Length polymorphisms, restriction site variation, and maternal inheritance of mitochondrial DNA of Drosophila melanogaster

PLASMID

3, 109-115 (1980)

Length Polymorphisms, Restriction Site Variation, and Maternal Inheritance of Mitochondrial DNA of Drosophila melanogaster J. G. REILLY’ Scripps

Clinic and Research 10666 North Torrey

AND C. A. THOMAS, Foundation, Pines Road,

JR.

Department of Cellular Biology, La Jolla, California 92037

Received November 19, 1979 We have studied the mitochondrial DNA in three wild type laboratory strains ofDrosophila ry+5 and two Oregon R-substrains, called here R and E. Lengths of the restriction bands for EcoRI, BglII, HpaII, MspI, HaeIII, and Hind111 were compared. The number of restriction sites was identical in all strains, with the exception of an extra HaeIII site in ry+§. Careful comparison of restriction fragment lengths showed that bands containing the AT-rich region were different in length among all strains. The laboratory strains, ry+5, proved to be a mixture of strains carrying different mtDNAs; these separated into substrains Gl and G2 in the progeny of single pair matings. Adult progeny of reciprocal crosses of Gl and R were analyzed byHaeII1 restriction digestion. The results demonstrated maternal inheritance for both the extra restriction site and band containing the AT-rich region. melanogaster,

The mitochondria of Drosophila melanogaster contain a small, 18-kb double-stranded circular DNA molecule that has received considerable attention in recent years since its first isolation by Bultmann and Laird (1973) and Polan ef al. (1973). The mitochondrial DNA (mtDNA) restriction patterns for numerous enzymes are known (Manteuil et al., 1975; Shen et al., 1976); in addition restriction maps have been made (Wolstenholme and Fauron, 1976; Klukas and Dawid, 1976; Bonner et al., 1978). On these maps the location of the ribosomal genes, the position of an AT-rich region (Klukas and Dawid, 1976), and the site of initiation and direction of DNA replication, within the AT-rich region, are known (Goddard and Wolstenholme, 1978). Circular concatemers have been identified (Shah and Langley, 1977) as well as monomers that differ in the number of superhelical turns (Rubenstein et al., 1977). The transcriptional pattern of the mtDNA is beginning to be understood (Bonner et al., 1978; Beminger et al., 1979). Perhaps most interesting is a region of the r T O whom correspondence

should be addressed.

molecule, about 20% of the genome (ca. 4 kb), that is very rich in AT and contains the origin of replication (Goddard and Wolstenholme, 1978). This region has been recognized by many workers, but studied most effectively by partial denaturation (Fauron and Wolstenholme, 1976; Bultmann et al., 1976) and by heteroduplexing between Drosophila species (Shah and Langley, 1979a; Zakour and Bultmann, 1979) and photochemical cross linking (Beminger et al., 1979) in the electron microscope. Fauron and Wolstenholme (1976) showed that the length of the AT region differed among species of Drosophila, but was thought to be of constant length in D. melanogaster. We have discovered that various laboratory strains of D. melanogaster have AT-rich segments of various lengths. In addition, one strain contains an additional, new, Hue111 cleavage site in a region .outside the AT-rich fragment. Using these differences we show here that the inheritance of mtDNA is primarily maternal. Even though included in the fertilized egg, the male mtDNA molecules make no detectable contribution to the progeny mitochondria. 109

0147-619x/80/02010%07$02.00/0 Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

110

REILLY

MATERIALS

AND

AND METHODS

Drosophila stocks and fly culture. Three Drosophila melanogaster stocks were used

in this study. Oregon R, called here E strain, was received from S. Elgin as frozen (-70°C) embryos. A second Oregon R line, called here R, was cultured in this lab and obtained from M. Messelson who received the strain in 1971 from S. Elgin (both at Harvard University). The third type, ry+5 (McCarron et al., 1974), was received from B. Gelbart (Harvard). This strain was found to be a mixed population consisting of sublines with two different length mitochondria DNAs that were separable by single pair matings. The two sublines are called here Gl and G2. Flies were grown on corn meal molasses by standard methods. Embryos were collected from population cages, 1 x 1 x 2 ft, containing >30,000 adults on grape agar plates (Kriegstein and Hogness, 1974) and stored at 5°C till extraction of mitochondria. Mitochondrial DNA extraction. Fresh embryos (5-10 g) were dechorionated and homogenized with a motor-driven Teflon pestle (10 strokes, full speed) in 0.01 M CAPS, pH 10.4 (Sigma), 1.0 M hexelyene glycol, 0.001 M CaCl, (Kriegstein and Hogness, 1974). Larvae were homogenized as above, then filtered through four layers of cheesecloth. Adults were ground in a handheld scintered glass mortar and pestle, then suspended in 0.3 M sucrose, 0.1 M Nacl, 0.03 M Tris, pH 7.4, and 0.01 M EDTA (Wolstenholme and Fauron, 1976) and homogenized with a motor-driven Teflon pestle described for embryos. Mitochondria were collected from all developmental stage homogenates by differential centrifugation (Wolstenholme and. Fauron, 1976). The semipurified mitochondria were then digested overnight with 50 pg/rnl Proteinase K (SO’C), nucleic acids extracted with chloroform:isoamylalcohol (24:l) and mtDNA purified by potassium iodide-ethidium bromide gradients (Rubenstein et al., 1977). Total DNA extraction was by the method of R. Lifton (personal communication).

THOMAS

Adult flies (l- 100) were ground in a cold (S’C) scintered glass homogenizer in 0.5 ml (l-25 flies) or 2 ml (25- 100 flies) of four parts NSET (0.1 M NaCl, 0.2 M sucrose, 0.01 M EDTA, 0.02 M Tris, pH 8.0), one part lysis buffer (0.25 M EDTA, 2.5% SDS, 0.5 M Tris, pH 9.2), l/100 volume diethylpyrocarbonate, mixed immediately before use. The homogenate was incubated at 65°C 30 min, then potassium acetate added to 1.0 M and the sample was incubated for 30 min at 5°C. The precipitate was removed by centrifugation in an Eppendorf centrifuge at 12,000g for 0.5 min and the supematant was allowed to precipitate at room temperature for 2 min after adding 2 volumes 100% ethanol. The ethanol pellet was collected by centrifugation 12,OOOg, 3 min, washed two times with 70% ethanol, and resuspended in 1.0 mM Tris, pH 7.5, 0.1 mM EDTA. DNA restriction HpaII, HpaI, DpnI,

and

electrophoresis.

HaeIII (Bethesda Research Laboratories), and MspI (New England BioLabs) were used as described by manufactures. BglII was a gift from J. Cregg. EcoRI was a gift from S. S. Smith. fiindII1 was prepared by an unpublished procedure of J. G. Reilly and S. S. Smith. The reaction conditions for these enzymes were described in Bethesda Research Laboratories’ catalog. Electrophoresis was with low EEO (Sigma) agarose in 0.04 M Tris, pH 7.9, 0.001 M EDTA, 0.005 M sodium acetate (Hayward and Smith, 1972). Horizontal submerged agarose slab gels were run at 3 V/cm for 12-16 h. Gels were then stained with 0.5 ,ug/ml ethidium bromide for 30 min and photographed after illumination with a short wave uv light using Polaroid type 665 film, a Polaroid 195 land camera, close-up lens, and wratten 23A filter (Kodak) behind a yellow filter (UV Products Co.) (Hamer and Thomas, 1975). RESULTS

AND DISCUSSION

As can be seen in Fig. 1 the three different laboratory strains of D. melanogaster mtDNA generate four different patterns of

MITOCHONDRIAL

111

DNA OF Drosophila ld

B 123456789

123456

FIG. 1. Gel electrophoresis of Gl, G2, R, and E mitochondrial DNA restricted with various enzymes. Mitochondrial (mt) DNA was restricted and electrophoresed in 0.8% agarose gels as described in Methods, stained with 0.5 &ml ethidium bromide, and photographed. The resulting segments were assigned lengths on the basis of A/Hind111 fragments (not shown) of 23.6, 9.64, 6.64, 4.34, 2.26, 1.98, and 0.56 kb and A/EcoRI fragments of 21.7, 7.52, 5.83, 5.64,4.85, and 3.48 kb (Philippsen ef al., 1978). A shows ry+5 (considered to be a mixed population containing sublines Gl and G2 as noted), R and E restricted with EcoRI (1, 2, 3) or EcoRI + BglIII (4, 5, 6). B is Gl + G2, R, and E restricted with HpaII (1, 2, 3), MspI (4, 5, 6), or Hue111 (7, 8, 9). C is the Hind111 restriction pattern for R adultsafter twosuccessive single pair matings(l), Gl + G2embryos(2), Gl + G2adults(3), Eembryos (4), R adults (5), and E embryos of a different preparation then 4 (6). Bands are lettered and correspond to those listed in Table 1. In A, EcoRI + &/II, a faint band of 3.1 kb can be seen in some preparations. The origin of this band is unknown; it is suspected to be satellite DNA which occasionally contaminates the sample as a resuh of the KI-ethidium bromide gradient purification step.

restriction segments. A summary of the lengths of the restriction segments appears in Table 1. It can be seen that the variation generally involves only a single restriction segment; these lengths are in italics in Table 1. In view of the fact that the measured lengths of these various restriction segments agree well with those previously published, it is reasonable to accept the restriction site mapping published from various groups

(Wolstenholme and Fauron, 1976; Klukas and Dawid, 1976; Bonner el al., 1978). This correlation shows that the segment of variable length always includes the AT-rich segment that was first found by denaturation mapping (Wolstenholme and Fauron, 1976). Since strain R has the shortest AT-rich segment, subtraction shows that strain Gl has 750 bases more, E has 570 bases more, and G2 has 240 bases more than strain R.

-GI

-I kb a

AT

RICH

REGION

t

T

FIG. 2. Restriction map summarizing length and restriction variants shown in Fig. 1. Restriction sites shown are BglII, used to linearize the normally circular DNA, Hind111 (q), HaeIII (F), and the new Hue111 site in the Gl and G2 strains ( Ta). The relative length and approximate map position oflength variants Gl, G2, and E are shown, with the map being drawn for R, the shortest length variant.

18.67

0.92

18.47

0.92

1.72

5.03

10.80

2

R

19.02

0.92

1.72

5.03

II.35

E 3

18.62

0.92

1.72

4.35

5.03

6.60

GI 4

OF RESTRICTION

18.12

0.92

1.72

4.35

5.03

6.10

G2 4

EcoRI

vahles

18.79

2.04

3.15

3.60

0.24 f 0.06 0 0.57 ? 0.05

18.48 i 0.25 18.24 f 0.25 18.82 + 0.27

R E

Differences in AT-rich segment”,P

18.19

2.04

3.15

3.60

10.0

9.40

G2

2 SD

18.54

2.04

3.15

3.60

E 3,6

Drosophila

R 2.5

or MspI

B

VARIOUS

0.75 t 0.07

lengths

Mean

19.04

2.04

3.15

3.60

9.75

G2 I,4

HpaII

FROM

19.00 c 0.23

Total

1.4

GI

10.25

OF mtDNA

Cl

18.42

0.92

0.92 17.92

I .72

4.35

5.03

6.40

E 6

1.72

4.35

5.03

5.90

R 5

+ &/II

A

FRAGMENTS

1

18.88

h

3.53

5.25b

9.50

GI 1

G2 7

18.33

h

3.53

5.25’

18.13

3.53

5.90

8.70

R 8

SHOWN B Hoe111

x.95

STRAINS

IN

18.73

3.53

5.90

9.30

E 9

FIG.

1

19.23

4.73

5.75

8.46

GI 293

18.74

c

4.73

5.26

8.46

G2 2,3

C Hind111

y All lengths are in kb, kilobases. b One (or more) additional Hoe111 sites near the end of segment b must cause this shorter length, the smaller of which (0.60 kb) is not seen but is added into the sums reported. HpaI cuts mtDNA only once, and cleaves the Hoe111 b segment into unequal lengths (Bonneret al., 1978). HaeIII-Hpul double digests show that HarIII b is broken into two segments: 2.34 and 3.27 kb (for E) and 2.34 and 2.70 kb (for Gl and G2). Thus the additional Hoe111 site is located near the right end of Hoe111 b as shown (Fig. 2). r A 0.29 segment reported by Banner ef al. (1978) was not seen in our gels, perhaps because of its small sire. However it is included in the sums reported. * Vanable bands are in Italics: these bands al1 contam the AT-rich region as determmed from the restriction maps of Wolstenholme and Fauron (1976). Kiukas and David (1976). Banner PI 0,. (1978). TheIengthsofthenonvariabIefragmentsandlengthsofvariabIe bands were incloseagreement with these mapsas wellas therestrictionfragment patternsof Mantueilrral. (1975), Shahand LangIey(I977), and Shen et al. (1975). p Differences from strain R. the smallest, calculated from variable segments (italics) only.

19.17

0.92

d

Sum

1.72

I.72

c

e

5.03

5.03

b

II.00

II.50

Band a

G2 1

GI I

Enzyme Strain Channel

A EcoRl

LENGTHS”

TABLE

18.51

c

4.73

5.03

8.46

19.13

c

4.73

5.65

8.46

E 4.6

R I.5

% u 4

2

E P

MITOCHONDRIAL

The nature of this extra DNA and its precise location(s) within the AT-rich segment is unknown. Several tempting hypotheses about duplications, deletions, or insertion sequences must wait until restriction sites are found, within both AT-rich region and the variant region, and sequencing done. The second kind of difference that can be found in Table 1 is a 5.25-kbHueII1 segment from Gl and G2 rather than a 5.90-kb segment found in R and E. This is due to the presence of one (or more) additional Hue111 sites located within Hind111 segment c, which is seen to be of constant length (Fig. lC, Table 1). The location of this site shown on the map in Fig. 2 was confirmed by HueIII&a1 double digestions (see Table 1, footnote b). Recently Shah and Langley (1979b) reported an additional Hue111 site which they locate close to the position shown in Fig. 2. Figure 1 shows the identical digestion patterns produced by HpuII (which breaks at CCGG but not CCmeGG) and MspI (which breaks at both sites) (Waalwijk and Flavell, 1978). DpnI (which cleaves only GAmeTC) does not break R and E mtDNA yet MboI (which cleaves at GATC) produces many fragments (results not shown). Thus methylation at those two sites does not occur in Drosophila mtDNA in agreement with the observations of Groot and Kroong (1979) on mtDNA in other species. Unfortunately, no analogous enzyme is known to test the Hue111 site (GGCC) for possible methylation. Finally, these results provide an opportunity to test whether the mtDNA is maternally inherited or not. Some legitimate question exists on this subject because the “nebenkern,” which is known to be derived from mitochondria during spermatogenesis, enters the egg during fertilization (Lindsley and Takuyasu, 1980). Does the paternal mtDNA contribute significantly to the progeny mtDNA? When the appropriate crosses are performed one finds that mtDNA from only the female parent is found in the immediate adult progeny (Fig. 3). Thus, Drosophiliu joins the numerous other higher

113

DNA OF Drosophila

870-

.8.95

5.90-5.25

3.53-

-3.53

FIG. 3. Demonstration of maternal inheritance of mitochondtial DNA in Drosophila. Total DNA extracted from parental adult flies (G2 and R) or mitochondrial DNA from these adults were prepared as described in Methods. Crosses were done by mating a virgin d with a virgin 0. Adult progeny from these crosses (0.1-0.2 g) were collected and total DNA extracted as described in the Methods section. These DNAs were digested with HaeIII, electrophoresed on 0.5% agarose gels, stained, and photographed as in Fig. 1. Channel 1 contains mtDNA from strain R prepared from the adult progeny of two successive single pair matings; Channel 2 contains total R DNA; Channel 3 contains total DNA from the progeny of Cl2 d x R 0 ; Channel 4 contains total DNA from the progeny of a duplicate cross as shown in Channel 3; Channel 5 contains total DNA from the progeny Rd x G20 ; Channel 6 contains total DNA from G2, a stock that was derived from Gl + G2 after two successive single pair matings which resulted in the separation of Gl from G2; Channel 7 contains mtDNA from the G2 stock. The segment lengths of mtDNA are listed on the left side for R and the right side for G2, these bands correspond to letters a, b, c in Fig. 1 and Table 1. In the total DNA digests a 4.35kb band is also seen (not labeled). This band is similar in length to band D found in HaeIII digests of total DNA, and is believed to be from satellite DNA (Manteuil et al., 1975). Notice that the 5.25kb and the 8.95-kb Hoe111 mtDNA restriction segment is found only in the progeny of G2 females, while the 5.90- and 8.7Skb segments are found only in the progeny of R females. This transmission pattern is irrespective of the male.

eukaryotes that display maternal inheritance of their mitochondrial genomes (Hutchinson et al., 1974; Buzzo et al., 1978; Francisco et al., 1979; Brown and Wright, 1979).

114

REILLY

AND

ACKNOWLEDGMENTS We thank Dr. S. Elgin for the Drosophila embryos, Drs. S. S. Smith and J. Cregg for restriction enzymes, M. Cook and R. Lee for technical assistance with fly cultures, and Dr. K. Saigo for critical reading of the manuscript. This work was supported by grants from NIH GM 25531; NSF PCM78-16282; and J.G.R. was an American Cancer Society Postdoctoral Fellow, California division (J-457-01).

REFERENCES BERNINGER, M., CECH, T., FOSTEL, J., POTTER, D., SCOTT, M., AND PARDUE, M. L. (1979). The structure and function of the mitochondrial DNA of Drosophila melanogaster. In “Specific Eukaryotic Genes, the Alfred Benzon Symposium 13” (J. Engberg, H. Klenow, and V. Leick, eds.), pp. 229-243. Scandinavian University Press, Munksgaard. BULTMANN, H., AND LAIRD, C. D. (1973). Mitochondrial DNA from Drosophila melanogaster. Biochim. Biophys. Aria 299, 196-209. BULTMANN, H., ZAKOLJR, R. A., AND SOSLAND, M. A. (1976). Evolution of Drosophila mtDNAs. Comparison of denaturation maps. Biochim. Biophys. Acfa 454, 21-44. BONNER, J. J., BERNINGER, M., AND PARDUE, M. L. (1978). Transcription of polytene chromosomes and the mitochondrial genome in Drosophila melanogaster. Cold Spring Harbor Symp. Quanf. Biol. 42, 803-814. BROWN, W. M., AND WRIGHT, J. W. (1979). Mitochondrial DNA anlayses and the origin and relative age of parthenogenetic lizards (genus Cnemidophorus). Science 203, 1247- 1241. Buzzo, K., FOUTS, D. L., AND WOLSTENHOLME, D. R. (1978). EcoRI cleavage site variants in mitochondrial DNA molecules from rats. Proc. Natl. Acad. Sci. U. S. A. 75, 909-913. FAURON, C. M.-R., AND WOLSTENHOLME, D. R. (1976). Structural heterogeneity of mitochondrial DNA molecules within the genus Drosophila. Proc. Natl. Acad. Sci. U. S. A. 73, 3623-3627. FRANCISCO, J. F., BROWN, G. G., AND SIMPSON, M. W. (1979). Further studies on types A and B rat mtDNAs: Cleavage maps and evidence for cytoplasmic inheritance in mammals. Pfasmid 2, 426-436. GODDARD, J. M., AND WOLSTENHOLME, D. R. (1978). Origin and direction of replication in mitochondrial DNA molecules from Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 75, 3886-3890. GROOT, G. S. P., AND KROONG, A. M. (1979). Mitochondrial DNA from various organisms does not contain internally methylated cytosine in -CCGGsequence. Biochim. Biophys. Acta 564, 355-357. HAMER, D. H., AND THOMAS, C. A., JR. (1975). The cleavage of Drosophila melanogaster DNA by restriction enzymes. Chromosoma 49, 243-267.

THOMAS HAYWARD, G. S., AND SMITH, M. G. (1972). The chromosome of bacteriophage T.5; 1. Analysis of the single-stranded DNA fragments by agarose gel electrophoresis. .I. Mol. Biol. 63, 383-395. HUTCHINSON, C. A., NEWBOLD. J. E., POTTER, S. S., AND EDGELL, M. A. (1974). Maternal inheritance of mammalian mitochondrial DNA. Nature (Loncfon) 251, 536-538. KLUKAS, C. K., AND DAWID, I. B. (1976). Characterization and mapping of mitochondrial ribosomal RNA and mitochondrial DNA in Drosophila melanogaster. Cell 9, 615-625. KREIGSTEIN, H. J., AND HOGNESS, D. (1974). Mechanism of DNA replication in Drosophila chromosomes: Structure of replication forks and evidence for bidirectionality. Proc. Natl. Acad. Sri. U. S. A. 71, 135- 139. LINDSLEY, D. L.. AND TOKUYASU, K. T. (1980). Spermatogenesis. In “The Genetics and Biology of Drosophila” (M. Ashbumer and T. R. F. Wright, eds.), Vol. 2b, pp. 225-294. Academic Press, New York. MANTEUIL, S., HAMER, D. H., AND THOMAS, C. A., JR. (1975). Regular arrangement of restriction sites in Drosophila DNA. Cell 5, 413-422. MCCARRON, M., GELBART, W., AND CHOUNICK, A. (1974). Intracistronic mapping of electrophoretic sites in Drosophila melanoga.ster: Fidelity of information transfer by gene conversion. Genetics 76, 289-299. PHILIPPSEN, P., KRAMER, R. A., AND DAVIS, R. W. (1978). Cloning of the yeast ribosomal DNA repeat until in SsrI and Hind111 lambda vectors using genetic and physical size selections. J. Mol. Biof. 123, 371-386. POLAN, M. L., FRIEDMAN, S., GALL, J. G., AND GEHRING, W. (1973). Isolation and characterization of mitochondrial DNA from Drosophila melanogasfer. J. Cell. Biol. 56, 580-589. RUBENSTEIN, J. L. R., BRUTLAG, D., AND CLAYTON, D. A. (1977). The mitochondrial DNA of Drosophila melanogaster exists in two distinct and stable superhelical forms. Cell 12, 471-482. SHAH, D. M., AND LANGLEY, C. H. (1977). Complex mitochondrial DNA in Drosophila. Nucleic Acid Res. 4, 2949-2960. SHAH, D. M., AND LANGLEY, C. H. (1979a). Electron microscopy heteroduplex study of Drosophila mitochondrial DNAs: Evolution of A + T rich region. Plasmid 2, 69-78. SHAH, D. M., AND LANGLEY, C. H. (1979b). Interand intraspecific variation in restriction maps of Drosophila mitochondrial DNA’s, Nature (London) 281, 696-699. SHEN, C. J., WIESEHAHN, G., AND HEARST, J. E. (1976). Cleavage patterns of Drosophila mekznogaster satellite DNA by restriction enzymes. Nucleic Acid Re.s. 5, 931-951.

MITOCHONDRIAL WAALWIJK, C., AND FLAVELL, R. A. (1978). MspI, an isoschizomer of HpaII which cleaves both unmethylated and methylated &a11 sites. Nucleic Acid

Res. 5, 3231-3236.

WOLSTENHOLME, D. R., and FAURON, C. M.-R. (1976). A partial map of the circular mitochondrial genome

115

DNA OF Drosophila of Drosophila

melanogaster.

.I. Cell Biol.

71, 434-

448. ZAKOUR, R. A., AND BULTMANN, H. (1979). Evolution of Drosophila mitochondrial DNA’s: Analysis of heteroduplex molecules. Biochim. Biophys. Acta 564, 342-351.