Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5254-5258, October 1979 Genetics
Organization of the multiple genes for the 70,000-dalton heat-shock protein in Drosophila melanogaster (DNA reassociation kinetics/DNA blots/restriction analysis/cloned DNA)
M.-E. MIRAULT, M. GOLDSCHMIDT-CLERMONT, S. ARTAVANIS-TSAKONAS*t, AND P. SCHEDL*t Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, 1211 Geneva 4, Switzerland; and *Department of Cell Biology, Biozentrum, University of Basel, 70, Klingelbergstrasse, 4056 Basel, Switzerland
Communicated by Niels Kaj Jerne, July 23, 1979
ABSTRACT The organization and number of 70,000-dalton heat-shock protein genes of Drosophila melanogaster has been investigated in a wild-type Oregon R fly stock and in a KC cell line. Six copies were found in the KC cells, and slightly more were found in the Oregon R population examined. In both cases, the basic gene element consisting of the mRNA coding region plus a short 5' "noncoding" sequence element was conserved. Two gene variants distinguished by specific restriction sites were found in both genomic DNAs. Restriction maps of the six genes in KC cells showed that these two gene variants are arranged differently. Restriction analysis of Oregon R embryonic DNA revealed polymorphism in the organization of the genes, which is not observed in KC cells. The data suggest that the arrangement as well as the number of genes for the 70,000-dalton heat-shock protein in D. melanogaster is subject to variations at both the 87A and 87C cytogenetic loci.
MATERIALS AND METHODS Tissue Culture Cells. The Dm KC161 cell line, an essentially diploid line derived from embryonic tissues (17), was a gift from G. Echalier. The cells were grown at 25°C in suspension as described (6). DNA Isolation. Hybrid plasmid DNA was isolated as described (8) and further purified by sucrose gradient centrifugation. Nuclear DNA from Dm (Oregon-R) and from KC cells was prepared according to Schachat and Hogness (18), including an additional phenol extraction (19) before consecutive CsCI density gradients. The DNA concentrations were measured by diphenylamine assay and by absorbance at 260 nm. All DNA preparations contained less than 2% RNA as measured by resistance to alkaline hydrolysis. Hybridization Probe for the hsp 70 Gene. The 2.15-kb Sal I fragment of 56H8 (see Fig. 4a) was isolated by gel electrophoresis and labeled by nick translation with [a-32P]dCTP [350 Ci/mmol (9); 1 Ci = 3.7 X 1010 becquerels]. The purity of the probe was ascertained by hybridization to DNA blots of 56H8 restriction digests according to Southern (20). DNA Reassociation Assayed by Nuclease Si Resistance. Dm DNA, 56H8 DNA, or 132E3 DNA was incubated for 20 min at 100°C in 0.13 M NaOH/0.1 mM EDTA/0.4% sodium dodecyl sulfate in the presence of carrier salmon sperm DNA (Sigma, type III) at 200 ,ug/ml. The 32P-labeled probe (about 4 X 107 cpm/,g) was then added and the mixture was incubated a further 4 min at 100°C and chilled in ice. These incubation conditions gave driver and probe DNAs of comparable size distribution (modal length, 300-400 nucleotides). Appropriate volumes of 1 M Tris-HCl, pH 7.4/5 M NaCl were added to give final concentrations of 1.5 M NaCl and 55 mM Tris-HCl. The DNA reassociation mixture was then incubated at 68°C; 100-,ul samples were removed every 20 min (including blanks at zero time), diluted into 0.5 ml of nuclease S1 buffer (30 mM NaOAc, pH 4.6/1 mM ZnCl2) and stored at -20°C. After digestion with nuclease S1 (16 units) for 2 hr at 37°C, the series of samples was assayed for acid-insoluble radioactivity. The nuclease S1 resistance background of the probe at zero time was usually 2-4% of the total radioactivity and was subtracted in the calculation of the ratio C/S in each sample. RESULTS AND DISCUSSION Measurement of Gene Number by DNA Reassociation Kinetics. The number of hsp 70 genes in Dm was estimated by determining the concentration of total genomic DNA required to give the same rate of reannealing with a gene-specific probe
The change in the pattern of gene expression after heat shock in Drosophila melanogaster (Dm) provides a system particularly suited for the investigation of the functional organization and expression of specific eukaryotic genes (1-7). We have recently characterized two cloned Dm DNA segments that contain genes for the major heat-induced polypeptide, the 70,000-dalton heat-shock protein (hsp 70) (8-10). The two hybrid plasmids 56H8 and 132E3 are derived from nonoverlapping regions of the Dm genome and contain, respectively, one and two hsp 70 gene copies (see Fig. 4a). Detailed analysis of these genes reveals that each is part of a 3-kilobase (kb) common unit composed of three contiguous sequence elements, x, y, and z (9). The mRNA coding region is restricted to a portion of the largest element (z), subdividing it into z coding (zc) and z noncoding (znj$; x, y, and Znc are located upstream of the gene (see Fig. 4a and ref. 10). Although the hsp 70 genes have a similar structure in the two hybrid plasmids, there are characteristic differences in their organization and their adjacent sequences. These differences may, in fact, reflect the chromosomal origin of the two Dm DNA segments. Functional hsp 70 genes are distributed at two loci, 87A and 87C, on the right of the third chromosome (3-6, 10-14), and 56H8 is found to be derived from 87A whereas 132E3 is from 87C. Cloned Dm DNA segments coding for hsp 70 have also been isolated in other laboratories (14, 15). Although the genes display many of the features described above, these segments differ in their overall sequence organization from 132E3 and 56H8. Such differences have also been observed in several new recombinant DNAs recently isolated in our laboratories (16). Most of these cloned Dm DNA segments do not appear to overlap. Here, we report the number of hsp 70 genes in a wild-type fly strain and in a diploid tissue culture cell line as well as their arrangement in total genomic DNA.
Abbreviations: kb, kilobase; hsp 70, 70,000-dalton heat-shock protein; Dm, Drosophila melanogaster. t Present address: Department of Biochemistry, Stanford University, Stanford, CA 94305. : Present address: Department of Biology, Princeton University, Princeton, NJ 08544.
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.
5254
Proc. Natl. Acad. Sci. USA 76 (1979)
Genetics: Mirault et al.
as observed when a reference DNA having a known number of gene copies was used as driver. At the point of gene-concentration equivalence, the DNA reassociation kinetics will coincide and this facilitates the titration. Fig. la shows the reassociation kinetics of the hsp 70 gene probe with increasing concentrations of 56H8 or 132E3 reference driver DNA. Because the 132E3 Dm segment has a duplication of the gene, the 132E3 DNA concentration used in these experiments was approximately one-half that of 56H8. Taking this into account, the reassociation kinetics observed with both driver DNAs are virtually identical. The concentration dependence of the slope, a, of the reassociation kinetics is essentially linear, and the calibration curve shown in Fig. Ic was used to calculate the number of gene copies in each driver DNA. Fig. lb shows the data obtained with Dm driver DNA prepared from either embryos, pupae, or KC cells. The DNA concentration used for each of these drivers was chosen to match the reference kinetics (Fig. la), and the reassociation kinetics slopes a are listed in Table 1. An average reiteration frequency N of the sequences complementary to the probe was calculated for each Dm DNA (Table 1). Assuming that the size of the Dm genome is 165,000 kb (24), these data indicate that our Oregon R population has between 7 and 11 hsp 70 gene copies per haploid genome. Moreover, no difference was evident between embryos and pupae. However, a lower value, six to seven copies, was obtained for the KC cell line. Measurement of Gene Number by Hybridization to DNA Blots. The DNA blotting technique (20) provides an alternative procedure for estimating the number of hsp 70 genes. Under appropriate experimental conditions, the hybridization signal detected by autoradiography is proportional to the concentration of the DNA sequences complementary to the probe (25). Cleavage of 56H8 (or 132E3) by Xba I yielded a 2-kb fragment a
5255
that hybridized to the probe (9) (Fig. 2). This fragment, derived from-the left side of the z element, overlaps with about 80% of the 56H8 Sal I/Sal I probe (see Fig. 4a) and accounts for the main hybridization signal (Fig. 2, lanes 1 and 2). A strong hybridization band was observed at the same position when embryonic or KC cell DNA was digested with Xba I. The additional weak bands correspond to fragments containing the end of the hsp 70 genes (unpublished data). The results suggest that all of the hsp 70 genes of Oregon R and KC cells contain the two Xba I restriction sites found in z of 56H8 and 132E3. From densitometric measurements of the autoradiographic signals on the 2-kb Xba I fragment, we deduce an average of 7.6 ± 0.4 copies (n = 4) in Oregon R embryos but only 5.8 I 0.6 in KC cells. This difference was observed in several experiments and, in agreement with the reannealing kinetics, suggests that the number of hsp 70 gene copies in Dm may be variable. Polymorphism in the Organization of the hsp 70 Genes. The apparent variation in the number of hsp 70 genes may be due to polymorphism. To explore this possibility, the distribution of the hsp 70 genes of Oregon R and KC cell DNA was examined by restriction enzyme digestion. Comparison of the patterns of hybridization obtained in parallel digestions of the two DNAs reveals a number of significant features (Fig. 3). First, five of the restriction sites in the z element of the common unit-Xba I, Xho I, BamHI, Xba I, and Sal I (see maps in Fig. 4a)-are present in all of the Oregon R and KC cell hsp 70 genes (Xba I digest in Fig. 2; Xho I, Sal I, and Xho I/Sal I digests in Fig. 3a; and Xba I/BamHI digests, not shown). This suggests that the sequence element (z, + Znc) defines a basic unit for the hsp 70 gene that is apparently conserved in both wild-type flies and cultured cells. Second, two different z variants are evident. The first type appears to be identical to that in 132E3 and is defined by the
b
C
1.6-
0.5
1.5
1.5-
~~~~~~~0
a
* -1.4
1.4 co
A
Time, hr
~~~~~0.4
0
"'ZOA~~~~
.
Time, hr
DNA, ng/ml
FIG. 1. Gene dosage by DNA reassociation kinetics. (a) 32P-Labeled Sal I/Sal I fragment from 56H8, a specific probe for the hsp 70 genes was reannealed at 8.8 X 104 cpm/ml in the absence (0) or in the presence of 56H8 DNA (-) at 14.2, 28.5, and 42.7 ng/ml or of 132E3 DNA (A) at 7.13, 14.3, and 21.4 ng/ml. Kinetics of duplex formation were followed by nuclease S1 assay and (C*/S*)2 values were plotted against time of reassociation. The least squares linear regression lines drawn here correspond to the probe alone (0) and to 56H8 DNA (-). (b) In parallel experiments, the same probe was reannealed in the presence of Dm embryonic DNA at 12.8 (0) and 25.7 (-) ,ug/ml, Dm pupae DNA (prep. 1) at 11.1 (3) and 22.2 (-) ,g/ml, Dm pupae DNA (prep. 2) at 12.9 (A) and 25.9 (A) ,ug/ml, Dm KC cell DNA at 12.9 (0) and 25.8 (*) ,g/ml. The least squares regression lines correspond to Dm embryonic DNA (0,O) and KC cell DNA (0, *); the bottom line corresponds to the probe alone as shown in a. (c) Concentration dependence of the slope a of the regression lines obtained in a with 56H8 DNA (*) and 132E3 DNA (0). The 132E3 DNA concentrations were converted to 56H8 equivalents in regard to the hsp 70 gene sequences, duplicated in 132E3. See Appendix for kinetics equation. The slope qD of the resulting least squares regression line was used to calculate the reiteration frequencies N presented in Table 1.
5256
Genetics: Mirault et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. Reiteration frequency of Dm hsp 70 genes by DNA reassociation kinetics Driver DNA N Source Yg/mi a, hr-' N (mean + SD) --
Embryos
-
0.107
-
16.3 32.6
8.92 7.82
25.7
0.203* 0.274* 0.194 0.260
11.1 22.2 12.9 25.9
0.185 0.256 0.177 0.255
12.9
Pupae
1
2
3
4
5
6
9.0 ± 1.0
10.23
9.14 10.65 10.28 8.12 8.77
9.5+ 1.2
KC cells
20.7 0.193* 6.35 41.3 0.264* 5.84 6.5 + 0.7 12.9 0.160 6.18 25.8 0.234 7.53 The data are from the experiments described in Fig. 1 and others. Reiteration frequency, N, the number of gene copies for the hsp 70 per haploid genome, was obtained by N=(a -a) GD 1 CD G 56H8 q 56H8 with the substitution a56H8 = a0 + q56H8 C56H8 (Fig. 1). The slope q was 5.525 X 103 liters-hr-'.g'- and the intercept a; was 0.109 hr-1. The value of N obtained for 56H8 and 132E3 was 0.98 i 0.02 (n = 5) and 2.06 i 0.16 (n = 5), respectively. The titration procedure appears to be insensitive to the slight nucleotide sequence differences observed between the 56H8 and 132E3 genes (F. Karch and I. Torok, personal communication). Incomplete homology was only detected at the reassociation plateau which gave 81% nuclease S1 resistance with 1:32E3 compared to the maximum of 86% driven by 56H8. Size distribution differences between reference and Dm DNAs entering reassociation kinetics were undetected by gel electrophoresis and thus are unlikely to contribute more than 10% error on (a - a*) values. Imprecision of DNA concentrations-not to exceed a few percent by diphenylamine assay-and in nuclease S1 assays are reflected in the SI)s. The error in the size of the hybrid plasmids (56H8 = 19.3 kb; 1 :32E.3 = 20.8 kb) is most likely 4
fi '-w
< -
A
a r--
1.5.
_
fpW_< a
4m
9 10 11 12 13 8 7 6 1 2 4 5 3 FIG. 3. Restriction fragments positive for hsp 70 gene sequences. (a) Pairwise comparison of restriction digests of embryonic (left gel) and KC cell DNA (right gel) hybridized to a 32P-labeled gene probe after transfer. The position of the restriction fragments found in 56H8 (
Organization of the multiple genes for the 70,000-dalton heat-shock protein in Drosophila melanogaster (DNA reassociation kinetics/DNA blots/restriction analysis/cloned DNA)
M.-E. MIRAULT, M. GOLDSCHMIDT-CLERMONT, S. ARTAVANIS-TSAKONAS*t, AND P. SCHEDL*t Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, 1211 Geneva 4, Switzerland; and *Department of Cell Biology, Biozentrum, University of Basel, 70, Klingelbergstrasse, 4056 Basel, Switzerland
Communicated by Niels Kaj Jerne, July 23, 1979
ABSTRACT The organization and number of 70,000-dalton heat-shock protein genes of Drosophila melanogaster has been investigated in a wild-type Oregon R fly stock and in a KC cell line. Six copies were found in the KC cells, and slightly more were found in the Oregon R population examined. In both cases, the basic gene element consisting of the mRNA coding region plus a short 5' "noncoding" sequence element was conserved. Two gene variants distinguished by specific restriction sites were found in both genomic DNAs. Restriction maps of the six genes in KC cells showed that these two gene variants are arranged differently. Restriction analysis of Oregon R embryonic DNA revealed polymorphism in the organization of the genes, which is not observed in KC cells. The data suggest that the arrangement as well as the number of genes for the 70,000-dalton heat-shock protein in D. melanogaster is subject to variations at both the 87A and 87C cytogenetic loci.
MATERIALS AND METHODS Tissue Culture Cells. The Dm KC161 cell line, an essentially diploid line derived from embryonic tissues (17), was a gift from G. Echalier. The cells were grown at 25°C in suspension as described (6). DNA Isolation. Hybrid plasmid DNA was isolated as described (8) and further purified by sucrose gradient centrifugation. Nuclear DNA from Dm (Oregon-R) and from KC cells was prepared according to Schachat and Hogness (18), including an additional phenol extraction (19) before consecutive CsCI density gradients. The DNA concentrations were measured by diphenylamine assay and by absorbance at 260 nm. All DNA preparations contained less than 2% RNA as measured by resistance to alkaline hydrolysis. Hybridization Probe for the hsp 70 Gene. The 2.15-kb Sal I fragment of 56H8 (see Fig. 4a) was isolated by gel electrophoresis and labeled by nick translation with [a-32P]dCTP [350 Ci/mmol (9); 1 Ci = 3.7 X 1010 becquerels]. The purity of the probe was ascertained by hybridization to DNA blots of 56H8 restriction digests according to Southern (20). DNA Reassociation Assayed by Nuclease Si Resistance. Dm DNA, 56H8 DNA, or 132E3 DNA was incubated for 20 min at 100°C in 0.13 M NaOH/0.1 mM EDTA/0.4% sodium dodecyl sulfate in the presence of carrier salmon sperm DNA (Sigma, type III) at 200 ,ug/ml. The 32P-labeled probe (about 4 X 107 cpm/,g) was then added and the mixture was incubated a further 4 min at 100°C and chilled in ice. These incubation conditions gave driver and probe DNAs of comparable size distribution (modal length, 300-400 nucleotides). Appropriate volumes of 1 M Tris-HCl, pH 7.4/5 M NaCl were added to give final concentrations of 1.5 M NaCl and 55 mM Tris-HCl. The DNA reassociation mixture was then incubated at 68°C; 100-,ul samples were removed every 20 min (including blanks at zero time), diluted into 0.5 ml of nuclease S1 buffer (30 mM NaOAc, pH 4.6/1 mM ZnCl2) and stored at -20°C. After digestion with nuclease S1 (16 units) for 2 hr at 37°C, the series of samples was assayed for acid-insoluble radioactivity. The nuclease S1 resistance background of the probe at zero time was usually 2-4% of the total radioactivity and was subtracted in the calculation of the ratio C/S in each sample. RESULTS AND DISCUSSION Measurement of Gene Number by DNA Reassociation Kinetics. The number of hsp 70 genes in Dm was estimated by determining the concentration of total genomic DNA required to give the same rate of reannealing with a gene-specific probe
The change in the pattern of gene expression after heat shock in Drosophila melanogaster (Dm) provides a system particularly suited for the investigation of the functional organization and expression of specific eukaryotic genes (1-7). We have recently characterized two cloned Dm DNA segments that contain genes for the major heat-induced polypeptide, the 70,000-dalton heat-shock protein (hsp 70) (8-10). The two hybrid plasmids 56H8 and 132E3 are derived from nonoverlapping regions of the Dm genome and contain, respectively, one and two hsp 70 gene copies (see Fig. 4a). Detailed analysis of these genes reveals that each is part of a 3-kilobase (kb) common unit composed of three contiguous sequence elements, x, y, and z (9). The mRNA coding region is restricted to a portion of the largest element (z), subdividing it into z coding (zc) and z noncoding (znj$; x, y, and Znc are located upstream of the gene (see Fig. 4a and ref. 10). Although the hsp 70 genes have a similar structure in the two hybrid plasmids, there are characteristic differences in their organization and their adjacent sequences. These differences may, in fact, reflect the chromosomal origin of the two Dm DNA segments. Functional hsp 70 genes are distributed at two loci, 87A and 87C, on the right of the third chromosome (3-6, 10-14), and 56H8 is found to be derived from 87A whereas 132E3 is from 87C. Cloned Dm DNA segments coding for hsp 70 have also been isolated in other laboratories (14, 15). Although the genes display many of the features described above, these segments differ in their overall sequence organization from 132E3 and 56H8. Such differences have also been observed in several new recombinant DNAs recently isolated in our laboratories (16). Most of these cloned Dm DNA segments do not appear to overlap. Here, we report the number of hsp 70 genes in a wild-type fly strain and in a diploid tissue culture cell line as well as their arrangement in total genomic DNA.
Abbreviations: kb, kilobase; hsp 70, 70,000-dalton heat-shock protein; Dm, Drosophila melanogaster. t Present address: Department of Biochemistry, Stanford University, Stanford, CA 94305. : Present address: Department of Biology, Princeton University, Princeton, NJ 08544.
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.
5254
Proc. Natl. Acad. Sci. USA 76 (1979)
Genetics: Mirault et al.
as observed when a reference DNA having a known number of gene copies was used as driver. At the point of gene-concentration equivalence, the DNA reassociation kinetics will coincide and this facilitates the titration. Fig. la shows the reassociation kinetics of the hsp 70 gene probe with increasing concentrations of 56H8 or 132E3 reference driver DNA. Because the 132E3 Dm segment has a duplication of the gene, the 132E3 DNA concentration used in these experiments was approximately one-half that of 56H8. Taking this into account, the reassociation kinetics observed with both driver DNAs are virtually identical. The concentration dependence of the slope, a, of the reassociation kinetics is essentially linear, and the calibration curve shown in Fig. Ic was used to calculate the number of gene copies in each driver DNA. Fig. lb shows the data obtained with Dm driver DNA prepared from either embryos, pupae, or KC cells. The DNA concentration used for each of these drivers was chosen to match the reference kinetics (Fig. la), and the reassociation kinetics slopes a are listed in Table 1. An average reiteration frequency N of the sequences complementary to the probe was calculated for each Dm DNA (Table 1). Assuming that the size of the Dm genome is 165,000 kb (24), these data indicate that our Oregon R population has between 7 and 11 hsp 70 gene copies per haploid genome. Moreover, no difference was evident between embryos and pupae. However, a lower value, six to seven copies, was obtained for the KC cell line. Measurement of Gene Number by Hybridization to DNA Blots. The DNA blotting technique (20) provides an alternative procedure for estimating the number of hsp 70 genes. Under appropriate experimental conditions, the hybridization signal detected by autoradiography is proportional to the concentration of the DNA sequences complementary to the probe (25). Cleavage of 56H8 (or 132E3) by Xba I yielded a 2-kb fragment a
5255
that hybridized to the probe (9) (Fig. 2). This fragment, derived from-the left side of the z element, overlaps with about 80% of the 56H8 Sal I/Sal I probe (see Fig. 4a) and accounts for the main hybridization signal (Fig. 2, lanes 1 and 2). A strong hybridization band was observed at the same position when embryonic or KC cell DNA was digested with Xba I. The additional weak bands correspond to fragments containing the end of the hsp 70 genes (unpublished data). The results suggest that all of the hsp 70 genes of Oregon R and KC cells contain the two Xba I restriction sites found in z of 56H8 and 132E3. From densitometric measurements of the autoradiographic signals on the 2-kb Xba I fragment, we deduce an average of 7.6 ± 0.4 copies (n = 4) in Oregon R embryos but only 5.8 I 0.6 in KC cells. This difference was observed in several experiments and, in agreement with the reannealing kinetics, suggests that the number of hsp 70 gene copies in Dm may be variable. Polymorphism in the Organization of the hsp 70 Genes. The apparent variation in the number of hsp 70 genes may be due to polymorphism. To explore this possibility, the distribution of the hsp 70 genes of Oregon R and KC cell DNA was examined by restriction enzyme digestion. Comparison of the patterns of hybridization obtained in parallel digestions of the two DNAs reveals a number of significant features (Fig. 3). First, five of the restriction sites in the z element of the common unit-Xba I, Xho I, BamHI, Xba I, and Sal I (see maps in Fig. 4a)-are present in all of the Oregon R and KC cell hsp 70 genes (Xba I digest in Fig. 2; Xho I, Sal I, and Xho I/Sal I digests in Fig. 3a; and Xba I/BamHI digests, not shown). This suggests that the sequence element (z, + Znc) defines a basic unit for the hsp 70 gene that is apparently conserved in both wild-type flies and cultured cells. Second, two different z variants are evident. The first type appears to be identical to that in 132E3 and is defined by the
b
C
1.6-
0.5
1.5
1.5-
~~~~~~~0
a
* -1.4
1.4 co
A
Time, hr
~~~~~0.4
0
"'ZOA~~~~
.
Time, hr
DNA, ng/ml
FIG. 1. Gene dosage by DNA reassociation kinetics. (a) 32P-Labeled Sal I/Sal I fragment from 56H8, a specific probe for the hsp 70 genes was reannealed at 8.8 X 104 cpm/ml in the absence (0) or in the presence of 56H8 DNA (-) at 14.2, 28.5, and 42.7 ng/ml or of 132E3 DNA (A) at 7.13, 14.3, and 21.4 ng/ml. Kinetics of duplex formation were followed by nuclease S1 assay and (C*/S*)2 values were plotted against time of reassociation. The least squares linear regression lines drawn here correspond to the probe alone (0) and to 56H8 DNA (-). (b) In parallel experiments, the same probe was reannealed in the presence of Dm embryonic DNA at 12.8 (0) and 25.7 (-) ,ug/ml, Dm pupae DNA (prep. 1) at 11.1 (3) and 22.2 (-) ,g/ml, Dm pupae DNA (prep. 2) at 12.9 (A) and 25.9 (A) ,ug/ml, Dm KC cell DNA at 12.9 (0) and 25.8 (*) ,g/ml. The least squares regression lines correspond to Dm embryonic DNA (0,O) and KC cell DNA (0, *); the bottom line corresponds to the probe alone as shown in a. (c) Concentration dependence of the slope a of the regression lines obtained in a with 56H8 DNA (*) and 132E3 DNA (0). The 132E3 DNA concentrations were converted to 56H8 equivalents in regard to the hsp 70 gene sequences, duplicated in 132E3. See Appendix for kinetics equation. The slope qD of the resulting least squares regression line was used to calculate the reiteration frequencies N presented in Table 1.
5256
Genetics: Mirault et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. Reiteration frequency of Dm hsp 70 genes by DNA reassociation kinetics Driver DNA N Source Yg/mi a, hr-' N (mean + SD) --
Embryos
-
0.107
-
16.3 32.6
8.92 7.82
25.7
0.203* 0.274* 0.194 0.260
11.1 22.2 12.9 25.9
0.185 0.256 0.177 0.255
12.9
Pupae
1
2
3
4
5
6
9.0 ± 1.0
10.23
9.14 10.65 10.28 8.12 8.77
9.5+ 1.2
KC cells
20.7 0.193* 6.35 41.3 0.264* 5.84 6.5 + 0.7 12.9 0.160 6.18 25.8 0.234 7.53 The data are from the experiments described in Fig. 1 and others. Reiteration frequency, N, the number of gene copies for the hsp 70 per haploid genome, was obtained by N=(a -a) GD 1 CD G 56H8 q 56H8 with the substitution a56H8 = a0 + q56H8 C56H8 (Fig. 1). The slope q was 5.525 X 103 liters-hr-'.g'- and the intercept a; was 0.109 hr-1. The value of N obtained for 56H8 and 132E3 was 0.98 i 0.02 (n = 5) and 2.06 i 0.16 (n = 5), respectively. The titration procedure appears to be insensitive to the slight nucleotide sequence differences observed between the 56H8 and 132E3 genes (F. Karch and I. Torok, personal communication). Incomplete homology was only detected at the reassociation plateau which gave 81% nuclease S1 resistance with 1:32E3 compared to the maximum of 86% driven by 56H8. Size distribution differences between reference and Dm DNAs entering reassociation kinetics were undetected by gel electrophoresis and thus are unlikely to contribute more than 10% error on (a - a*) values. Imprecision of DNA concentrations-not to exceed a few percent by diphenylamine assay-and in nuclease S1 assays are reflected in the SI)s. The error in the size of the hybrid plasmids (56H8 = 19.3 kb; 1 :32E.3 = 20.8 kb) is most likely 4
fi '-w
< -
A
a r--
1.5.
_
fpW_< a
4m
9 10 11 12 13 8 7 6 1 2 4 5 3 FIG. 3. Restriction fragments positive for hsp 70 gene sequences. (a) Pairwise comparison of restriction digests of embryonic (left gel) and KC cell DNA (right gel) hybridized to a 32P-labeled gene probe after transfer. The position of the restriction fragments found in 56H8 (
