J Mol Evol (1994) 39:473477 Journal of Molecular Evolution @ Springer-Verlag New York Inc. 1994

A Repetitive DNA Family, Conserved Throughout the Evolution of Free-Living Nematodes

Adriana La Volpe

CNR International Institute of Genetics and Biophysics, 10, Via Marconi, 80125 Napoli, Italy

Received: 23 February 1994 / Accepted: 27 April 1994

Abstract. This paper concerns the molecular evolu- tion of a tandemly repeated DNA family, RcC9, origi- nally identified in Caenorhabditis elegans. The mini- mum unit of pe r iod ic i ty of this fami ly is the pentanucleotide [nGAAn] and its complement [nTTCn] recurring several times in alternating tandem arrays. This consensus sequence is identical to that of the heat-shock element (HSE). Multiple HSEs are present in the regu- latory regions of heat-inducible genes in a wide range of eukaryotic species; HSEs mediate transcriptional activa- tion through the binding of the heat-shock factor (HSF). I describe some repeated DNA families sharing this same consensus and found in nematode species other than C. elegans. Although the consensus is conserved, the re- peated sequence diverged between species to the point that cross-hybridization is abolished. Evolutionary impli- cations will be discussed.

Key words: Repetitive DNA - - C. elegans - - Free- living nematodes - - Heat-shock element - - Heat-shock factor - - Concerted evolution

Introduction

The repetitive DNA family RcC9 was originally identi- fied in C. elegans (La Volpe et al. 1988). It is composed of the reiteration of a highly conserved tandemly re- peated block of 40 base pairs. Loci containing RcC9 have been mapped on the C. elegans physical map (Na- clerio et al. 1992) and appear to be uniformly distributed in the genome. Analysis of the tandem repetition has shown that the minimal repeated unit is the pentanucle- otide [nGAAn] and its complement [nTTCn] reiterated

several times in alternating tandem arrays. This consen- sus coincides with that generally accepted for the heat- shock element (HSE) (Amin et al. 1988), the binding site for the heat-shock factor (HSF), a transcriptional activa- tor. Various copies of the HSE minimal unit are present in the promoter of genes (heat-shock genes) whose tran- scription is induced by a sudden increase of temperature. The palindromic pattern of repetition in the HSE is ex- plained by the binding of HSF monomers on opposite strands of DNA (Cunniff and Morgan 1993; Xiao et al. 1991). This sequence, present in the heat-shock promot- ers of C. elegans (Kay et al. 1986), is also found as very long arrays (RcC9 family) in nontranscribed portions of the C. elegans chromosome (La Volpe et al. 1988; Na- clerio et al. 1992). Although the function, if any, of the RcC9 family is unknown, a subfragment of such a tan- dem array is sufficient to confer in vivo heat inducibility on a reporter gene in yeast (La Volpe et al. 1988; Mo- racci et al. 1992).

RcC9-specific probes, such as those of other repeti- tive DNA families from C. elegans, do not cross- hybridize with genomic DNA from other free-living nematodes (Cangiano and La Volpe 1993; La Volpe et al. 1988). I speculated that if the DNA module conserved among different species is a reiteration in alternate ori- entations of the pentanucleotide [nGAAn], one would not expect cross-hybridization if the choice of the first and fifth nucleotides followed different drifts in different species.

I describe here the cloning and sequencing of ele- ments belonging to repetitive DNA families of Cae- norhabditis briggsae and of Rhabditis maupasi that share the RcC9 consensus.

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Materials and Methods

N2-JW-H, the wild-type C. elegans strain, was obtained from the Cae- norhabditis Genetics Centre. DNA from C. briggsae, PG-2, and R. maupasi, three free-living nematode species, was a gift from L.W. Nawrocki. Routine strain maintenance and DNA purification from liq- uid cultures was performed as described (Sulston and Hodgkin 1988).

Genomic DNA was digested with 10 us per ~tg of the restriction enzyme XbaI for 2 h at 37°C. Digested DNA was end-labeled with Klenow-fragment polymerase in presence of 20 ~tCu of 32p-dCTP and an excess of cold TTP. End-labeled products were denatured and sep- arated by 6% polyacrylamide-urea gel electrophoresis and autoradio- graphed. The digestion products were ligated at low efficiency to de- phosphorylated XbaI-digested plasmid vector (pGEM4 Amersham). Presence and size of the genomic inserts were monitored by polymer- ase chain reaction (PCR) with all four deoxynucleotides and 0.1 ~Cu of 32p-dATP using T7 and SP6 promoter primers situated at the sides of the insert on the plasmid. PCR products were analyzed by polyacryl- amide-urea gel electrophoresis and autoradiographed. Chosen clones were sequenced on both strands using the UBS Sequenase kit.

The sequences obtained during this work have been submitted to the Genome Sequence Database. Accession numbers are indicated in the text. Sequences of several elements belonging to the C. elegans RcC9 family had already been deposited in the EMBL Data Bank (ac. no.: X07581, ac. no.: X07582, ac. no.: X07583, ac. no.: X07584, ac. no.: X61241, ac. no.: X61245, ac. no.: X61247, ac. no.: X61248, ac. no.: X61249, ac. no.: X61252, ac. no.: X61254, ac. no.: X61255, ac. no.: X61257, ac. no.: X61261). Sequence data were analyzed with the FASTA software package (Pearson and Lipman 1988).

100bp~,

80bp ~,

Results

Identification and Cloning of Low-Molecular-Weight X b a / G e n o m i c Fragments from Free-Living Nematodes Species

The restr ict ion endonuclease XbaI recognizes the six-

base-pa i r - long pa l indromic sequence T C T A G A ; this is

expec ted to occur every few ki lobases in an ideal unbi-

ased genomic sequence. Never theless , since the XbaI re-

s t r ic t ion endonuc l ea se r ecogn i t ion site is compa t ib l e

wi th the R c C 9 consensus sequence, it is also found every

few tens o f nucleot ides wi thin the over 60 R c C 9 genomic

loci present in the C. elegans g e n o m e (Nacler io et al.

1992). This pattern can be easi ly v isual ized by digest ing

C. elegans genomic D N A with XbaI and end- label ing the

digest ion products (La Volpe et al. 1988; Fig. 1). I have

pe r fo rmed the same exper iment on the genomic D N A of

three o ther species o f nematodes : R. maupasi, PG-2 (Fig.

1), and C. briggsae (Fig. 1; see also La Volpe et al.

1988). In R. maupasi a per iodici ty o f 50 nucleot ides is

observed; in PG-2 the smallest XbaI band is around 160

bp in length, whi le in C. briggsae a 10-nucleot ide peri-

odici ty is evident . These results suggest the presence o f

t andemly repea ted genom ic sequences conta in ing the

XbaI site. I do not know whether the per iodic patterns

observed in R. maupasi and C. briggsae lanes are due to

50bp

40bp ~,

i i

ii i i

ii:= i ~ ~ =

iiii~ ~ ~!i(:/::i

iill :::iii% if! ̧

Fig. 1. Genome distribution of XbaI sites: Genomic DNA from R. maupasi, PG-2, C. briggsae, Saccharomyces cerevisiae (as negative control), and C. elegans was digested with the restriction endonuclease XbaI; restriction products were end-labeled with Klenow fragment polymerase (see Materials and Methods), analyzed on a 6% polyacryl- amide-urea gel, and autoradiographed. Size markers are in base pairs. Signals marked by an asterisk are electrophoresis artefacts also present in the control lane.

incomple te restr ict ion digest ion or to r andom loss o f the

XbaI site in some intervals by random mutation.

In order to invest igate the molecu la r nature of these

low-molecu la r -we igh t XbaI genomic fragments , I c loned

them by l igat ing the same digest ion products used for the

end- label ing react ion with a p lasmid vec tor digested with

XbaI and dephosphoryla ted. The l igat ion react ion was

pe r fo rmed at low eff ic iency in order to favor small and

abundant fragments . I obtained 58 colonies in the C.

briggsae minil ibrary, and 26 and 62 colonies , respec-

t ively, in PG-2 and R. maupasi libraries. I moni to red the

presence and the size o f the c loned inserts by the poly-

A RCC9

GGAA~ TIL-T OGAACATFIU AGAATITTCU £V~AAGTnL~

~AATITICr OGAA~ AGAAGGITCT TGAACATIL~ AGAATITrCT

AGAAGTTIt~ ~-~AT/TIL~ A~T- - - TGAACATrlC AGAATI'ITAT (~x~A,I.I • i,i.i, I ,

GC.AACATIgC AGAcI'I'I'ICC CC~I.I.I.I'It~T AGAAAGTIL~ GGAATATIE~ /XEAAqTITCC CCAATIqTCT AGAAAATIET GGACCATIO3 AGAACI'I'IL-~ CGC-~TITIL~ AGAAAATIL-T GGACCATTCC GGAATI'ITCT (~I~ATITICf AGAAACTIt~ GGACCATIL~ AGAACTI'IL-T AGAATITIL-T AGAAAATIL~ GGACCATIL'C GGA~TITIL~ CGA~TITIt? AGAAACIffE? GCAACAT~

8 RcbC9

AAAATTCC AGAAAATCCT AGGAAGTTCC AGAACATTCT AG

CT AGAAAATICC AGTAAATCTC AGAACATTCT AGAAAATTCC AGTAAATCTCT AGAAAATTCC AG-TAATTCC AGAAAGATCC AGAACATTCT AGAAAATI'CC AGTAATCTCA GAAC

TCT

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AGAATI'I'IL~ AGAACITIL~ AAAGTlX2T AGAAGITIL~ C_AAA~ AGAAAATIU? GGAACATIL~ AGAA-TTIL-T AGAIACITCT GGACCATIL~ AGAAA~ GC3XATGTIX3C AGAAAATIL'T GGAACAT?GC GGAATITIXZT CGA:ri'i'.'it~ AGAACTI'IL-T GG"IL-C_ATIDC AGACAT/'IU2 CGAAETI'IL-T AGAAAG'IqL'T AGAAAATIL~ GGAATI'I'i'C~ C~ i'l'l'l'l~a~ AGAAACI~ GG~xCAq'I~ ~TTCC CC~AAqVlvI~ AGAAAGT~ GC~AACAq~ AG~I'I'I'It3C GGAATC-~ AGAAACITCT GGAATATI~C CG~ITITICT AGAATITTCT GGAAC~TT AGAA~ AC~AA AG

CG~'ITI'iL'T CT AGAA~ AC4k~IA~K]G

AGAA~ ~TIL'T AGAAAATIL-T AGACi'iTit-E CT

TGAATIqT(~ GGA~i'i'i'ICT AGAAC- TCL'T A~AAAQI'IL-T TGAAAA~ ACC4~A~ CGAA'I'I'I'I t~F AGAAAAATCG GGAATATIEU GC_AATGqTCC ACA- -TI'I~- AGAAACTI~ AGAAAATIL-T ~Q_~I'I'I'IL~C i~Cri~ AGAATIqTAT GC-mA GGAATGTIL-~ CGAA~ AAAAAATIEG ~ T I ' I ' r l T AGAACTlqL'T ABAAAAT1WI' ATAAAATIL-T AGAGAGTIL-T AGAAAAATIg (~C~_TlX3C - C,A.~TGEqC CC.AATATIL'U GGAA-~ ~ AGAACI'I'IL-T - - -AATI'ILXT~ AGAATI'ITCC CC.AA~ GGAATGTIL-U AGAA'r I'I'ICT GGAAAAAE'I~ CC~ATFITC2 AGA%AC~-Cr AGAACITILT AGAAAATIL~ AGAAACTIET AGAAAATIL~ GGACCAq~CC AGAAAAATCG CGACCATIL-'C GC~ACCA'I'IL~ AGAA~ AGAAAATIC_A GGAA'iTriCD

CGAATFI'IL~ TGAAAATIL~ AGAACTITCT AGAAACTIET AGAAAAATCU A_GAAACTIL~ GGACC_ATT(X] /xDAAA_AATTG GGACCATIL'C AGAAATG'It~ (r=AAC~TIX~ GGAATITIL~ GGAAAG'IL~ ~F.AACTITCT GCAATGTIL~ AC~q'I'I~ CGA~I'I'I'I'I~.'T AGAA'/'I'I'II~'T AGAAG~ CGki'i'i'i'it? AGAAAG"IqL~ AGAAAATIL~ -GAA~ETIL'T AGAAAGTIL'T AGAACAqTAC CT GGACCATIL'C GGAATATIEI2 GGAATA~ CGACq'ITIE]C A ~ GG~I'I'I'It~i ~ AGAAVlVI~ 3BA£rI'Iff~IEE (]GRi'i'i'riCA (W~/~kCATICC CGAki'i'±ut~ CGN~PI'IET C~E'I~ TC~G~ AG~C'I~ A G / ~ AC,~IET AGAA GGAATATIL'~ C~i'iTi'IL-T (~CATD GGACCATTCC

AGAA~ AGAAAGTIL-T GGAAqTI'IL~ CAAATI'ITC4~ GGAACATTC~ CGAA~

CT TCAAACTIL-T AGAATITIL~ TCG AC~G~TD ~[AAAAT~ GGAACAT~ CGI~I'I'I'I'I ~rl ~ AGAA T~IL'T AGAACATIDC ~TI'CC AGACATI'IL~ ~ GCAAEQTIL72 ( 2 G ~ C G A ~ G~A~TIEC /bSAA~ CE~z~i'i'i'riCf CGAATITIL'T AGAAAGT21~ AGAATITIET AGAAAA-TC- AGAAAGTIL~ AGAAACTIL-T GGAATATIL~ CG/{ITI'I'ICT GGAAA- -TC- GGAATATICA AGAACATIS~ A ~ AGAAAGTIET GC-CATGTICT -GAATITIt'C

CGA%TITIV_-A GGAACATIL~ /X~TIL~ CGAATITIC~ CC=~TI'I'I'It-T TCAAAAETCr AGAt'I'I'I'f GGA_ATC~ ~X~AAAC~ AGAIA~ GGACCATIX~ AGAATI'ITCT GGAACAET GGAATATIE32 AGAACI'ITC? AGAAAATIL~

AGAAAATTCC AG-AAATCCT AGGAAGTI~C TGAACATTCT TGAAAATTCC AGTAAATCTC AGAAAATTCC ACgt~CATTCC AGTTITTTCC AGAAAATTCC A~TTCT AGAAAATTCC AGAAAAGATT CAGAAC-ATT C

AGAACATTCT AGAAAGTTCC AGA~AAATCC AGAACATTCT AGA

RcmC9 TCT

AGAAGTTTCT GGAAG-TTCT GGAC~IATTCT AGA

CTTC TGAAAATTCC AGAAA TCCC AGAAAATTCC AGAACATTCC AGAAAATTCC AGAAAATTCT AGAAAGT'I~C

Fig. 2. A Fifteen individual clones containing the RcC9 repeat of C. elegans. (Sequences here reported are adjacent or overlapping in part with the repetitive clusters discussed in Naclerio et al. 1992.) B Four individual clones containing the RcbC9 repeat of C. briggsae and one RcmC9 clone (R. maupasi).

merase chain reaction (PCR) using vector primers flank- ing the insert site (not shown).

Sequencing Analysis of the Repetitive DNA Elements

Four clones in the C. briggsae minilibrary (ac. no.: L26057, L26058, L26114, L26115) and one in the R. maupasi minilibrary (ac. no.: L26116) contained DNA inserts characterized by the presence of the pentanucle- otide [nGAAn] and its complement [nTTCn] recurring

several times in alternating tandem arrays. Sequences are shown in Fig. 2B. I named the C. briggsae and R. mau- pasi repetitive DNA families RcbC9 and RcmC9, respec- tively. Individual cloned elements belonging to each spe- cies were used as probes in a Southern blot experiment on genomic D N A from all the three nematode species. Each probe hybridized over a wide range of molecular sizes with the homologous genomic DNA, confirming the repetitive nature of the DNA elements, but not with genomic D N A of the other species (not shown).

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Table 1. Nucleotide choices in positions 1, 2, 3, 4, and 5 of the cryptic repeat [nGAAn]

RcC9 (C. elegans) RcbC9 (C. briggsae)

1 2 3 4 5 2 2 3 4 5

G 150 454 6 8 22 G 29 76 9 2 1 A 263 15 487 435 294 A 50 8 65 73 33 T 19 4 1 21 187 T 4 / 7 4 33 C 56 20 1 30 87 C 2 / / 2 23 tot 488 493 495 494 489 tot 85 84 82 81 80 G% 32 92 2 2 4 G% 34 90 11 2.5 1 A% 54 3 98 88 40 A% 59 10 80 90 41 T% 4 1 <1 4 38 T% 5 / 9 5 41 C% 11 4 <2 6 18 C% 2 / / 2.5 16

"Invariable" nucleotides are indicated in bold face.

The C. briggsae consensus sequence is A~GAAAT re- peated in tandem in alternate orientation. The nucleotide choice for positions 1 and 5 is no different from that observed in C. elegans (Naclerio et al. 1992; Table 1), but once again these positions have a lower constraint than positions 2, 3, 4. As a consequence of alternative choices in consecutive repeats, although the consensus between the two families is conserved, there are no long stretches of sequence identity between the two species.

While C. briggsae sequences have a period of 10 bp (Fig. 2B), C. elegans have a supraperiodicity of 40 bp (Fig. 2A); homogeneity between supraperiodic repeats in each species increases the divergence between RcC9 and RcbC9.

The most common repeat in C. briggsae (likely to be the ancestor repeat) is [AGAAA][ATTCC]. The next- most-frequent repeat is [AGAAC][ATTCT]. Surpris- ingly, other repeats, diverging from the "ancestor" by a single mismatch, are less abundant.

Discussion

Coding sequences are often conserved between closely related species (Heschl and Baillie 1990; Kennedy et al. 1993; Zucker-Aprison and Blumenthal 1989). Only a few regulatory elements in some promoters are main- tained throughout evolution and can be identified by se- quence comparison between related species (Heschl and Baillie 1990; Zucker-Aprison and Blumenthal 1989); re- petitive DNA families instead are subject to a faster turn- over resulting in a loss of identity between close species (Kennedy et al. 1993). Transposable elements can be conserved or not according to the different rules of hor- izontal evolution (Kidwell 1992). In this paper I study the conservation between related species of nematodes of a repetitive DNA family of the nontransposable type. This family, named RcC9, was first identified and de- scribed in C. elegans. RcC9 is characterized by the rep- etition in alternate orientation of a short consensus se-

quence [nGAAn] (La Volpe et al. 1988). A puzzling sequence identity between elements of the RcC9 family and the consensus of the HSE (a conserved transcrip- tional regulatory element) was noted.

I have shown in this work that repetitive DNA fami- lies with similar features are present in at least two other species of related free-living nematodes: C. briggsae and R. maupasi. I named these two families RcbC9 and RcmC9, respectively. Both families share the [nGAAn] consensus with the previously identified repetitive DNA family RcC9 of C. elegans.

Tandemly repeated sequences evolve under the pres- sure of several mechanisms of genomic turnover such as unequal sister-strand exchange, slippage replication, and gene conversion within and between loci (Dover 1982; Dover and Flavell 1984). It is therefore easy to under- stand how each species has developed different supra- periodic patterns superimposed on the minimal repeti- tion: repeated 40-bp RcC9 modules in C. elegans (see both Figs. 1 and 2), 10-bp RcbC9 modules in C. brigg- sac, and probably 50-bp RcmC9 modules in R. maupasi (Fig. 1).

Correction by crossing-over (or other mechanisms) will tend to homogenize the supraperiodic modules within each species (i.e, the 40mer in C. elegans and the decamer in C. briggsae). Homogenization, however, did not interfere with the conservation of the minimal-repeat [nGAAn]. The repetitive families described here must be under some sort of selective constraint. Nucleotides of the repeat that have diverged most are by definition the less constrained while conserved nucleotides are those most likely to be of structural or functional significance. Remarkably, the same nucleotides that are conserved in the evolution of the repeated DNA families I describe (nucleotides 2, 3, and 4) have also been conserved in the transcriptional regulatory element HSE throughout phy- logeny.

Perhaps these repetitive DNA families have somehow evolved from the HSEs: amplification events might have initially conferred a selectable advantage to genomes by providing a more efficient response to stress. After fur- ther amplification, the original function might have been lost and new structural features might have been gained. Alternatively, in these nematodes species the sequence fraction here described might still serve for storage of the inactive HSF or of some other related DNA-binding pro- tein.

Acknowledgments. I thank Massimo Di Giulio, Clara Frontali, and John F. Pulitzer for helpful discussions and critical reading of the manuscript and Leon W. Nawrocki for C. briggsae, PG-2, and R. maupasi DNAs. The wild-type C. elegans strain N2-JW-H used in this work was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. This work was supported in part by Commission of European Communities con- tract SC2"-CT92-0633, CNR Progetto Speciale Bioinformatica and CNR Target Project Biotechnology and Bioinstrumentation.

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