-i5(n765) were determined by using the standard deviate, as before (MENEELY and WOOD 1987). Statisticalanalysisof the levelsof acetylcholinesterase activity was done by pairing the sammples at random within a run, a sample of the experimental with one of the standard (for instance, dpy-21 hermaphrodites with N2 hermaphrodites), then using standard procedures for comparing paired samples. From this, the student t value was calculated and the probability P assigned from a table. Because the total activity varied from run to run, only worms done within a run were paired. Thus, the tables list the means and the standard deviation used to compute the t value and the probability that the means were different. However, means between runs are not comparable because the specific activity of the substrate may have been different. As a control for our statistical procedure, N2 hermaphrodites (in two separate runs) and him-5 hermaphrodites (in one run) were compared to N2 hermaphrodites; no significant differences were found. RESULTS Assays using h15(n765): The phenotype of a hypomorphic mutation is a classical assay for the level of gene expression (MULLER 1950). By definition, the phenotype of a hypomorph is more severe when the mutation is placed hemizygous with a deficiency than when the mutation is homozygous. The phenotype of many hypomorphic mutations is less severe (that is, the mutant is suppressed) when three copies of the mutant allele are present or when the level of expression of the gene is increased. The phenotype of 1i~-I5(n765) has been described in detail by FER- GUSON and HORVITZ (1985) and was used previously as a measure of X chromosome expression (MENEELY and WOOD 1987).Briefly, lin-15(n765) results in a mutant multivulva (Muv) phenotype that is virtually 100% penetrant in homozygous hermaphrodites at 20". At this temperature, the penetrance of the Muv phenotype, as measured by counting the fraction of animals with at least one additional pseudovulva, is sensitive to allele dosage and to extragenic suppressors (MENEELY and WOOD 1987; P. M. MENEELY, unpublished data). Increasing the dose of the mutant allele, as in a triplo-x strain, decreases the penetrance (Table 1; also MENEELY and WOOD 1987). Likewise, mutations that raise overall X chromosome expression, such as dpy-21, suppress lin-i5(n765) in 2X animals at 20". Conversely, at the permissive temperature of 15", almost no animals are mutant. This temperature can be used to look for enhancement of the mutant phenotype, since decreasing the dose using a deficiency increases the penetrance (seen as mutant animals at the permissive temperature). Effect of X duplications in 2X animals. The penetrance of the Muv phenotype resulting from lin-

4 368 P. M. Meneely and K. D. Nordstrom TABLE 1 Effect of X chromosome duplications on the phenotype of Iin-l5(n765) hermaphrodites Strain Muv/Total 7% Muv interval (%)" him-5; lin-15 2X him-5; lin-i5 3X unc-2 lin mdf571+ ; unc-2 lin mdp57lmnll$57;unc-2 lin-i unc-20 lin mndp331+ ;unc-20 lin-i UTU-27 lin-i stdp21+ ;UTI&" lin unc-3 lin mndp.251 i- ;unc-3 lin-i mndp.25lmdfi25; unc-3 lin a The 95% confidence limits are the mean % Muv plus or minus the confidence interval. 15(n765) was used to determine what effect, if any, X chromosome duplications have on the level of X chromosome expression. Four duplications were tested for their effect on lin-i5(n765). Each duplication is stably translocated to an autosome; mndp33 is attached to linkage group (LG) ZV, mndp25 and mndp57 are attached to LGZ, and stdp2 is attached to LGZZ. The approximate extent of each duplication, including the genes duplicated and used here, is shown in Figure 1. The duplications represent three non-overlapping regions of the X chromosome genetic map. In allcases, the duplication carries the wild-type alleles of thex-linked genes, and the mutant recessive alleles are present on the X. None of the duplications duplicates lin-15 + itself. This conclusion is based on the published map positionof lin-i5 (FERGUSON and HORVITZ 1985). To demonstrate directly that lin-15 is not duplicated by mndp25, and to rule out gene-specific effects on its phenotype, we tested the effects of mndp25 and mndp57 on lin-i5(n309), an allele that is insensitive to gene dosage (MENEELY and WOOD 1987). Neither duplication suppressed lin-i5(n309). For mndp57, hermaphrodites of genotype mndp571+ ; unc-2 n309 were Muv. For mndp25, hermaphrodites of genotype mndp251-t ; unc-3 n309 were Muv. Thus, lin-15 + is not present on either mndp25 or mndp57 (or on stdp2 or mndp33 based on map position), and the Muv phenotype is not suppressed in a dose-insensitive allele. We then tested the hypomorphic allele Zin- 15(n765), with the results shown in Table 1. The presence of three copies of the mutant allele itself suppresses the Muv phenotype, from 100% to 55%. Likewise, of the four duplications tested, all except mndp33 suppress the Muv phenotype of lin-i5(n765). The amount of suppression varied depending on the duplication and strain. On average, about half the hermaphrodites were mutant in strains heterozygous for a duplication, compared with 100% of the hermaphrodites in strains lacking a duplication. Thus, the suppression of n765 resides in at least three sites of the X chromosome, distinct from Zin-15 itself. In fact, genes duplicated by mndp57 are at least 30 map units, or more than half the chromosome, away from lin-15 (Figure 1). The suppression of lin-l5(n765) was also tested in strains with two copies of a single duplication. Both map25 and mndp57 are viable when present in two copies (HERMAN, MADL and KARI 1979; R. HERMAN, personal communication). For each duplication, suppression is stronger when the duplication is homozygous than when it is heterozygous (Table 1). For example, about 50% of animals heterozygous for mndp25l-t were mutant, but only 21% of those homozygous for mndp25 were mutant. Similar results were seen with mndp57: mndp57lmndp57 strains had just over half as many mutant animals as the corresponding mndp57/+ strain. The effect of two copies of the other duplications was not tested since mndp33 is not viable as a homozygote and stdp2 grows extremely slowly as a homozygote and is quite sick. Effects of X duplications in X0 males: About half the males hemizygous for Zin-I5(n765) have an easily recognized protrusion on the ventral surface near the tail (Figure 2). While it has not been proved that the penetrance of this mutant phenotype is dosedependent, it is temperature-dependent like the Muv phenotype in hermaphrodites and it is suppressed by dpy-21 which raises X chromosome expression in 1X animals (MEYER and CASSON 1986; MENEELY and WOOD 1987; see below). The ability of the X chromosome duplications to suppress this protrusion was tested, with the results shown in Table 2. Three of four duplications, mndp25, mndp57, and stdp2, significantly suppressed the penetrance of this phenotype; the penetrance in mndp33 is not significantly different from the control strain lacking the duplication. For the others, the penetrance in a strain with the duplication is consistently less than half the penetrance in the strain without the duplication. The effect of being homozygous for a duplication in a male was tested by making strains of the general genotype DpIDp; him-5; unc, and examining the male self-progeny. [Male self-progeny are expected because him-5 causes X chromosome non-disjunction resulting in nullo-x gametes in each germline of the hermaphrodite (HODGKIN, HORVITZ and BRENNER 1979).] However, in duplication homozygotesvery few male self-progeny are seen (MENEELY and WOOD 1984; and P. M. MENEELY, unpublished data). Those that were present had a lower penetrance of lin-

5 -- Dosage Compensation in C. elegulls 369 Unc-2 dpy-23 ~m-6 uno27 dpy-22 /in- 15 unc-20 Ion-2 HH unc-3 ace- 1 / / mndp57 stdp2 mndp25 H mndp33 TABLE 2 Effect of X chromosome duplication on the phenotype of Iin-IS(n765) males Strain Muvrrotal c/c Muv interval (F)" him-5; urn9 lin mndp57/+; him-5; urn-2 lin mndp57lmndp57; him-5;urn lin- 15 unc-20 lin mndp33l+ ;urn-20 lin unc-27 lin stdp21+ ;unc-27 lin him-5; urn-3 lin rnndp251-k ;him-5; urn-3 lin mndp25lmndp25; him-5; urn lin-15 a See footnote a, Table 1. FIc.Lw 2,"Brightfield photomicrographs of Iin-l5(n765) animals. A, A hermaphrodite; B, a male. The small arrowheadr indicate pseudovulvae; the large arrowhead in A points to the vulva. (Photographed with a Zeiss Universal microscope, scale is 0.5 mm.) 15(n765) than did the corresponding duplication heterozygote (Table 2), consistent with a dose effect in males, as in hermaphrodites. For example, 20% of mndp25/+ males were mutant, but less than 1% of mndp25lmndp25 males were. A similar, but less pronounced effect was seen with mndp57 (22% us. 10%). Effect of X-dependent dpy mutations, dpy-22 and dpy-23: In this section, we consider the effect of varying the dose of different X-linked genes duplicated by mndp57 and stdp2. This is to test the hypothesis that the dose of either of two X-linked genes, dpy-23 and dpy-22, has a major effect on the penetrance of lin-i5(n765). The conclusion is that the dose of these two genes is no more important than the dose of other X-linked markers varied for controls. This conclusion is presented first to aid the reader. It is possible that the duplications are affecting the penetrance of lin-l5(n765) because they are duplicating a positive regulator either of Zzn-15 specifically or of X-linked gene expression generally. In particular, two X-linked genes, dpy-22 and dpy-23, have been suggested to be positive regulators of X chromosome expression based on the mutant phenotype of a single allele (see the Introduction; MENEELY and WOOD 1987). The wild-type alleles of these genes are duplicated by stdp2 and mndp57, respectively. Thus, it is possible that the effect of these duplications is due to the elevated dose of one of these regulators. In order to test that, the dose of dpy-22+ and dpy was varied, with the results shown in Tables 3 and 4. The effect of varying the dose of dpy-23 + will be described first. The strains heterozygous for mndp57 have three copies of dpy-23 +, two on the X chromosome and one on the duplication. If the dose of dpy-23+ is important in controlling the phenotype of lin-15, then changing the dose of this one gene should have more dramatic effects than changing the dose of any

6 370 P. M. Meneely and K. D. Nordstrom TABLE 3 Effect of dfy-23 + dose on the phenotype of lin-ls(n765) hermaphrodites and males Strain Muvmotal % Muv interval (%)" A. HERMAPHRODITES unc-2 lin mndp57/+ ;unc-2 lin unc-20 lin mndp57l+;unc-20 lin dpy-23 lin mndp57/+ ;dpy-23 lin lon-2 lin mndp57l+ ;lon-2 lin B. MALES him-5; unc-2 lin mndp57/+ ;him-5; unc lin-i5 lin-15 unc mndp57l+ ;unc-20 lin mndp571+ ;dpy-23 lin lon-2 lin mndp57i + ; lin-15 lon a See footnote a, Table 1. other gene duplicated by mndp57. In particular, since dpy-23 + is postulated to be a positive regulator, decreasing its dose should result in a higher penetrance of the Muv phenotype. To check the effect of other markers, the doseof three other genes was varied, unc-2, unc-20, and lon-2. None of these mutations alone affects the phenotype of lin-l5(n765) in hermaphrodites (Table 3A) or males (Table 3B). The penetrance when each was used as the X-linked marker for mndp57 in hermaphrodites is shown in Table 3A. Regardless of which marker is used, mndp57 suppresses the phenotype of lin-l5(n765), ranging from 38% Muv in strains marked with unc- 2 to 56% Muv in dpy-23 strains. The difference between unc-2 strains and dpy-23 strains is significant. However, the penetrance of strains marked with unc- 20 is not different from those with dpy-23, and strains marked with lon-2 on the X chromosome are not different from any of the others. This suggests that dose of dpy-23 + is unimportant in determining the penetrance of the Muv phenotype of lin-l5(n765) hermaphrodites, or atleast that the difference in penetrance is no more than seen with markers which do not affect X-linked gene expression. The effect of varying the dose of dpy-23 + in males is shown in Table 3B; since dpy-23 males are inviable (MENEELY and WOOD 1987), they are not listed in the table. Again, mndp57 suppresses the phenotype in males regardless of the marker on the X chromosome, ranging from 18% mutant to 31% mutant. The TABLE 4 Effect of dpy-22 + dose on the phenotype of lin-li(n765) hermaphrodites and males Strain MuvKotal % Muv interval (%)" A. HERMAPHRODITES unc-27 lin stdpz/+ ;unc-27 lin dpy-22 lin stdp2l+ ;dpy-22 lin dpy-22 unc-27 lan stdp2/+ ;dpy-22 unc-27 lin B. MALES stdp2/+ ;unc-27 lin stdp2/+ ;dpy-22 lin stdpzl+ ;dpy-22 unc-27 lin a See footnote a, Table 1. penetrance in strains with dpy-23 on the X chromosome is not different from strains with any of the other genes. For dpy-22 +, a slightly different approach was used. This was necessary because one convenient marker, unc-6, proved to decrease the penetrance of lin-15 even in the absence of stdp2 (our unpublished data). Thus, for dpy-22, the comparison is among dpy- 22, unc-27, and the double mutant in cis, dpy-22 unc- 27. The results are given in Table 4 for hermaphrodites (Table 4A) and males (Table 4B). In hermaphrodites, stdp2 suppresses lin-15(n765) in all three strains, ranging from 59% in a strain with unc-27 to 79% in the strain with dpy-22. The penetrance in these two strains is significantly different. If the difference is due to dpy-22 + dose and not to a nonspecific marker effect, a strain of genotype stdp2/+ ; unc-27 db-22 lin-l5(n765) is expected to have the same penetrance as stdp2i-t ; dpy-22 lin-15 and a higher penetrance than stdp2/+ ; unc-27 lin-15. In fact, 61% of stdp21 + ; unc-27dpy-22 lin-l5(n765) hermaphrodites are Muv, consistent with a marker effect rather than a specific effect of dpy-22 dose (Table 4A). The dose of dpy-22+ allso does not affect the penetrance of lin-l5(n765) in males (Table 4B). In summary, with the marker effects taken into account, neither the dose of dpy-22 nor dpy-23 appears to affect the penetrance of Zin-15(n765) beyond the difference seen when apparently innocuous markers are varied. The results are still somewhat ambiguous and do not rule out a role for either of these genes in the regulation of X chromosome expression. Part of the ambiguity with these mutations may be unavoidable, since the mutations themselves are quite variable in mutant morphology and viability (ME- NEELY and WOOD 1987), and there is only one known mutant allele of each gene.

7 Compensation Dosage in C. eleguns 371 TABLE 5 Effect of dpr-21 and X chromosome duplications on the phenotype of lin-ls(n765) hermaphrodites MuvlTotal Strain % Muv interval (%)" dpy-2l;unc-2 lin mndp571+ ;unc-z lin mndp571+ ;dpy-zl; unc lin-15 dpy-2 1 ; urn-20 lin mndp331+ ;unc-20 lin mndp331+ ;dpy-21; unc lin-15 dpy-21; urn-27 lin stllp21+;unc-27 lin stllpz/+;dpy-21 unc-27 lin dpy-21; unc-3 lin mndp251+ ;unc-3 lin mnd@251+ ;dpy-21; unc lin-15 a See footnote a, Table 1. Effect of X chromosome duplications and X-dependent dpy mutations, dpy-21 in 2X hermaphrodites: Recessive mutations in the autosomal gene dpy- 21 result in abnormally high expression of X-linked genes, leading to the postulate that db-21 is a negative regulator of X chromosome expression (MEYER and CASSON 1986; MENEELY and WOOD 1987). The interaction between dpy-21 and X duplications in hermaphrodites was tested by making strains of the general genotype Dpl+ ; dpy-21; unc lin-l5(n765), and determining the penetrance of the Muv phenotype in the Dpy non-unc hermaphrodite progeny. The results are shown in Table 5. The control comparisons in each case are the corresponding dpy-21; unc lin- 15(n765) strains without the duplication, which have a penetrance of about 40%, and the Dp/ + ; dpy-21+ ; unc lin-l5(n765) strains. (The data on the strains with a duplication and dpy-21+ are reproduced from Table 1 for ease of comparison.) In all cases except mndp33, the combination of dpy-21 and an X chromosome duplication suppressed lin-l5(n765) better than either dpy-21 or the duplication alone. For example, 50% of hermaphrodites heterozygous for mndp25 and homozygous for dpy-21+ were Muv, and 37% of dpy-21; unc-3 lin-l5(n765) hermaphrodites were Muv, but the penetrance is 10% when dpy- 21 is mutant and mndp25 is present (Table 5). An attempt was made to test the homozygotes for mndp25 and mndp57 in combination with dpy-21 as follows. Hermaphrodites of genotype mndp25/+ ; dpy-21; unc-3 lin-l5(n765) (and the corresponding strain with mndp57, dpy-21 and unc-20 or unc-2) were allowed to self-fertilize, and their Dpy progeny picked one to a plate. A duplication homozygote would have TABLE 6 Effect of dpy-21 and X chromosome duplications on the phenotype of lin-l5(n765) males" MuvlTotal Strain % Muv interval (56)" dpy-21; urn2 lin mndp57l+;dpy-21; unc-2 lin dpy-21; unc-20 lin mndp33l+ ;dpy-21; unc-20 lin dpy-21; unc-27 lin-i stdp21+ ;dpy-21; unc-27 lin dpy-21; unc-3 lin mndp25/+;dpy-21; unc-3 lin a All males were the self-progeny of hermaphrodite strains homozygous for him-5(e1490). been recognized by the failure to segregate Dpy Unc hermaphrodite progeny. No such hermaphrodites were seen among more than 50 animals for either mndp25 or mndp57,implying that the duplication homozygotes are inviable when dpy-21 is also homozygous. This had been seen previously with mndp25 and dpy-21 (MENEELY and WOOD 1984). Effect of X chromosome duplications in dpy-21 1X animals: A dpy-21 mutant raises the level of X chromosome expression in 1X animals (MEYER and CAS- SON 1986; MENEELY and WOOD 1987), and dpy-21 has been shown to interact with X chromosome duplications to affect the morphology of 1X animals (ME- NEELY and WOOD 1984). The combined effect of an X chromosome duplication and dpy-21 on the level of X chromosome expression is shown in Table 6. In most cases, the 1X males with both dpy-21 and the duplication had a lower penetrance of Zin-l5(n765) than did either dpy-21 or the duplication alone. For example, 22% of mndp57/+ ; dpy-21+ ; unc 2 males were mutant (Table 2) and 25% of dpy-21; unc-2 males were mutant (Table 6), but only 4% of mndp57/ + ; dpy-21; unc-2 males were mutant. Similar results were seen with stdp2 and mndp25. Assays with ace-1: Although the genetic assay using lin-l5(n765) appears to reflect the level of X-linked gene expression (MENEELY and WOOD 1987; VILLE- NEUVE and MEYER 1987), it is indirect and subject to unexpected marker effects, as seen above. A more direct assayis the levelofacetylcholinesterase encoded by the gene ace-1 (what wewillcall for simplicity, ace-1 activity). We assayed both total acetylcholinesterase activity and ace-1 activity in wild-type, mutant, and duplication strains (see MATERIALS AND METHODS). Because ace-1 is tightlylinkedto lin-15, the same duplications can be tested with it as were tested for suppression of lin-i5(n765). mndp33 was not tested since it had no effect on lin-l5(n765). Our results with the other three duplications are shown in Table 7.

8 372 P. M. Meneely and K. D. Nordstrom TABLE 7 Acetylcholinesterase activities of wild-type, mutant, and X chromosome aneuploid strains Experimental Assaf (n) Experimental mean Control mea& S.d Ratid p h N2 males him-5 3X dpy-21 herm. dpy-21 male mndp251+ mndp25lmndp25 stdp21 + mndp571+ mndp57lmndp57 ace-1 (28) Total (28) ace-1 (8) Total (8) ace-1 (7) ace-1 (28) Total (28) ace-1 (28) Total (28) ace-1 (21) ace-1 (29) Total (20) ace-1 (23) ace-1 (20) ace-1 (17) ** 2.33* 2.88** 3.81** 2.80*** *** ** a ace-1 activity was assayed as DOC-resistant; total activity was water-soluble activity * Number of sample vials, each with two worms The mean, in cpm. The control animals were N2 hermaphrodites for all experiments except for him-5 3X, which are compared to him-5 2X hermaphrodites. e Computed as ( ~~ln)~.~, where s2 is the pooled variance and n the sample size. f The ratio of the experimental mean to the control mean. g The Student t, computed as the difference in the means divided by Sx,-xo. *, **, *** the difference in means is significant at the 5%, 1%, or 0.1% level, respectively, in a two-tailed t-test with n - 1 degrees of freedom. J. G. DUCKETT and R. L. RUSSELL (unpublished, but cited in BULL 1983; and R. L. RUSSELL, personal communication) had shown previously that ace-1 is compensated between males and hermaphrodites, although the level of acetylcholinesterase activity is higher inmales than in hermaphrodites probably due to physiological differences. We find a similar result; there is no significant difference between ace- 1 activity in wild-type males and hermaphrodites, but males do have about 25% more total acetylcholinesterase activity than hermaphrodites. We next assayed both total and ace-1 activities in 3X hermaphrodites to be sure that elevated levels of activity could be seen. Activity due to ace-1 was elevated about 50% in two different runs, as expected in strains with three doses of the gene rather than two. Total activity is also elevated significantly, about 30% higher in 3X hermaphrodites. We tested whether d@-21 elevates ace-1 activity by comparing dpy-21 hermaphrodites and males in pairwise samples with wild-type hermaphrodites and males. For hermaphrodites, the result was unequivocal: dpy-21 elevates ace-1 activity, by an average of about 50%. For males, some elevation was seen but the activity was not significantly different from wildtype males. The result with hermaphrodites is consistent with other assays for X chromosome expression (MEYER and CASSON 1986; MENEELY and WOOD 1987). Other assays have shown that dpy-21 elevates X-linked gene expression in males also (MEYER and CASSON 1986; MENEELY and WOOD 1987), which we do not see with ace-1 activity, possibly because of the high variation among samples in this run. We note a difference between dpy-21 hermaphrodites and 3X hermaphrodites. For ace-1, dpy-21 results in higher activity of the X-linked gene in hermaphrodites, but does not elevate total acetylcholinesterase activity. In 3X animals, both are elevated, although not perhaps to the same extent. This suggests that the levelof ace-1 activity is controlled by means other than dosage compensation especially when the activityis high, although the gene is clearly compensated. We asked if duplications elevate the level of ace-1 activity by comparing strains homozygous for mndp25 and mndp57, and heterozygous for stdp2, mndp25, and mndp57. The results are also shown in Table 7. Clearly, an mndp25; unc-3 homozygote and an stdp21 + ; dpy-7(e88) heterozygote have much higher levels of ace-1 activity than does wild type, or than do unc- 3 and dpy-7 which are not different from wild type (data not shown). For mndp25lmndp25, ace-1 activity is elevated by about 60%, and for stdp2/+ the elevation is more than twofold. As with dpy-21, the mndp25 homozygoteelevates ace-1 activity but not the total acetylcholinesterase activity. A slight, but not significant, elevation is seen in mndp25/+ het-

9 erozygotes. No elevation of ace-1 activity is seen in mndp57imndp57 homozygotes. In mndp57/+ heterozygotes, ace-1 activity is slightly but not significantly lower than wild type. DISCUSSION We have shown that duplications of the X chromosome in C. elegans affect the expression of at least two X-linked genes they do not duplicate. Three nonoverlapping X chromosome duplications suppress the phenotype of the X-linked hypomorphic mutation Zzn-l5(n765), although none of the duplications includes Zin In addition, by assaying the level of activity of the X-linked acetylcholinesterase encoded by ace-1, we find that mndp25 and stdp2, two duplications which do not include ace-1 +, elevate its activity. For mndp25, significantelevationof ace-1 activity is seen in duplication homozygotes, although some slight (nonsignificant) increase is seen in duplication heterozygotes. The effect of stdp2 is quite striking; ace-1 activity is more than twice as high in stdp2/+ hermaphrodites as in wild-type hermaphrodites. The third duplication, mndp57, did not elevate ace-1 activity but did suppress lin-l5(n765). The effect of mndp25 on ace-1 activity illustrates a second important result: the magnitude of the effect depends on the dose of the duplication. Homozygotes for mndp25 have a higher level of ace-1 activity that do mndp251+ heterozygotes; and both mndp25l mndp25 and mndp57lmndp57 homozygotes suppress Zin-l5(n765) better than the corresponding heterozygotes. This may indicate that the effect depends on the amount of X chromosome material added. Consistent with this is that mndp57 suppresses Zin- 15(n765) while mndp33, a smaller duplication of the same region, does not. Of course, in this latter case, it may be that mndp57 duplicates some important genes that mndp33 does not, so the argument is not strong. We think that suppression of Zin-l5(n765) and elevation of ace-1 activity reflect increased expression of many or most X-linked genes in duplication strains. However, we have data for only two genes and even for ace-1, mndp57 has no effect. Another caution about suggesting global effects is that both genes we tested are at the right end of the X chromosome genetic map, as drawn in Figure 1. Furthermore, the genes affected are to the right of the duplications used. Thus, there is a formal possibility that only genes in this region of the X chromosome are affected, or that the effect is somehow polar over the chromosome and that only genes located to the right of a duplication are affected. Experiments with another X chromosome dupli- cation support our thinking, however. mndpl0 homozygotes have abnormally high levels of RNA from Dosage Compensation in C. elegam 373 two cloned X-linked genes not duplicated by mndplo (B. MEYER, personal communication); mndplo was not tested for its effect on Zzn-l5(n765) or ace-1 because it includes the wild-type allele of these genes. The effect of mndplo on transcription eliminates the arguments about regional specificity and polarity: the clones assayed, uvt-4 and wt-1, are to the left of mndplo and in the left half of the chromosome (MEYER and CASSON 1986). Combining this result with the results from lin-15 and ace-1, a tentative conclusion can be drawn: X chromosome duplications result in elevated levels of global X-linked gene expression. What might be the basis for such an effect? At least two general models can be imagined: increasing the levelof an activator or decreasing the levelof a repressor. In either case, the regulator has to be limiting. Under the first model, the X chromosome is postulated to have genes that are positive regulators of X-linked gene expression, and the duplications we used include the wild-typeallelesof these positive regulators. Under the second, a negative regulator (either X-linked or autosomal) is titrated by some X- linked feature such as a sequence; the duplications elevate X-linked gene expression by competing with the normal X chromosome for the repressor. In fact, candidates for both classes of mutations are known in C. elegans. Two X-linked genes, dpy-22 and dpy-23, have been suggested to be positive regulators of X-linked gene expression based on evidence that each mutant has abnormally low expression of X-linked genes (MENEELY and WOOD 1987). As noted previously (MENEELY and WOOD 1987; and see Intro- duction), the evidence is at best suggestive. However, dpy-22 + is duplicated in stdp2 strains and dpy-23 + is duplicated in mndp57 strains, two of the duplications used here. We tested the possible involvement of these two genes by asking if a mutation in the proposed regulatory gene had synergistic or antagonistic effects with the duplications. For dpy-22 and dpy-23, there was no effect of mutant dose other than what was seen by varying other X-linked markers. This argues against a dose-dependent role for dpy-22 or dpy-23 in dosage compensation. In addition, both dpy-22 and dpy-23 mutant hermaphrodites have the same level of ace-1 activity as wild-type hermaphrodites (our unpublished data). Bothofthese mutants are sick and variable in phenotype, which may be compounding the inconsistencies in the results from different assays. In contrast to the weak evidence implicating these two X-linked genes, there is good evidence that at least four autosomal genes, dpy-21, dpy-26, dpy-27, and dpy-28, are negative regulators of X-linked gene expression (MEYER and CASSON 1986; MENEELY and WOOD 1987). A recession mutation in any one of

10 374 P. M. Meneely and K. D. Nordstrom these four genes elevates the expression of many X- linked genes in 2X animals, in some cases as much as twofold. We tested the role of dpy-21 by constructing strains mutant in it and containing an X chromosome duplication. There was stronger suppression of lin-i5(n765) in strains with both a duplication and dpy-21 mutation than in strains with the duplication alone or with the dpy-21 mutation alone. This supports an interpretation in terms of a titration or competition model, and suggests thathe dpy-21 product (or something it interacts with) may be the negative regulator being titrated. The synergistic effect of dpy-21 and the X chromosome duplications on 2X animals could also account for the observed inviability of X chromosome duplication homozygotes in dpy-21 hermaphrodites (MENEELY and WOOD 1984; and RESULTS); the level of X chromosome expression in these strains may be so high as to be lethal. Likewise, a 3X dpy-21 hermaphrodite is inviable (HODGKIN 1983), and the presence of a third X chromosome, like the duplications, elevates X chromosome expression (Tables and 1 7; see also MENEELY and WOOD 1987). The duplications suggest that there are at least three sites that interact with the (postulated) repressor, and there may well be more. In fact, as a working model, we favor the idea that all compensated X-linked genes compete for the repressor. A titration model is appealing for its simplicity and may be correct in outline, but is probably not correct in detail. For example, in order to achieve dosage compensation, a negative regulator is expected to work preferentially if not exclusively in 2X animals. However, dpy-21 appears to affect both 2X and 1X animals (MEYER and CASSONS 1986; MENEELY and WOOD 1987; and RESULTS), and it interacts with X chromosome duplications in both 2X and 1X animals. Thus, the actual repressor may not be dpy-21 but something it controls. In addition, not all the duplications appear to affect all genes consistently; mndp57 affected lin-15 but not ace-1, and stdp2 affected ace- 1 much more strongly than it did lin-15. This may simply reflect strain differences, or it may indicate a more complicated regulation than we envision. Also, the duplication strains appear to have as high or higher levels of X-linked gene expression than do triplo-x animals; a simple titration model predicts that the triplo-x animals, with the entire X-chromosome duplicated, should have the highest expression. We suggest two explanations for this last point. It may be that X-linked gene expression in 3X animals is subject to somewhat different controls that in 2X + X-duplication animals. Another possibility is that the additional X chromosome has duplicated regulators (either specific to a particular gene or general to the X chromosome) that an individual duplication has not. The biggest shortcoming of any model for dosage compensation in C. elegans is that there are other genes affecting dosage compensation besides these dpy genes mentioned (VILLENEUVE and MEYER 1987; WOOD et al. 1987; J. MANSER and W. B. WOOD, personal communication; B. MEYER, personal communication; P. M. MENEELY, unpublished data). The interactions of these genes with each other and with X chromosome duplications or aneuploids are not known, and it is not clear which are of major importance and which, if any, are of minor importance. Furthermore, relatively few mutant alleles are known for each of the X-dependent dpy genes and the other genes, and it is possible that the known alleles have atypical affects. Regardless of the mechanism by which the effect is occurring, the most important conclusion from this work is that X chromosome duplications can apparently affect the expression of genes they do not duplicate. We see this effect by two independent assays, the suppression of an X-linked hypomorphic mutation and the elevation of enzyme activity from an X-linked gene. The elevated expression in duplication strains has implications in several areas. Duplications are used in mapping X-linked genes. If the mutation being mapped is hypomorphic, it may be suppressed by the duplication even when not duplicated. Experiments that map X-linked genes by measuring differences in expression in duplication strains are also subject to this problem. Two other sets of experiments using duplications are more immediately relevant for understanding the control of X chromosome expression and the role of the X chromosome in sex determination. We previ- ously (MENEELY and WOOD 1984) used X chromosome duplications to investigate the interaction between the X chromosome and dpy-21, specifically asking if a duplication could mimic an X chromosome in producing the Dpy (in 1X animals) and inviability (in 2X animals) phenes of dpy-21 strains. The conclusion was that the Dpy-21 phenotypes depend on several different regions of the X chromosome. This conclusion is probably stillvalid if, as we postulate here, the dpy-21 gene product (or some gene product it interacts with) is being titrated. In an earlier set of experiments, MADL and HERMAN (1979) used X chromosomes duplications to vary the dose of different X-linked regions in studying sex determination. The experiment was to ask if an X chromosome duplication affected sexual development of a chromosomal male in triploids. Intersexes were found, and the effect was attributed to the duplication introducing additional hermaphroditedetermining sites. However, the duplications may have had an additional effect: they could have been raising the expression of a distant X-linked gene controlling hermaphrodite development. In fact, in

11 an extreme view, none of those duplications contains a sex determination site or gene, but instead all affected the expression of the sex determination gene by titration. We thank BARBARA MEYER, BILL WOOD, and BOB HERMAN for sharing unpublished data, and CARL JOHNSON and JIM RAND for advice with the enzyme assays. We also thank BOB HERMAN and the Caenorhabditis Genetics Center (CGC) for strains used in this work. We thank BILL MCCOUBREY for extensive comments on the manuscript. The CGC is supported by a contract between the National Institutes of Health and the Curators of the University of Missouri. This work was supported by grants from the National Science Foundation (DCB ) and the Basil OConnor Starter Research Grant program of the March of Dimes Foundation (5-525). LITERATURE CITED BAKER, B. S., and J. M. BELOTE, 1983 Sex determination and dosage compensation in Drosophila melanogaster. Annu. Rev. Genet. 17: BELOTE, J. M., and J. C. LUCCHESI, 1980 Male-specific lethal mutations of Drosophila melanogaster. Genetics 96: BRENNER, S., 1974 The genetics of Caermhubditis elegans. Genetics 77: BULL, J. J., 1983 Evolution of Sex-Determining Mechanism. Benjamin-Cummings, Menlo Park, Calif. CULOTTI, J. G.,G. VON EHRENSTEIN, M.R. CULOTTI and R. L. RUSSELL, 1981 A second class of acetylcholinesterase-deficient mutants of the nematode Caenorhabditis elegam. Genetics 97: FERGUSON, E. L., and H. R. HORVITZ, 1985 Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode Caenurhubditis elegans. Genetics 110: GARTLER, S. M., and A. D. RIGGS, 1983 Mammalian X-chromosome inactivation. Annu. Rev. Genet. 17: HERMAN, R. K Analysis of genetic mosaics of the nematode Caenorhabditis elegans. Genetics 96: HERMAN, R. K., J. E. MADL and C. K. KARI, 1979 Duplications in Caenorhabditis elegans. Genetics 92: HODGKIN, J., 1983 X-chromosome dosage and gene expression in Caenorhubditis elegam: two unusual dumpy genes. Mol. Gen. Genet. 192: Dosage Compensation in C. elegans 375 HODGKIN, J. A., H. R. HORVITZ and S. BRENNER, 1979 Nondisjunction mutants of the nematode Caenorhabditis elegans. Genetics 91: JAFFE, E., and C. LAIRD, 1986 Dosage compensation in Drosophila. Trends Genet. 2: JOHNSON, C. D., and R. L. RUSSELL, 1975 A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64: JOHNSON, C. D., J. G. DUCKETT, J. G. CULOTTI, R. K. HERMAN, P. M. MENEELY and R.L. RUSSELL, 1981 An acetylcholinesterase-deficient mutant of the nematode Caenorhabditis e2egam. Genetics 97: MADL, J. E., and R. K. HERMAN, 1979 Polyploids and sex determination in Caenorhubditis elegans. Genetics 93: MARONI, G., and J. C. LUCCHESI, 1980 X-chromosome transcription in Drosophila. Chromosoma 77: MENEELY, P. M., and R. K. HERMAN, 1979 Lethals, steriles and deficiencies in a region of the X-chromosome of Caenurhabditis elegans. Genetics 92: MENEELY, P. M., and W. B. WOOD, 1984 An autosomal gene that affects X-chromosome expression and sex determination in Caenorhabditis elegam. Genetics 106: MENEELY, P.M., and W. B. WOOD, 1987 Genetic analysis of X- chromosome dosage compensation in Caenorhubditis elegans. Genetics 117: MEYER, B. J., anmd L. CASSON, 1986 Caenorhubditiselegans compensates for the difference in X-chromosome dosage between the sexes by regulating transcript levels. Cell 47: MULLER, H. J Evidence for the precision of genetic adaptation. Harvey Lect. 43: VILLENEUVE, A. M., and B. J. MEYER, 1987 sdc-i: a link between sex determination and dosage compensation in Caenorhubditis elegans. Cell 48: WOOD, W. B., P. MENEELY, P. SCHEDIN and L. DONAHUE, 1985 Aspects of dosage compensation and sex determination in Caenorhabditis elegans. Cold Spring Harbor Symp. Quant. Biol. 50: WOOD, W. B., C. TRENT, P. MENEELY, J. MANSER, and S. BURGESS, 1987 On control of X-chromosome expression and sex determination in embryos of Caenorhubditis elegans. Pp In: Genetic Regulation of Developmnt (45th Symposium of the Society for Developmental Biology), Edited by W. LOOMIS. Alan R. Liss, New York. Communicating editor: R. K. HERMAN