Satellite DNA in the crustacean Artemia

341

Gene, 44 (1986) 341-345 Elsevier GENE 1648

Satellite DNA in the crustacean Artemiu (Recombinant DNA; heterochromatin; cloning vector; sequencing; isopycnic centrifugation; CsCl gradient; Hoechst 33258; tandem repeats; brine shrimp)

Jesis Cruces b, Maria Luisa G. Wonenburger a, Margarita DLaz-Guerraa, Jestis Sebastian a and Jaime Renart a* o Instituto de Investigaciones Biomkdicas de1 C.S.I.C., Universidad Authoma

and b Departamento de Bioquimica, Facultad de Medicina de la de Madrid, Arzobispo Morcillo. 4.28029~Madtid (Spain) Tel. 733-0100 (Ext. 300)

(Received November 27th, 1985) (Revision received and accepted April 16th, 1986)

SUMMARY

We have isolated a satellite fraction from the Artemiu genome by both restriction endonuclease digestion and equilibrium density centrifugation in CsCl gradients containing ligand dye Hoechst 33258. Satellite DNA was arranged in long stretches (approx. 23 kb) of tandem repeats of a basic unit of 113 bp. The basic unit has been sequenced, showing a G + C content very close to that of total DNA. Different amounts of satellite were present in several populations of Artemia, whereas it was absent from others.

INTRODUCTION

Highly repeated DNA is present in all eukaryotic organisms (Brutlag, 1980; Singer, 1982) in proportions varying from < 1 to > 66% of the genome (Skinner, 1977). This DNA can usually be isolated by isopycnic centrifugation, although some species have cryptic satellites, with the same density as main

* To whom correspondence addressed.

and reprint requests should be

Abbreviations: bp, base pair(s); Hoechst 33258, bisbenzimide H33258; kb, 1000 bp; nt, nucleotide(s); PA, polyacrylamide; SSC, 0.15 M NaCl, 0.015 M Na,.citrate, pH 7.6. 0378-l 119/86/$03.50

0

1986 El sevier Science Publishers

B.V. (Biomedical

band DNA, which cannot be separated by these methods. Some cryptic satellites can be resolved by centrifugation in the presence of base-specific ligands, that increase the difference in density between the main and satellite bands. Artemiu (class Crustacea, order Anostraca) has 16% of highly repeated DNA (measured by reassociation kinetics; Vaughn and Petropoulos, 1979), although no satellite fractions can be detected by thermal denaturation or equilibrium density centrifugation in CsCl gradients (Cruces, 1982). Recently, Barigozzi et al. (1984) showed that heterochromatin of some Artemia species contained a repetitive sequence that could be isolated by AM digestion of genomic DNA. Division)

342

In this report we demonstrate the existence of a satellite fraction in Artemia DNA, as well as its organization and sequence. In addition, we have determined the proportion of this DNA fraction in different populations of Artemia.

kb 23.13

EXPERIMENTAL

9.42

AND DISCUSSION

6.56 4 36

(a) Methods

Artemia cysts came from San Francisco Bay Brand, unless stated otherwise. Different San Francisco batches were obtained from the same supplier. Salt Lake (UT, U.S.A.) cysts were from Long Life Aquarium Products. Other populations were a generous gift from Dr. F. Amat, Instituto de Acuicultura de1 C.S.I.C. Newly hatched nauplii were grown as described by Osuna and Sebastian (1980). DNA from larvae or cysts was obtained by a moditication of the procedure of Cruces et al. (198 1).

2.32 2.02

0.56

0

(b) Isopycnic centrifugation Fig. 1. Equilibrium

When Artemia DNA is analyzed by CsCl gradient centrifugation, a single, well defined peak is obtained, with a density of 1.692 g/cm3, slightly lower than that of bacteriophage T4 DNA @ = 1.694 g/cm3). This density corresponds to 32.6% G + C. This result, lower than others previously described for Artemia, has been confirmed by direct chemical analysis (32.5 f 1.5) and by thermal denaturation in both 1 x SSC (32.2 f 2.5) and 0.1 x SSC (32.3 f 2.3) (not shown), which give a mean value of (32.3 f 2.5)% G + C. When the gradients are made in the presence of the dye Hoechst 33258 (obtained from Riedel-de Haan AG, cat. No. 33217), as described by Manuelidis (1977) a clear satellite band is obtained (Fig. 1). These results confirm the presence in the Artemia genome of a cryptic satellite, not previously described. The characterization of this satellite band is shown below. (c) Restriction analysis When Artemia DNA is digested with a wide variety of restriction endonucleases, a resistant fraction is obtained, of around 23 kb in length, that

presence

Artemia DNA NaCI,

33258. An aliquot

(in 1 ml of 1OmM

0.5 mM EDTA)

through

ofAr?emia DNA in the

density centrifugation

of Hoechst

was

containing

Tris.HCI,

sheared

by passing

a 22G needle. It was mixed gradually

200 pg/ml of Hoechst and incubated

solid

CsCI.

at

35 000 rev./min for 64 h at 20°C. The upper band corresponds

to

was

then

DNA, and the lower one to the main DNA band.

Fig. 2. Restriction

analysis

a 0.8%

gel at

undigested

It

to a density of centrifuged

the satellite

with

: dye was 1: 1)

The mixture was

diluted with 5 ml of the above buffer and brought 1.638 g/cm3

it ten times

with 0.49 ml of

33258 (final ratio of DNA

for 10 min at room temperature.

1OOng of

pH 8.0, 1OmM

agarose

of Artemia DNA. Electrophoresis

(lane 1) and digested

(lane 3) or Suu3A

of Ar?emia DNA

1 V/cm

with Hind111 (lane 2), HpaII

(lane 4). Size markers

with Hind111 (indicated

in

(2-5 pg),

on margins

were 1 DNA digested

in kb).

represents about 2% of the genome. Fig. 2 shows three representative digestions, with HindIII, HpaII and Sau3A. The resistant fraction is not sensitive to double digestions (not shown). However, digestion by two restriction endonucleases, Ah1 or Mb011 (Fig. 3), permits to determine how the resistant fraction is organized, and shows an enrichment in the llO-120-bp band; when the digestion is only partial, a ladder with a periodicity of 1lo-120 bp is visible. These results indicate that

343

the resistant fraction is composed of tandem repetitions of this 1lo-120-bp unit. The experiment shown in Fig. 3 also demonstrates that the satellite fraction obtained by isopycnic centrifugation in the presence of Hoechst 33258 is enriched in the llO-120-bp band. Taken together, these findings strongly suggest that the satellite observed by the two methods is the same. Although AluI cuts the satellite very efficiently (see also Barigozzi et al., 1984), we prefer to call this satellite “satellite I”, to distinguish it from the well-known Ah family of interspersed repetitive DNA in man (S&mid and Jelinek, 1982). (d) Sequence of the satellite I basic unit

Fig. 3. Restriction analysis of the satellite band obtained by isopycnic centrifbgation. DNA samples (3 pg) were digested as described below and electrophoresed in a 10% PA gel, at 100 V for 11 h. Lanes: (1) total DNA; (2) satellite band; (3) main band (all digested with MboII); (4) total DNA; (5) satellite band; (6)main band (all digested with AluI); (7)markers: pBR322 DNA digested with HpaII.

pMWOO2

To sequence the satellite I basic unit, we obtained several recombinants. In one experiment, total DNA was digested with AM. The llO-120-bp band was purified from a 2% agarose gel and ligated directly to M13mp8 (Messing and Vieira, 1982) in the SmaI site. Four random phages were purified and sequenced by the dideoxy procedure of Sanger et al. (1977) (clones pMWOO1 to 004). In another experiment, the resistant fraction obtained by HaeIII digestion of Artemia DNA was isolated, digested with Ah1 and ligated to pUC9 (Vieira and Messing, 1982). The four clones selected (pMWlO1, 103, 115 and 116) were sequenced by the chemical method (Maxam and Gilbert, 1980).

pMW003

CTATTAGCCT __________

CGAAAACTAA __________

AACTTTTGAC ____-_____

ATAGGAAAAG __________

AGCCTTTAAT ________--

CACATTCTTT ----------

pMW103

----__C___

--_-------

----------

-_--------

_________3

----------

pMWl15

------T--m

----------

----------

----------

__________

----------

PMWll6

----w-T---

---------_

-----_----

---------_

-------_--

--__-----_

TCAACATACG __________

TAG ___

MbolI

pMW003

ACCATTTGCA -----G----

ATCATAAAAT -____-_--_

AGTCTAATAZATTTTT T_---___-________-_

pMW103

-----G----

-__-------

T---------

--_------_

--C---G--A

A--

pMWl15

-----G----

_______-__

T---------

-__-------

--C---G---

PMWl16

__________

-__------_

____-___--

-__-_----_

__---_----

A-A--

pMW002

Fig. 4. Sequence of different satellite DNA clones. The sequence is given for pMWOO2; for the other clones, only the variant nt are specified.

344

The sequence of several of these clones are shown in Fig. 4. The repeat is of 113 bp. The only changes detected between them are always in the same positions: nt 7,66,81,103,107, 110 and 111 starting at the AluI cutting site. Besides the restriction sites for AluI and MboII, the sequence shows a recognition site for TaqI, which has been confirmed by digestion of total DNA (not shown). The base composition of the repeat is 29.8% G + C, very close to the G + C content of Artemiu DNA, which could explain the failure to visualize the satellite band in the absence of Hoechst 33258.

_”

Another of the phages sequenced (pMW001) contains an insert of 122 bp, with recognition sites for Ald, Mb011 and TuqI, plus an additional AvuII site. Its sequence, however, is not related to the five clones presented (not shown). Therefore, it could represent another satellite. (e) Occurrence of satellite I in other Artemiu populations

Barigozzi et al. (1984) have reported that heterochromatin is missing from some Artemiu populations, based on chromosome staining and in situ hybridization. We have checked whether different Artemiu populations have satellite I. The results are presented in Fig. 5. Satellite I is present in Artemiu from Yucatan (Mexico), Bocachica (Venezuela), the Salt Lake (UT, U.S.A.) and San Francisco Bay (CA, U.S.A.), the latter one being used throughout this study (lane 7). The Utah population has more satellite I than does that from San Francisco. Artemiu from Alcochete (Portugal), Tianjin (China) and a different batch (No. 3462) from San Francisco do not contain satellite I, although in the latter a very weak signal can be observed when the film is overexposed (not shown). We also find that seven different populations from Spain, do not have the satellite (not shown). Fig. 5 shows that the populations which do not have satellite I seem to have another satellite with a repeat unit of about 130 bp. As can be seen from the hybridization data, this new satellite is not homologous with satellite I. (f) Conclusions

Fig. 5. Occurrence Artemiu.

DNA

[I: Alcochete

of satellite I in different (5 pg)

(Portugal);

co); 4: Bocachica

from

different

2: Tianjin

(Venezuela);

populations

Artemia

(China);

3: Yucatan

were digested

with HaeIII

trophoresed

in a 0.6% (A) or 2% (C) agarose to nitrocellulose [3*P]pMW003

are L DNA digested and pBR322

digested

membranes

with AhI,

San

gel. Both gels were

and hybridized

(B and D, respectively).

with HindIII,

batch,

(A)or Ah1 (C) and elec-

transferred nick-translated

(Mexi-

5: batch No. 3462, San Francisco

(CA, U.S.A.); 6: Salt Lake (UT, U.S.A.); 7: standard Francisco]

of

populations

with

Markers

in kb (A and B; not shown) in bp (C and D).

In this paper we report the characterization of a satellite DNA in the crustacean Artemiu. This satellite I appears as long stretches of approx. 23 kb formed by 200 tandem repeats of the 113-bp basic unit. Assuming 1.64 x lo9 bp per haploid genome (Cruces, 1982), there should be around 140 copies of the 23-kb array, and 2.8 x lo5 copies of the basic unit. Satellite I can be resolved from total DNA only when the isopycnic centrifugation is done in the presence of Hoechst 33258, a dye that binds preferentially A + T-rich regions (Muller and Gautier, 1975). It does not seem to be the result of amplification

345

and divergence of a small sequence, as is the case for other common satellites in Drosophila or mouse (Brutlag, 1982). In this respect, several satellites of the same or greater complexity have been found in crustaceans (see, for instance, LaMarca et al., 198 1; Fowler and Skinner, 1985, and Fowler et al., 1985), although always in members of the subclass Mulucostruceu (crabs) and not in lower crustaceans such as Arfemiu (subclass Branchiopoda). The satellite I we have characterized and sequenced seems to be the same as that which Barigozzi et al. (1984) have studied. Their results about the presence of heterochromatin in two different Artemiu populations have been recently extended by their group (Barigozzi, C., Badaraco, C., Baratelli, L., Plevani, P. and Ginelli, E., Second International Symposium on the brine shrimp Artemiu, Antwerp, 1985) and by ourselves. We show here that only American populations have satellite I, whereas Eurasian ones do not. The presence or absence of satellite I has been implicated by the group of Barigozzi in the problem of speciation of Artemiu. Abreu-Grobbois and Beardmore (1982) have suggested that American populations of Artemiu are derived from those living in Eurasia. If this were the case, the evolution of the former should involve the acquisition and amplification of satellite I.

ACKNOWLEDGEMENTS

We thank Elvira Dominguez for technical help and Juan Ortin for helpful discussions and suggestions. Dr. R. Garesse helped us with the dideoxy sequencing method. Dr. F. Amat gave us Artemiu cysts of the different, non-commercial, populations studied. This investigation was supported by grants from the Comision Asesora para la Investigation Cientilica y Tecnica.

REFERENCES Abreu-Grobbois, F.A. and Beardmore, A.: Genetic differentiation of the brine shrimp Arremia. Prog. Clin. Biol. Res. 96 (1982) 347-375.

Barigozzi, C., Badaraco, G., Plevani, P., Baratelli, L., Profeta, S., Ginelli, E. and Meneveri, R.: Heterochromatin in the genus Arfemia. Chromosoma 90 (1984) 332-337. Brutlag, D.L.: Molecular arrangement and evolution of heterochromatic DNA. Annu. Rev. Genet. 14 (1980) 121-144. Cruces, J.: Caracterizaci6n de1 genoma y de 10s genes ribos6micos de Artemia. Ph.D. Thesis. Universidad Aut6noma de Madrid, 1982. Cruces, J., Sebastian, J. and Rena& J.: Restriction mapping of the rRNA genes from Artemia larvae. Biochem. Biophys. Res. Commun. 98 (1981) 404-409. Fowler, R.F. and Skinner, D.M.: Cryptic satellites rich in inverted repeats comprise 30% of the genome of a hermit crab. J. Biol. Chem. 260 (1985) 1296-1303. Fowler, R.F., Bonewell, V., Span, M.S. and Skinner, D.M.: Sequence of three closely related variants of a complex satellite DNA diverge at specific domains. J. Biol. Chem. 260 (1985) 8964-8972. LaMarca, M.E., Allison, D.P. and Skinner, D.M.: Irreversible denaturation mapping of a pyrimidine-rich domain of a complex satellite DNA. J. Biol. Chem. 256 (1981) 6475-6479. Manuelidis, L.: A simplified method for preparation of mouse satellite DNA. Anal. Biochem. 78 (1977) 561-568. Maxam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65 (1980) 499-560. Messing, J. and Vieira, J.: A new pair of M 13 vectors for selecting either DNA strand of double digest restriction fragments. Gene 19 (1982) 269-276. Muller, W. and Gautier, F.: Interactions of heterochromatic compounds with nucleic acids. A.T-specific non-intercalating DNA ligands. Eur. J. Biochem. 54 (1975) 385-394. Osuna, C. and Sebastian, J.: Levels of the RNA polymerases during the early larval development of Arfemia. Eur. J. Biochem. 109 (1980) 383-389. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5476. Schmid, C.W. and Jelinek, W.R.: The Ah family of dispersed repetitive sequences. Science 216 (1982) 1065-1070. Singer, M.F.: Highly repeated sequences in mammalian genomes. Int. Rev. Cytol. 76 (1982) 67-l 12. Skinner, D.M.: Satellite DNA’s. Bioscience 27 (1977) 790-796. Vaughn, J.C. and Petropoulos, C.J.: DNA sequence organization in the genome ofthe brine shrimp Artemiu salina. In Bagshaw, J.C. and Warner, A.H. (Eds.), Biochemistry of Artemia Development. University Microfilms International, Ann Arbor, MI, 1979, pp. 190-208. Vieira, J. and Messing, J.: The pUC plasmids: an Ml3mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19 (1982) 259-268. Communicated by H.G. Zachau.