shown by acid hydrolysis of labelled DNA. .... After acid hydrolysis, 100. fig ...... A after alkaline hydrolysis and subsequent electrophoresis on 3MM paper at.
EVOLUTION OF POLYPYRIMIDINES IN DROSOPHILA Y. M. T. CSEKOllzJ, N. A. DOWER2, P. MIN002, L. LOWENSTEIN1, G. R. SMITH&, J. STONE2 AND R. SEDEROFF1*2s5 IDepartment of Biological Sciences, Columbia University, New York, New York 2Department of Biology, University of Oregon, Eugene, Oregon AND
Department of Genetics, North Carolina State University, Raleigh, North Carolina Manuscript received May 17, 1978 Revised copy received December 27, 1978 ABSTRACT
We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/ polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D . simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. uarians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, but the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polypurine sequence, with an oligonucleotidecomposition different from that of D . melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.-These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA. These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed.
INCE the discovery of satellite DNA in 1961 (SUEOIIA1961; KIT 1961), this unusual kind of DNA has been detected in a wide variety of eukaryotic organisms. Significant advances in our understanding of the organization and possible function of satellite DNA have come from the demonstration that these DNAs TRAUTNER and KORNare often composed of short repeating sequences (SWARTZ, BERG 1962; WARING and BRITTEN1966; SOUTHERN 1970) and that these sequences are usually a major component of constitutive heterochromatin (SCHILDKRAUT and MAIO1968; PARDUE and GALL1970; JONES1970). Current address Divlslon of Molecular and Cellular Biology, Natlonal Jewlsh Hospital and Research Center, Denver, Colorado. Institute of Molecular Biology, Umverslty of Oregon, Eugene, Oregon To whom correspondence should be addressed at North Carolina State University.
'
Genetics 92: 459-484 June, 1979.
460
Y. M. T. CSEKO
et al.
In previous work, we have characterized an unusual repetitive DNA sequence in D.melanogaster containing poly-pyrimidinejpolypurine tracts that range from 300 to more than 1000 nucleotides long (BIRNBOIM,STRAUS and SEDEROFF 1975; BIRNBOIMand SEDEROFF 1975). These polypyrimidine/polypurine tracts are composed of a predominant sequence of (TCTCT/AGAGA) (SEDEROFF, LOWENSTEIN and BIRNBOIM 1975) and are now known to be a major fraction of satel1975; ENDOW, lite IV, also called the 1.705 satellite (BRUTLAGand PEACOCK POLAN and GALL1975; BRUTLAG et al. 1977). Restriction enzyme digestion by Mbo ZZ (ENDOW 1977) and psoralen cross-linking studies (SHEN and HEARST 1977) have shown that satellite IV DNA is extremely homogeneous, as might be expected for a satellite composed of exceedingly long pyrimide tracts. These DNA sequences are located at a small number of heterochromatic sites, primarily in the second chromosome and in the Y chromosome ( SEDEROFF, LOWENSTEIN and BIRNBOIM1975; PEACOCK and STEFFENSEN 1976). A major purpose of this work was to examine the evolution of the polypyrimidine sequences found in D.melanogaster. This study was made possible by the detailed knowledge of the phylogeny of Drosophila accumulated over many years (PATTERSON and STONE1952; BOCKand WHEELER 1972; THROCKMORTON 1976). Several mechanisms have been proposed for the evolution of satellite DNAs involving gradual changes by unequal exchange (SOUTHERN 1975; SMITH1976) or by more drastic changes based on replicational mechanisms (BRITTENand KOHNE1968). Long pyrimidine tracts offer advantages for the study of satellite evolution in that their detection and quantitation may be done by acid hydrolysis rather than by traditional methods based on DNA densities. In addition, we have developed a technique f o r the detection of specific repeated sequences in Drosophila by carrying out nucleic acid hybridizations in crude extracts (CSEKO et al., in preparation). With this technique it has been possible to screen 101 Drosophila species for the presence of the polypyrimide sequence found in D. melanogaster. An identical or similar sequence has been found in a small number of the species tested, and where possible, the new repetitious sequence has been isolated and characterized. We have carried out these experiments in order to answer the following questions with regard to the evolution and possible function of satellite DNA. (1 ) How widespread is a specific satellite sequence within the genus Drosophila? (2) Do closely related species often contain a small amount of the same sequence that is not detected as a distinct satellite? ( 3 ) Is there special evolutionary significance to the unusual asymmetric DNA structure found in polypyrimidine/ polypurine DNA? MATERIALS AND METHODS
Crude extract preparation and hybridization reactions: The preparation of crude extracts, annealing conditions and thermal chromatography are described in detail in a separate paper, et al., in preparation). which is available on request (CSEKO DNA labelling in larval brains with [gH]-thymidine: The labelling of larval brains was
EVOLUTION O F POLYPYTIMIDINES
461
done as described by LEE (1974). Fifty to 100 brains with associated imaginal discs from third instar larvae were dissected in 0.1 M NaC1, then transferred to reduced Schneider's medium (SEDEROFF and CLYNES1974) containing 100 units per ml penicillin, 100 pg per m l of streptomycin and 50 pCi per ml [3H]-thymidine. After one to two hr, the brains were washed in 0.1 M NaCl and pooled for DNA extraction. Purification of labelled DNA from larval brains: DNA extraction was modified from BIRNBOIM, STRAUSS and SEDWOFF(1975). The labelled brains, in a drop of 0.1 M NaC1, were lysed in one ml of a solution containing 1 M LiC1, 0.1 M Tris-HC1 (pH 8.0), 0.1 M EDTA and 2% SDS. One-hundred micrograms of E. coli DNA was added as carrier. The mixture was transferred to 50" and incubated, with 500 fig predigested pronase, for one hr. The mixture was then put on ice and extracted three times with an equal volume of chloroform-isoamyl alcohol (24:l). The DNA was ethanol precipitated and resuspended in 0.5 ml of water. Isolation of [SH] labelled polypyrimidines as acid-resistant acid-precipitable (ARAP) radioactivity: To the [3H] labelled DNA and carrier DNA, in 0.5 ml of water, 1.5 ml of 88% formic acid with 2% diphenylamine was added. Acid hydrolysis proceeded at 30" for 18 hr. The isolation of long pyrimidine tracts was done by the ARAP procedure, as described by BIRNBOIMand SEDEROFF (1975). Briefly, 50 pg of carrier DNA was added to the hydrolysate, as well as 0.2 ml of 0.1 N sodium pyrophosphate, and the mixture was precipitated by the addition of 0.2 ml of 3 N HCl and 6 ml of ethanol. After centrifugation, the pellet was dissolved in 0.8 ml of 0.1 M sodium pyrophosphate and reprecipitated with 0.4 ml of 3 N HC1. This precipitate was again collected, redissolved, and reprecipitated with 0.2 ml of 3 N HCI, after which the long pyrimidine tracts were collected by centrifugation. Preparation of DNA from adult flies for t h isolation of unlabelled polypyrimidines: For the isolation of unlabelled polypyrimidines, DNA from D. simulans, D. varians and D. hydei was prepared according to MAYFIELDand ELLISON(1974). Flies were first homogenized in 6 M guanidine-HC1, 1% Triton-X-100 and 0.1 M 2-mercaptoethanol. After centrifugation at 12,000 for ten min, the pellets were homogenized again, recentrifuged, and the resulting supernantants were ethanol precipitated. The resulting pellets were dissolved in 0.1 M glycine buffer pH 10.5 with 2% sodium sarkosyl. Cesium chloride was added to give a density of 1.55 g per ml. Ethidium bromide was added to a concentration of 0.5 mg per ml. The mixture was centrifuged in a Beckman Ti-60 rotor for 48 hr at 40,000 rpm (160,000 x g). The red band, formed by the DNA, was removed. After extracting the ethidium bromide with isopropyl alcohol, the DNA was wound out of solution after addition of two volumes of 2-ethoxyethanol. Preparation of unlabelled long pyrimidine tracts: Polypyrimidines were prepared for transcription from 250 pg of D. simulans DNA, 150 ,pg of D. varians DNA, 350 fig of D . hydei DNA or 150 pg of D. repzeta DNA as described above for labelled DNA. After acid hydrolysis, 100. fig of E. coli tRNA was added as carrier and the long pyrimidine tracts were precipitated by the ARAP method described earlier. The carrier tRNA was removed by the addition of 0.1 ml of 0.4 N KOH to the pellet. After 30 min at 60" this solution was neutralized with HCl. Transcription of polypyrimidines: The transcription of D. simulans, D. varians, D. hydei and D. repleta polypyrimidines was done as described previously ( SEDEROFF, LOWENSTEIN and BIRNBOIM 1975), with some modifications. The reaction volumes were 0.5 ml, and template polypyrimidines were added to the following concentrations: D. repzeta and D. hydei 10 pg per ml, D. simulans and D.varians 0.5 Bg per ml. [dZP]-GTP (New England Nuclear) was used at a concentration of 50 pCi per ml (specific activity 50 Ci per "ole). The specific activity of the product was estimated to be about 109 dpm per fig. The transcribed polypurines were purified on hydroxyapatite and precipitated with cetyl trimethyl ammonium bromide (CTAB), as described by SEDEROFF, LOWENSTEIN and BIRNBOIM (1975). Digestion of polypurine RNA by T,RNase: The samples of [32P] polypurine RNA dried under vacuum were resuspended in water, boiled and quenched on ice to dissociate any polypyrimidine/polypurine hybrid present. The solution was then brought to 10 mix tris pH 7.5, 1 mM EDTA. T,RNase (Calbiochem) was added to a concentration of 0.5 mg per ml. The mixture was incubated for one h r at 37". TWOdimensional amlysis of T,RNAase digests: The RNA oligonucleotides resulting from
462
Y. M . T. CSEKO
et al.
complete digestion with T,RNase were fractionated by a two-dimensional method described by BROWNLEE (1972), using high-voltage electrophoresis on cellulose acetate paper, at pH 3.5, fdlowed by DEAE-cellulose thin-layer homochromatography using homo-mix C at 60" for six hrs. Kodak Blue Brand X-ray film BB-5 was used for autoradiography of the [szP] labelled oligonucleotides. Analysis of specific oligonucleotides: The oligonucleotides of interest were scraped from the DEAE plates, counted by Cerenkov radiation (efficiencyof counting 35%), washed with ethanol and then eluted with 30% triethylamine carbonate (pH 10). The samples were dried under yacuum, resuspended with water and dried three times, to remove all triethylamine carbonate. The samples were retreated with T,RNase, rerun under various conditions or hydrolysed in 0.2 N KOH for 18 h r at 37". The resulting samples were spotted on DEAE paper or Whatman 3MM paper, and subsequent electrophoresis was done either at p H 3.5 or with 7% formic acid (FA). Alkaline sucrose gradients: Alkaline sucrose gradients, for the determination of sizes of polypyrimidines, were done as described by BIRNBOIM and SEDEROFF (1975). Samples were dissolved in 0.2 ml of 0.2 M sodium pyrophosphate, loaded onto a 12 ml linear 5 to 20% sucrose gradient in 0.1 N NaOH, 0.9 N NaCl and centrifuged in a Beckman SW 40 rotor at 38,000 rpm for 17 h r at 20". The average molecular weights (weight average) were calculated using a computer program developed by R. HOLFORD (1975) and kindly provided by H. C. BIRNBOIM. Agczrose gel electrophoresis: Samples of labelled polypyrimidines were dissolved in 0.005 M sodium acetate and then the pH was lowered with addition of an equal volume of 0.005 M acetic acid. In some cases, samples were boiled in 0.003% formaldehyde for five min. The samples were then diluted into a final buffer at pH 7.8 containing 13 mM Tris base, 2 mM sodium acetate, 0.1 mM EDTA, then made up to 7.5% sucrose and layered on the gel. The gel was 1.4% agarose and made with the pH 7.8 running buffer, which contained 0.04 M Tris, 5 mM sodium acetate, 0.25 mM EDTA. The gel was run for three to four hrs at 5 mA per tube at 4" (until the bromphenol blue dye marker ran about 8 cm). Slices (2 mm) were counted in Triton-X-I00 aqueous scintillation fluid. Markers for molecular weight estimates were obtained using Hin I1 restriction fragments of bacteriophage X (b2 p r n l 1 6 cZ857 susS7) (gift of C. BLASCOW). The position of the marker DNAs were determined by staining with ethidium bromide. The molecand VAN DE ular weights of the fragments were estimated as described by MANIATIS,JEFFREY SANDE(1975) and ALLETand BURHARI(1975). RESULTS
Survey of 101 Drosophila species for the D. melanogaster polypyrimidine DNA sequences by molecular hybridization: The presence of the polypyrimidine DNA sequence was assayed by nucleic acid hybridization and hydroxyapatite chromatography, using the crude extract technique described and characterized in a separate paper ( C S E K Oet al., in preparation). We studied species primarily in the subgenus Sophophora, which include; the melanggaster group. A list of tested species is shown in Table 1 and the results are pres-nted in Table 2. Up to 100 flies of a given species were homogenized in a lysis solution, LS (8 M urea, 1 M sodium perchlorate, 0.5% SDS and 0.12 M sodium phosphate pH 6.8), and deproteinized by chloroform extraction. After centrifugation, the resulting aqueous phase may be used directly as a source of DNA. Labelled polypurine RNA or polypyrimidine DNA was added, the solution was boiled briefly and incubated at 55" for various periods of time (up to 1000 minutes), and then assayed for the presence of stable hybrid by hydroxyapatite thermal chromatography. Most experiments were done with [3H]-ATP polypurine (complementary RNA) because of the very high specific activity of this material.
463
EVOLUTION O F POLYPYTIMIDINES TABLE 1 Species
D.affinis D.ambigua D.anunassae D.athabaska D.atripex D.austroslatans D.aztecrr D.baimaii D.barbarae D.biauraria D.bicornuia D.bifasciata D.bifurca D.bipectinata D.birchii D.busckii' D.capricorni D.denticulaia D.dominicrrna D.elegans D.emarginata D.eohydei D.equinoxialis D.erecta D.eugracilis D.fieusphila D.fumipennis D.funebris D.hydei D.immigrans D.iambulina D.kanapiae D.kikkawni D.lactaicornis D.lini D.lucipennis D.lusaltans D.lutea D.malerkotliana D. mayri D.mauriiiana D.melanogaster D.micromelanica D.milleri D.mimetica D.miranda D.mulleri D.narraganseti
Group
obscura obscura melanogaster obscura melanogaster saltans obscura melanogaster melanogaster melanogasier melanogaster obscura repletrr melanogaster melanogaster
Subgroup
afinis obscura ananassae afinis ananassae saltans afinis montium montium montium montium obscura hydei ananassae montium
__
willistoni melanogasier melanogaster melanogaster saltans repleta willisioni melanogaster melrrnogaster melanogaster willistoni funebris repleta immigrans melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster saltans melanogaster melanogaster melanogarter melnnogaster melanogaster melanica saltans melanogmter obscura repleta obscura
deniiculata montium elegans elliptica
hydei
melanogaster eugracilis ficusphila
-
Place
Stock number
Stock center
Nebraska England Hawaii Manitoba Thailand Brazil Mexico Thailand Malaya Korea Cambodia Japan
2069.2 2529.7 2370.11
UTDSC UTDSC UTDSC UTDSC UTDSC CUI UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC
of origin
New Guinea Australia Montana Panama New Guinea New Guinea Philippines Peru Salvador Brazil
Thailand Taiwan Colombia
__
3256.9 2536.4 2266.3 3250.9 3033.10 3255.1 3120.5 309.0.7 A8.13 3007.3 3007.1 1763.8 3015.3 3021.9 3029.4 3140.2 2536.6 H62.57 1975.3 154.1 3251.7 3075.8 H191.4.F
__
-
hydei
Mexico
1797.8
montium montium montium montium montium suzukii saltans takahashii ananassae montium melanogaster melanogaster
India Philippines Korea Okinawa Taiwan Taiwan Haiti Japan India New Guinea
3253.8 3138.6 2210.1
-
sturtevanti suzukii obscura mulleri afinis
--
Oregon (R) Arizona Puerto Rico
__ __
Nebraska
__
-
3146.11 3068.3 H411.20 3040.13 3253.5 3020.6 163.1 2160.12 H129.17 3033.29 3328.1
__
2528.9
UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC CE UTDSC UTDSC UTDSC CUI BGOSC BGOSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC CE BGOSC UTDSC UTDSC UTDSC UTDSC CUI UTDSC
464
Y. M. T. CSEKO
et al.
TABLE I-Continued Species
Group
SUbUOUD
D.nasutoides D.nebulosa
immigrans willistoni
-
D.necordata D.neoelliptica D.neoshydei D.neseotes D.nigrens D.nigrohydei D.nigrosaltans D.nikananu D.orosa D.pallens D.pallidosa D.parabipectinata D.paranaensis D.paralutea D.parvula D.paulistorum D.pennae D.persimilis D.phaeopleura D.prosaltans D.prostipennis D.pseudoananassae D.pseudoobscura D.pseudosaltans D.pulchrella D.punjabiensis
saltans saltans repleta me lanogaster melanogaster repleta saltans melanogaster melanogaster melanogaster me lanogaster melunogaster repleta melanogaster melanogaster willistoni melanogaster obscura melanogaster saltans m elanogaster melanogaster obscura saltans melanogaster melanogaster
cordata elliptica hydei ananassae ananassae hydei saltans montium montium montium ananassae ananassae mercatorum takahashii montium
D.quadraria D.rajasekari D.repleta D.ruja D.saltans D.seguyi D.septentriosaltans D.serrata D.simulans D.sturteuanti D.subobscura D.sucinea D.suzukii D.takahashii D.teissieri D.tolteca D.triaurari D.trilutea
melanogaster melanogaster repleta melanogaster saltans melanogaster saltans melanogaster melanogaster saltans obscura willistoni melunogaster melanogaster melanogaster obscura melanogaster melanogaster
Place of origin
Tutuila Barbados Brazil __
Venezuela Caroline Islands Brunei Guatemala Panama Africa Thailand Brunei Tutuila Cambodia
Stock number
3059.4 H119.6
UTDSC UTDSC
2536.7
UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC
-
H207.26 3069.1 1 3122.7 2510.1 H360.92 2371.5 3250.17 3122.10 305 7.6A
-
-
--
Thailand Malaya Brazil
montium obscura ananassae saltans takahashii ananassae obscura saltans suzukii montium
-
Ques ne11 Fiji Guatemala Taiwan Australia Colombia Brazil Japan Malaya
3250.7 3033.9 1975.21 3028.1 2529.6 30M.4 1911.5 3146.7 2372.11 2542.2 2536.10 3M.11A 3033.4
montium suzukii melanopalpa montium saltans montium saltans montium melanogaster sturteuanti obscura
Taiwan India Australia Japan Costa Rica Rhodesia Colombia Australia Amherst Colombia Iran Salvador Japan Taiwan
suzukii takahashii melanogaster affinis montium takahashii
-
Bolivia China Alishan
Stock center
3075.1 325'3.9 2372.15 3040.16 HI 80.40 3254.1 H103.21 2404.10
-
H101.2 3115.2 H62.39 3040.11 3075.4 144.3 H346.50 1736.1 3066.9
UTDSC UTDSC U0 UTDSC UTDSC UTDSC UTDSC UTDSC BGOSC UTD UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC UTDSC
465
EVOLUTION O F POLYPYTIMIDINES
TABLE 1-Continued Species
willistoni melanogaster repleta melanogaster uirilis melanogaster willistoni melanogaster
D . tropicalis D. tsacasi D . tumidi D.ucrrians D . uirilis D. uulcana D.willistoni D. yakuba
Place
Subgroup
Group
of ongin
-
Stock number
Stack center
1975.1 2371.4 3160.29
UTDSC UTDSC UTDSC UTDSC BGOSC UTDSC UTDSC UTDSC
montium
Brazil Africa
ananassue
Philippines
-
-
montium
Rhodesia Mexico Africa
3254.2 1802.2 2371.6
-
-
-
melanogaster
O NSTONE(1952), The group and subgroup classifications are given as described by P A ~ E R Sand
BOCK and WHEELER (1972) and THROCKMORTON (1962). The stock numbers are those given by the University of Texas Drosophila Species Collection. The following abbreviations indicate the source of the stocks: (CE) University of Cambridge, Department of Genetics, England; (BGOSC) Bowling Green, Ohio Stock Center; (UTDSC) University of Texas Drosophila Species Collection; (UO) University of Oregon; (CUI) Cornel1 University, Ithaca, New York. An asterisk (*) indicates the subgenus Dorsilopha.
The highly repetitive polypyrimidine DNA sequence reanneals with total
D.melanogaster D N A with an equivalent Cot 1/2 of approximately (CSEKO et al., in preparation). An incubation mixture prepared from ten D.melanogaster in 1 ml and annealed for one hour would have an estimated Cot equivalent of 5 1.0 x For purposes of a screening procedure, we have presented OUT TABLE 2
A survey of Drosophila species for the presence of the D. melanogaster polypyrimidine sequence Species
D. bifurca D. eohydei D. fumipennis D. hydei D . mcluritiana D.melanogaster D.neohydei D. nigrohydei D . simulans D. uarians
Males
3
Time of annealing Number of flies Females Mixed sexes (min) Total % hybrid
7 25
18 10
17 70
5 21 10 40
51
65
60 60 70 5 75 5 70 60 60 60
45 41 69 65 7.5 79 56 62 63 77
T,,,
72 63 67 67 80.5 81 71 72 82 82
Data are presented for selected species that form hybrid with D.melanogaster polypurine RNA. The phylogenetic relationships of the species are shown in Figure 2. The conditions of annealing and hydroxyapatite chromatography with DNA in crude extracts have been described (CSEKO et al., in preparation). Total % hybrid represents the fraction of the input 3H-ATP or 3H-GTP polypurine RNA that was bound to the column and eluted between 55 and 95". The melting temperature is calculated from the midpoint of the elution profile of the hybrid formed. All polypurine RNA preparations were usually 34 mCi per mg when 3H-ATP was used in synthesis, but were 6.8 mCi per mg when 3H-GTP was used. Each hybridization reaction contained approximately 104 cpm of probe. For further details of experiments with species where no hybrid was detected, see CSEKO (1976).
466
Y. M. T.CSEKO
et al. Subgenus DrosophiJ&
bafmaii barbarae biauraria bicornuta birchii dominicana jambulina kanapiae kikkawai lacteicornis lini
nikananu orosa pallens parvula pennae punjabiensis quadraria rufa seguyi serrata triauraria tsacasi vulcana
Arepleta
7 repleta group
montium subgroup ~
neohydei nigrohydci 4virilis
virilis group
micromelanica
melanica group
funebris group
ananassae subgroup
neseotes
hydei subgroup (repleta group)
Subgenus Dorsilopha
phaeopleura pseudoananassae
Ibuskii
I
I
1
suzukii sub group
pulchrella rajasekari
takahashii subgroup takahashii necordata I
mauritiana iranda
teissieri
emarginata
I (
4 denticulata
I
I eleaans I -
11
fiscusphila
I
eugracilis
1
I
pseudoobscura subobscura athabaska azteca narragansett tolteca
septentrio-
I
melanogaster group
obscura group
willistoni group
saltans group
FIGURE 1.-Species tested for the presence of the D. melanogaster polypyrimidines. The species were grouped based on the work of THRWKMORTON (1962), PATTERSON and STONE (1952) and BOCKand WHEELER (1972). Hybridizations were done to D. melanogaster polypurine RNA or polypyrimidines as described in MATERIALS AND METHODS and Table 1. The species that cross-reacted with the D.melanogaster sequence with a T, above 65" are marked with an arrow. In organizing species in the table, the relationship of some subgroups are presented in only one of a number of possible relationships. In the melanogaster group, the montium subgroup and the amnmsae subgroup are more closely related to each other than to the other subgroups within
EVOLUTION O F POLYPYTIMIDINES
467
results on a per fly basis for each species, keeping in mind that the DNA content per fly may vary several-fold between distantly related species. Of the 101 species tested, eight were found to form stable hybrid with labelled polypurines or polypyrimidines from D.melanogaster (Figure 1) . DNA preparations from these nine species formed hybrid that was stable above 65" in 0.18 M sodium phosphate buffer pH 6.8 (PB) (Table 2 ) . In the melanogaster group, three of 55 species tested formed stable hybrid (D.simulans, D.mauritiana and D.uarians) . One species, D.fumipennis, in the willistoni group formed hybrid and four of 5 species in the hydei subgroup formed stable hybrid (D.bifurca, D.nigrohydei, D.neohydei, and D.hydei). A small number of species formed some hybrid with low thermal stability (57" to 62"). These were not studied further at this time, with the exception of D.eohydei and D.repleta, because of their close relationship to other species in the hydei subgroup (WASSERMAN 1962). In one case, D.mauritiana, we have been able to detect the presence of the same polypyrimidine sequence at a level of 0.03% of the genome, that is, at a level 50-fold less than that present in D.melanogaster. Highly repetitious sequences with short repeating units show great differences in their ability to hybridize to related sequences as compared to homologous reassociation. BLUMENFELD,Fox and FORREST (1973) demonstrated that the related satellites of D.uirilis differing from one another by changes of one nucleotide in the seven-nucleotide repeat, reassociate to form hybrid that melts at a depressed temperature of 12" to 30" below the optical density temperature transition of the fully complementary strands. Consequently, the T , of the hybrid formed in cross-hybridizations can be a sensitive indicator of DNA homology between related species. We infer that any sequence that formed hybrid with a T , above 65" would be similar to the polypyrimidine sequence of D.melanogaster, which melts at 82" under the same conditions. Conditions of annealing (criterion): We have shown that hybridization at 55" in 0.12 M PB with 8 M urea is equivalent to annealing at 75" without urea (CSEKOet al., in preparation). These stringent conditions were selected because they enable us to detect even small amounts of hybrid among very nearly homologous sequences. Even though the conditions employed should not detect partially homologous hybrid, five species were found to form considerable amounts of hybrid with melting temperatures of 65" to 75". Under the conditions used, we would not have expected these hybrids to be found. Subsequent experiments determined that these species contain sequences related to the long pyrimidine tracts that formed hybrid, not during the annealing at 55" in LS, but in the five to ten min of annealing in diluted lysis solution at 45" or 55" during the loading and equilibrating of the hydroxyapatite columns. Low-melt hybrid can be obtained with these species when the total annealing is carried out in diluted lysis solution (four-fold) . We have therefore detected two types of cross-hybridithe group. The close placement of the other subgroups in the figures does not imply closeness of relationship in all cases.
468
Y. M. T. CSEKO
et al.
zation: those that form hybrid under stringent conditions and are presumably homologous, and those that represent related, but not identical, sequences present in sufficient amounts to form hybrid under less stringent conditions. A method to test for long pyrmidine tracts in other species of Drosophila: In order to determine if the cross-reacting species contained an identical or similar polypyrimidine sequence, labelled DNA from larval brains was purified, hydrolyzed with acid and tested directly for long pyrimidine tracts (Table 3 ) . In all previous experiments, labelled polypyrimidines were obtained from cultured cells of D. melanogaster. In order to detect and measure the amount of long pyrimidine tracts in other Drosophila species, we incubated larval neural ganglia in organ culture with 3H-thymidine, using the procedure of LEE (1974), and extracted the DNA. Purified DNA was then hydrolyzed to completion with formic acid diphenylamine and the resulting long pyrimidine tracts selectively precipitated. This acid-resistant, acid-precipitable (ARAP) material represents 1975). Our criteria purified long pyrimidine tracts (BIRNBOIMand SEDEROFF for polypyrimidines have been modified by including a hybridization step; therefore, we consider polypyrimidines to be bona fide if they are recovered as ARAP DNA that forms high melting hybrid with homologous DNA at low Cot.Even TABLE 3 Direct assay for polypyrimidines in various Drosophila species
Species
1. D. melanogaster 2. D. sinulnns
Total cpm in DNA
4.5 X
IO5
4.6 X 106 3. D.mauritiana males 1.2 X 106 4. D . mauritianafemales 9.9 X IO5 5. D . erecta 2.5 X l o 5 6. D.teissieri 2.5 x lo5 7. D.yakuba 1.9 x IO5 8. D.varians 1.4x 106 9. D. fumipennis 2.1 x 106 10. D. bijurca 6.2 x 105 11. D.eohydei 5.4 x 105 12. D.mohydei 5.6 x 105 13. D. hydei 7.0 x 105 14. D . nigrohydei 1.0 x io6 15. D.repleta 1.1 x io6
% of total resistant DNA as DNA (AI") AFL4P.DNA cpm in acid-
1.7 X 2.4 X 4.8 x 7.9 x
104 IO4 IO3
lo3 1.6 X lo3 3.2 x IO3 1.4 x lo3 9.8 x 103 4.2 x 104 5.6 104 2.7 x 103 3.9 x 103 4.5 x 104 1.1 x 105 8.0 x 104
x
3.8%
0.5% 0.4% 0.8%
0.6%
1.3% 0.7% 0.7%
2.0% 9.1% 0.5% 0.7% 6.4%
% of A W DNA forming hybrid at Cot of
50% 26% 7.5% n.d. (50.4%) n.d.(
