Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6795-6799, August 1991
Genetics
Dosage compensation of the Drosophila pseudoobscura Hsp82 gene and the Drosophila melanogaster Adh gene at ectopic sites in D. melanogaster (gene control/position effect/P-element transformation/RNA probe protection)
HEINZ SASS* AND MATTHEW MESELSON Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138
Contributed by Matthew Meselson, April 8, 1991
Measurements were made of the amounts of ABSTRACT larval RNA transcribed from the autosomal Adh gene of Drosophila melanogaster and the X chromosomal Hsp82 gene of Drosophila pseudoobscura carried on the same P-element transposon inserted at various sites in the D. melanogaster genome. Both genes were fully compensated at sites in euchromatic regions of the X chromosome but neither was compensated at a site in the centric 8-heterochromatin of the X chromosome. No compensation of the D. pseudoobscura Hsp82 gene was found at any of 10 autosomal insertion sites tested. The compensation behavior of the transposed genes was, therefore, not determined by closely linked sequences but instead was determined in each case by their new chromosomal environment.
The expression of most X chromosome-linked alleles, including hypomorphs, is essentially the same in male and homozygous female Drosophila, even though males have one and females have two X chromosomes. Such gene-dosage compensation is a specific response of the expression of X chromosome-linked genes to the ratio of X chromosomes to autosomes (1-4) and is manifested at the level of transcript accumulation (5-9). Although the mechanism of dosage compensation is unknown, it is almost certainly mediated by cis-acting determinants on the X chromosome. It may therefore be asked whether such determinants are closely linked to the individual gene, are more widely dispersed, or both. P-elementmediated transformation provides a method for testing these possibilities. Genes cloned from the X chromosome may be inserted into autosomes and, conversely, autosomal genes may be inserted into the X chromosome. The activity of the transposed gene in males may then be compared with that in females for evidence of compensation. Such comparisons have been published for two X chromosome-linked genes inserted into autosomes and four autosomal genes inserted into the X chromosome. Autosomal insertions of the X chromosome-linked white gene with as little as 0.2 kilobase (kb) of upstream DNA had greater activity in heterozygous males than in heterozygous females, as indicated by eye color or the amount of pigment in adult heads (10-12). This result, expected for compensation, contrasted with observations involving females homozygous for the inserted white gene. Such females usually had 2-3 times as much pigment as males heterozygous for the same insertion and had approximately the same amount of pigment as homozygous males (11, 12). Autosomal insertions of the X chromosome-linked Sgs4 gene with 2.5 kb of upstream DNA had an average male/female (m/f) activity ratio per gene of 1.3-1.9, measured as specific larval protein
or RNA, normalized to the corresponding products of the endogenous autosomal Sgs3 gene (13, 14). Insertions with 0.84 kb of upstream DNA were more active in males in some transformed lines but in other lines were more active in females, as determined by assays of larval transcripts of the transposed gene, normalized to transcripts of an endogenous Sgs4 allele (15). In all three studies, the normalized Sgs4 gene activity varied widely among individual larvae of the same sex and transformed line. Insertions of the autosomal Xdh gene into the X chromosome displayed a higher m/f ratio of enzyme activity per gene in adults, normalized to total protein, than did autosomal insertions, corresponding to 60% average compensation (16, 17). In contrast, one study of the autosomal Adh gene inserted at X chromosome sites gave no indication of compensation in larvae or adults (18) and another showed only weak compensation, averaging -30% in larvae (17). For the autosomal Ddc gene transposed to the X chromosome, the m/f ratio of enzyme activity per gene in older adults, normalized to total protein, was higher than in autosomal transformants, but the effect was weak or absent in prepupae and in newly eclosed adults (19, 20). Lastly, larval transcripts of the autosomal Sgs3 gene, normalized to RNA from an allele of the endogenous gene, were more abundant per gene in males than in females in only one of four X chromosome insertion lines tested and there were unexplained differences between males and females with autosomal insertions (21). At least some of the variability in the above results may have resulted from inaccuracies in measurement of gene activity. The experiments we report were designed to minimize such effects by quantifying transcripts directly, using RNA probe protection. Also, to observe the compensation behavior of an X chromosome-linked gene and an autosomal gene inserted at the same chromosomal sites, we employed a P-element transposon containing both genes. We measured the amounts of larval RNA transcribed from the autosomal Adh gene of Drosophila melanogaster and the X chromosome-linked Hsp82 heat shock gene of Drosophila pseudoobscura, carried on a P-element transposon inserted at various sites in the genome of D. melanogaster. The D. pseudoobscura Hsp82 gene was ligated in its second exon to the coding region of a bacterial neomycin-resistance (neo) gene, giving the Hsp82-neo fusion gene. In each determination, the amount of transcript of the inserted Hsp82-neo or Adh gene was normalized to the amount of transcript of the endogenous Hsp82 gene. The X chromosome of D. pseudoobscura is metacentric, with one arm homologous to the D. melanogaster X chromosome and the other, on which the Hsp82 gene is located, Abbreviations: m/f, male/female; neo, neomycin resistance; nt, nucleotide(s). *Present address: Institute of Genetics, Gutenberg University, P.O. Box 3980, 6500 Mainz 1, Federal Republic of Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6795
67%
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Sass and Meselson
homologous to the left arm of D. melanogaster chromosome 3. The Hsp82 gene is, therefore, autosomal in D. melanogaster and X chromosome-linked in D. pseudoobscura, where it has been shown by transcription autoradiography to be compensated (22). We found that the Hsp82-neo gene and the Adh gene gave twice as much RNA per gene in males as in females when inserted at either of two sites in euchromatic regions ofthe D. melanogaster X chromosome, corresponding to essentially complete dosage compensation. Both genes were uncompensated, however, when inserted at a site in the ,B-heterochromatic most proximal section of the X chromosome. The Hsp82-neo gene was not compensated in any of the seven autosomal insertion lines examined, nor was the intact D. pseudoobscura Hsp82 gene compensated in any of three autosomal transformant lines of D. melanogaster made by other investigators. In all cases examined, therefore, the compensation behavior of the transposed genes was independent of their autosomal or X chromosomal origin but was determined instead by their new chromosomal environment.
Table 1. Sex-specific amounts of Hsp82 RNA in D. pseudoobscura RNA, Hsp82/control cpm x 10-3 Male Female Male Female Probe
MATERIALS AND METHODS Transformation. The P-element transformation plasmid pHS22 (23) was coinjected (300-500 ,ug/ml) with helper plasmid pir25.7wc (70-100 ,ug/ml) (24) into embryos of Adh-null recipient strain Adhf4 pr cn (25). Flies from injected embryos were individually back-crossed to the recipient strain and the progeny were cultured for five or six generations in the presence of the neomycin analogue Geneticin (G418, GIBCO), selecting the earliest eclosing flies at each generation. This procedure efficiently selects for transformants homozygous for Hsp82-neo since they eclose earlier than heterozygotes in the presence of the antibiotic. The selective medium was unyeasted instant Drosophila food (Carolina Biological Supply) with 0.1% G418, a concentration -5 times greater than the maximum tolerated by the recipient line. Transformants were kept on standard cornmeal/ molasses food without antibiotic. Adults of all transformed lines survived overnight exposure to 6% ethanol and each line was still resistant to G418 2 years after transformation. Insertions were mapped by hybridization in situ of female larvae with biotinylated pHS22 DNA. Both homologues invariably hybridized, in contrast to the asymmetric labeling seen in larvae from a back-cross to the recipient line. RNA Isolation and Probe-Protection Analysis. Total cell RNA of actively feeding nonshocked or heat-shocked (20 min, 36°C) third instar larvae was prepared and analyzed by RNA probe protection (23). After gel electrophoresis and 10 5 20 AJp fIt
Compensation of the Endogenous Hsp82 Gene in D. pseudoobscura. We first sought to confirm, by RNA probe protection, the earlier finding (22) by transcription autoradiography that the Hsp82 gene of D. pseudoobscura is dosage compensated. Fig. 1 shows the analysis of RNA from male and female larvae of D. pseudoobscura using antisense RNA probes transcribed from a 366-base-pair (bp) segment of the D. pseudoobscura Hsp82 intron and, for normalization, a 430-bp segment of exon VII of the autosomal D. pseudoobscura Gart gene. It is seen in lanes 3-8 that the amount of probe protected was proportional to the amount of RNA in the hybridization mixture and that the ratio of Hsp82 RNA to Gart RNA was independent of sex. The normalized m/f ratio of Hsp82 RNA was 0.97 (Table 1). An independent experiment, using probes transcribed from a 280-nucleotide (nt) segment of D. pseudoobscura Hsp82 exon II and a 160-nt segment of exon II of the D. melanogaster a-tubulin gene, also summarized in Table 1, gave a m/f ratio of 0.81. These results confirm that the X chromosome-linked H1sp82 gene in D. pseudoobscura is approximately twice as active per gene in males as in females. Transformed Lines. All transformations were done with the P-element transformation plasmid pHS22, containing the
430
m
-
Om
OW-
m
-
-m
q.".
_m
1
2
3
4
5
f
m
f
6 rI
7 f
.Sp 82
8 rn
FIG. 1. Probe-protection analysis of endogenous Hsp82 and Gart transcripts in D. pseudoobscura. RNA of nonshocked D. pseudoobscura larvae was hybridized with a 366-nt antisense RNA probe (nt 514-880) for the D. pseudoobscura Hsp82 intron (26) and/or a 430-nt probe (nt 10,277-10,707) for the D. pseudoobscura Gart gene exon VII (27). Lanes: 1, Gart probe; 2, D. pseudoobscura Hsp82 probe; 3-8, both probes hybridized with 10, 5, or 20 Zg of RNA from female (f) or male (m) larvae, as indicated. The appearance of two protected lengths of the Gart probe may reflect polymorphism in our D. pseudoobscura stock. gene
Hsp82 exon
1.44 2.07 4.90
Tubulin
5.66
Gart
1.31 1.81 5.08 4.70
0.70 08
0.87
0.72 .8
08
1.08
0.81
autoradiography, bands of protected probe were excised and radioactivity was measured for 10-20 min per sample by liquid scintillation counting. The activity ofa sample from the region between the bands on a representative lane of each gel, generally
Genetics
Dosage compensation of the Drosophila pseudoobscura Hsp82 gene and the Drosophila melanogaster Adh gene at ectopic sites in D. melanogaster (gene control/position effect/P-element transformation/RNA probe protection)
HEINZ SASS* AND MATTHEW MESELSON Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138
Contributed by Matthew Meselson, April 8, 1991
Measurements were made of the amounts of ABSTRACT larval RNA transcribed from the autosomal Adh gene of Drosophila melanogaster and the X chromosomal Hsp82 gene of Drosophila pseudoobscura carried on the same P-element transposon inserted at various sites in the D. melanogaster genome. Both genes were fully compensated at sites in euchromatic regions of the X chromosome but neither was compensated at a site in the centric 8-heterochromatin of the X chromosome. No compensation of the D. pseudoobscura Hsp82 gene was found at any of 10 autosomal insertion sites tested. The compensation behavior of the transposed genes was, therefore, not determined by closely linked sequences but instead was determined in each case by their new chromosomal environment.
The expression of most X chromosome-linked alleles, including hypomorphs, is essentially the same in male and homozygous female Drosophila, even though males have one and females have two X chromosomes. Such gene-dosage compensation is a specific response of the expression of X chromosome-linked genes to the ratio of X chromosomes to autosomes (1-4) and is manifested at the level of transcript accumulation (5-9). Although the mechanism of dosage compensation is unknown, it is almost certainly mediated by cis-acting determinants on the X chromosome. It may therefore be asked whether such determinants are closely linked to the individual gene, are more widely dispersed, or both. P-elementmediated transformation provides a method for testing these possibilities. Genes cloned from the X chromosome may be inserted into autosomes and, conversely, autosomal genes may be inserted into the X chromosome. The activity of the transposed gene in males may then be compared with that in females for evidence of compensation. Such comparisons have been published for two X chromosome-linked genes inserted into autosomes and four autosomal genes inserted into the X chromosome. Autosomal insertions of the X chromosome-linked white gene with as little as 0.2 kilobase (kb) of upstream DNA had greater activity in heterozygous males than in heterozygous females, as indicated by eye color or the amount of pigment in adult heads (10-12). This result, expected for compensation, contrasted with observations involving females homozygous for the inserted white gene. Such females usually had 2-3 times as much pigment as males heterozygous for the same insertion and had approximately the same amount of pigment as homozygous males (11, 12). Autosomal insertions of the X chromosome-linked Sgs4 gene with 2.5 kb of upstream DNA had an average male/female (m/f) activity ratio per gene of 1.3-1.9, measured as specific larval protein
or RNA, normalized to the corresponding products of the endogenous autosomal Sgs3 gene (13, 14). Insertions with 0.84 kb of upstream DNA were more active in males in some transformed lines but in other lines were more active in females, as determined by assays of larval transcripts of the transposed gene, normalized to transcripts of an endogenous Sgs4 allele (15). In all three studies, the normalized Sgs4 gene activity varied widely among individual larvae of the same sex and transformed line. Insertions of the autosomal Xdh gene into the X chromosome displayed a higher m/f ratio of enzyme activity per gene in adults, normalized to total protein, than did autosomal insertions, corresponding to 60% average compensation (16, 17). In contrast, one study of the autosomal Adh gene inserted at X chromosome sites gave no indication of compensation in larvae or adults (18) and another showed only weak compensation, averaging -30% in larvae (17). For the autosomal Ddc gene transposed to the X chromosome, the m/f ratio of enzyme activity per gene in older adults, normalized to total protein, was higher than in autosomal transformants, but the effect was weak or absent in prepupae and in newly eclosed adults (19, 20). Lastly, larval transcripts of the autosomal Sgs3 gene, normalized to RNA from an allele of the endogenous gene, were more abundant per gene in males than in females in only one of four X chromosome insertion lines tested and there were unexplained differences between males and females with autosomal insertions (21). At least some of the variability in the above results may have resulted from inaccuracies in measurement of gene activity. The experiments we report were designed to minimize such effects by quantifying transcripts directly, using RNA probe protection. Also, to observe the compensation behavior of an X chromosome-linked gene and an autosomal gene inserted at the same chromosomal sites, we employed a P-element transposon containing both genes. We measured the amounts of larval RNA transcribed from the autosomal Adh gene of Drosophila melanogaster and the X chromosome-linked Hsp82 heat shock gene of Drosophila pseudoobscura, carried on a P-element transposon inserted at various sites in the genome of D. melanogaster. The D. pseudoobscura Hsp82 gene was ligated in its second exon to the coding region of a bacterial neomycin-resistance (neo) gene, giving the Hsp82-neo fusion gene. In each determination, the amount of transcript of the inserted Hsp82-neo or Adh gene was normalized to the amount of transcript of the endogenous Hsp82 gene. The X chromosome of D. pseudoobscura is metacentric, with one arm homologous to the D. melanogaster X chromosome and the other, on which the Hsp82 gene is located, Abbreviations: m/f, male/female; neo, neomycin resistance; nt, nucleotide(s). *Present address: Institute of Genetics, Gutenberg University, P.O. Box 3980, 6500 Mainz 1, Federal Republic of Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6795
67%
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Sass and Meselson
homologous to the left arm of D. melanogaster chromosome 3. The Hsp82 gene is, therefore, autosomal in D. melanogaster and X chromosome-linked in D. pseudoobscura, where it has been shown by transcription autoradiography to be compensated (22). We found that the Hsp82-neo gene and the Adh gene gave twice as much RNA per gene in males as in females when inserted at either of two sites in euchromatic regions ofthe D. melanogaster X chromosome, corresponding to essentially complete dosage compensation. Both genes were uncompensated, however, when inserted at a site in the ,B-heterochromatic most proximal section of the X chromosome. The Hsp82-neo gene was not compensated in any of the seven autosomal insertion lines examined, nor was the intact D. pseudoobscura Hsp82 gene compensated in any of three autosomal transformant lines of D. melanogaster made by other investigators. In all cases examined, therefore, the compensation behavior of the transposed genes was independent of their autosomal or X chromosomal origin but was determined instead by their new chromosomal environment.
Table 1. Sex-specific amounts of Hsp82 RNA in D. pseudoobscura RNA, Hsp82/control cpm x 10-3 Male Female Male Female Probe
MATERIALS AND METHODS Transformation. The P-element transformation plasmid pHS22 (23) was coinjected (300-500 ,ug/ml) with helper plasmid pir25.7wc (70-100 ,ug/ml) (24) into embryos of Adh-null recipient strain Adhf4 pr cn (25). Flies from injected embryos were individually back-crossed to the recipient strain and the progeny were cultured for five or six generations in the presence of the neomycin analogue Geneticin (G418, GIBCO), selecting the earliest eclosing flies at each generation. This procedure efficiently selects for transformants homozygous for Hsp82-neo since they eclose earlier than heterozygotes in the presence of the antibiotic. The selective medium was unyeasted instant Drosophila food (Carolina Biological Supply) with 0.1% G418, a concentration -5 times greater than the maximum tolerated by the recipient line. Transformants were kept on standard cornmeal/ molasses food without antibiotic. Adults of all transformed lines survived overnight exposure to 6% ethanol and each line was still resistant to G418 2 years after transformation. Insertions were mapped by hybridization in situ of female larvae with biotinylated pHS22 DNA. Both homologues invariably hybridized, in contrast to the asymmetric labeling seen in larvae from a back-cross to the recipient line. RNA Isolation and Probe-Protection Analysis. Total cell RNA of actively feeding nonshocked or heat-shocked (20 min, 36°C) third instar larvae was prepared and analyzed by RNA probe protection (23). After gel electrophoresis and 10 5 20 AJp fIt
Compensation of the Endogenous Hsp82 Gene in D. pseudoobscura. We first sought to confirm, by RNA probe protection, the earlier finding (22) by transcription autoradiography that the Hsp82 gene of D. pseudoobscura is dosage compensated. Fig. 1 shows the analysis of RNA from male and female larvae of D. pseudoobscura using antisense RNA probes transcribed from a 366-base-pair (bp) segment of the D. pseudoobscura Hsp82 intron and, for normalization, a 430-bp segment of exon VII of the autosomal D. pseudoobscura Gart gene. It is seen in lanes 3-8 that the amount of probe protected was proportional to the amount of RNA in the hybridization mixture and that the ratio of Hsp82 RNA to Gart RNA was independent of sex. The normalized m/f ratio of Hsp82 RNA was 0.97 (Table 1). An independent experiment, using probes transcribed from a 280-nucleotide (nt) segment of D. pseudoobscura Hsp82 exon II and a 160-nt segment of exon II of the D. melanogaster a-tubulin gene, also summarized in Table 1, gave a m/f ratio of 0.81. These results confirm that the X chromosome-linked H1sp82 gene in D. pseudoobscura is approximately twice as active per gene in males as in females. Transformed Lines. All transformations were done with the P-element transformation plasmid pHS22, containing the
430
m
-
Om
OW-
m
-
-m
q.".
_m
1
2
3
4
5
f
m
f
6 rI
7 f
.Sp 82
8 rn
FIG. 1. Probe-protection analysis of endogenous Hsp82 and Gart transcripts in D. pseudoobscura. RNA of nonshocked D. pseudoobscura larvae was hybridized with a 366-nt antisense RNA probe (nt 514-880) for the D. pseudoobscura Hsp82 intron (26) and/or a 430-nt probe (nt 10,277-10,707) for the D. pseudoobscura Gart gene exon VII (27). Lanes: 1, Gart probe; 2, D. pseudoobscura Hsp82 probe; 3-8, both probes hybridized with 10, 5, or 20 Zg of RNA from female (f) or male (m) larvae, as indicated. The appearance of two protected lengths of the Gart probe may reflect polymorphism in our D. pseudoobscura stock. gene
Hsp82 exon
1.44 2.07 4.90
Tubulin
5.66
Gart
1.31 1.81 5.08 4.70
0.70 08
0.87
0.72 .8
08
1.08
0.81
autoradiography, bands of protected probe were excised and radioactivity was measured for 10-20 min per sample by liquid scintillation counting. The activity ofa sample from the region between the bands on a representative lane of each gel, generally
