Repetitive DNA Sequences Located in the Terminal Portion of the Caenorhabditis Elegans Chromosomes

Nucleic Acids Research, 1993, Vol. 21, No. 5 1133 -1139

Repetitive DNA sequences located in the terminal portion of the Caenorhabditis elegans chromosomes

Giuseppina Cangiano and Adriana La Volpe* CNR International Institute of Genetics and Biophysics, 10 Via Marconi, 80125 Naples, Italy

Received December 14, 1992; Revised and Accepted January 28, 1993 EMBL accession nos+

ABSTRACT

We describe the distribution along the chromosomes chromosomes, 4, 5, 6). However, when the chromosomes of Caenorhabditis elegans of two repetitive DNA segregate in meiosis they align parallel to the spindle axis such families, RcS5 and Cerep3 and interstitial telomeric that one end or the other points towards the spindle pole (Donna sequences. Both families show, among other Albertson personal communication). interesting features, a preferential location in the In an effort to resolve the chromosome structural and finctional terminal 30% of the chromosomes. It is known that in organization in C elegans, we have recently described the these regions of the genome the frequency of distribution of clusters of satellite-like DNA sequences and short recombination is much higher than in the central interspersed elements (7). We observed a uniform distribution portion, genes are rarer and sequences important for of five repetitive DNA families along the chromosome. Here we chromosome disjunction may lie. identify and describe a family ofrepetitive DNA elements, RcS5, which in contrast is preferentially located at the pro-terminal INTRODUCTION portion of the chromosomes. We fur-ther show that another repetitive DNA family previously As a result of the intensive and multifaceted analysis of genomes, described, Cerep3 (8) is similarly distributed. Associated with it is becoming increasingly manifest that most chromosomal DNA these two repetitive DNA families we find a high concentration performs functions other than those involved in pure coding of interstitial telomeric repeats. activity. Chromosomes need to attach to the scaffold and to the nuclear matrix, to duplicate with fidelity and to segregate MATERIALS AND METHODS efficiently. Homologous and non homologous recombination is active in somatic tissues and germ line cells. Specific sequences N2-JW-H, W-PA-1H, W-PA-2H, DH424, RW7000, RC301, along the chromosome guarantee the binding of specific matrix TR403, CB4507, CB4555, CB4853, CB4854, CB4855, CB4856, proteins, proper DNA duplication and segregation during the cell CB4857 are wild type, independently isolated C.elegans strains, cycle, and probably the enhancement or the repression of CB1392 is a nucOOl mutant, they were all obtained from the recombination. C.elegans is a well known model system for Caenorhabditis Genetics Centre. DNA from C. remanei, PG-3, developmental genetics and genomic studies. The physical map P. redivivus, C.briggsae, PG-2, C.marpasi , all closely related is close to completion and a pilot project to sequence the nematode species, was a gift from L.W.Nawrocki. All the strains C.elegans third chromosome has been undertaken (1). were raised at 18°C. Routine strain maintenance was as described Comparison between the physical map and the genetic map (2) by Sulston and Hodgkin 1988 (9). has confinned a non uniform distribution of crossovers along the C. elegans DNA purification from liquid cultures was chromosomes. Crossovers are relatively rare in the central part performed according to Sulston and Hodgkin (9). of the chromosomes, in the so called 'gene clusters' (where most The cosmid library, from which the initial representative of mutants map), but their frequency is highly enhanced in the pro- the RcS5 family (RcS5 # 1) was obtained, was a gift from Silvana terminal region of the chromosomes, where genetic loci defined Gargano. It was made by inserting genomic DNA, partially by mutations are rarer (fig 1). A survey of the distribution of digested with SaufIA, into the BamHI site of tetra-cos vector. the coding regions in the genome (by cDNA analysis) has shown A subset of clones (250), corresponding to about 10% of the that although variation in the frequency of recombination accounts C.elegans genome, was screened by hybridisation with labelled for the clustering of most genes in the centre of the chromosomes total genomic DNA as probe. We obtained 12 positive clones, on the genetic map, nevertheless coding sequences are physically two of which contain ribosomal genes, three contain the somewhat rarer in the so called 'chromosomal arms' (3). transposable element Tcl, two contain elements belonging to the Cytological and genetic studies indicate that C. elegans same repetitive DNA family, the others all represent individual centromeres are diffuse along the chromosomes (holocentric elements belonging to independent repetitive DNA families. All

* To whom correspondence should be addressed +X69085, X69082, X68553 and X68498 1134 Nucleic Acids Research, 1993, Vol. 21, No. 5 the other cosmid clones derive from a library also constructed from a partial SaulIA digest, using the vector Lorist6 (10). The genomic DNA library in yeast artificial chromosomes was that made for tho C.elegans mapping project and has been ,LT A previously described (11, 12, 13, 7). The vector was pYAC4 TCHU B dyl TI OC and the Saccharomyces cerevisiae host strain was AB1380 ddy- 10 T tOL6 n -1 04 t- 23 CAPZ/B (MAMTa, y+t ura-3, trp-1, ade2-1, can-i 100, lys 2-1, his-5). o-61 k n-1i cey-1 0e -2 - II YPP/1 Southern transfer was performed as described by Southern (14) Ii n-31 aAA zg tc TCIOTI ISTOSEll I LIN3 as; 4 or 5 1 3 kin-6 the DNA oftotal genomic BWO 10S5 HyoD 1 n 1'Cll 1 1+* 4n -S3 1ST1 301l2 with following modifications: the (1 ltg ,..IO us 3 n TC aP p- 1IST07E7 per was to membrane and 11T21 aS -1 t n | I i- IS1F25H2 DNA lane) transferred Hybond-N LISM 5 a -7 r p0j rsn-2 Ill of a - / g NFjj 613 immobilized by UV treatnent. Hybridisations of libraries and tiPlOl St 6 at I 1 4 2 iN 2lrsn-3 Southern nylon filters were performed in 6 x SPC (20 x SPC = 01SK105 C2 S X H 2 unII~c - 52 2M NaCl, 0.6M Na2HPO4, 0.02M EDTA pH 6.2), 0.9% dextran sulphate and strand DNA Sarkosyl, 10% 100l,g/ml single 0 10 20 30 40 50 60 70 80 90 100 carrier at 65°C for 6-16 hours. We used approximately 107 cpm of oligolabelled DNA probes prepared as described by Feinberg and Vogelstein (15, 16) with a specific activity of greater Figure 1. Comparison between the genetic map and the physical map of than 109 cpm/tg per hybridisation bag. The telomeric probe was chromosome 2. On the genetic map, top, all the genetic loci (unspecified) are a single strand 48mer containing eight copies the TTAGGC repeat shown as vertical bars. The bolded box repesents the region named 'gene cluster' identified as the Ascaris telomeric repeat (17). It was purchased and the bare terminal portions ofthe chromosome are evident. Slanted lines point from Eurogentech (Belgium) and end-labelled with T4 at corresponding loci in the genetic map and physical map undernath. Variations in the frequency ofrecombiation account for the discrepancies between genetic polynucleotides kinase and [y 32P]-ATP. Hybridisation was and physical distances. In order to allow the comparison, the scale used for both performed in the mixture described above at 60°C. maps is expressed in percent ofthe chromosome length. Genetic data were derived Following the hybridisation, filters were washed four times from Edgley et al. (2). Physical data were derived, with permission, from physical either in 1xSPC, 1%SDS at 50°C or in 2xSPC, 1%SDS at map version 920628 (physical map data may be subjected to changes, Coulson 420C (relaxed conditions) and air dried. and Sulston personal communication). Removal of probe: In order to reuse the filters, after each independent hybridisation, probes were removed from filters by repeating the following steps twice: 10 min room temp. in 0.2 cosmids cover about 80% of the genome in a non-overlapping M NaOH, 0.5 M NaCl, 10 min room temp. in 0.5 M Tris pH7.4, manner. The second kind of grid is a set of overlapping YAC 0.5M NaCl and 10 min room temp. in 1 xSSPE (20xSSPE=3M clones representing almost 95% ofthe genome. We demonstrated NaCl, 0.2M NaH2PO4.H20, 0.02M EDTA, pH 7.4). Efficiency elsewhere that all the cloned members of a repetitive family can of removal was monitored by re-exposing used filters before be positioned in this way on the already mapped portion of the reuse. genome (7). The use of two indexed libraries carried by a Cosmids and plasmids used as probes were maintained in E.coli prokaryotic and a eukaryotic host may overcome problems of rec A strain HB101 (F-, hsd S20, (rBmB), recA13, ara-14, instability (in one host or the other) of some DNA sequences. proA2, lacYl, galK2, rpsL20 (Smr), xylS, mntl-, supE-44, X-) In figure 2 we report the physical distribution ofRcS5 elements selecting for kanamycin or ampicillin resistance. The strain was on the physical map of the five autosomes and on the X checked periodically for the recA phenotype. chromosome. RcS5 elements appear to be preferentially located All RcS5 subclones were constructed in pGEM3/4 or outside the 'gene clusters'. Another family with a similar pUC18/19. Cerep3 plasmid was a gift from Scott Emmons. distribution is the Cerep3 family. This latter family has been Sequencing was performed according to Sanger and Coulson identified by Felsentein and Emmons in 1988 and comprises (18). The complete sequences obtained during this work have elements about one kb long, some of which have been shown been sent to the EMBL Data Library, and only relevant portions to affect replication and segregation ofp ids inSaccharomyces are shown. Accession numbers are indicated in the legend of cerevisiae . The distribution of Cerep3 elements along the fig. 5. chromosomes is also shown in figure 2. Computer programs used in the physical mapping analysis are described in Sulston et al. (19). Sequence data were analysed Strains and species comparison with the GCG (20) software package. We have compared the patterns obained by digesting the genomic DNA ofseveral independendy isolated C.elegans wild type strains RESULTS with the restriction endonuclease Eco RI and hybridising the Southern transfer with RcS5 or Cerep3 probes (figure 3). The Physical distribution of RcS5 and Cerep3 elements in the RcS5 probe is an Eco RI-Sac I fragment from the genome RcS5(ZK1091) element containing only RcS5 sequences (see The first element belonging to the RcS5 family, called RcS5 # 1, figure 5 for details), the Cerep3 probe is an EcoRI-BamHI from was isolated from a genomic DNA library using as probe total pCel7(Cerep3. 1) containing a Cerep3 entire element (Felsenstein genomic C.elegans DNA; clones containing DNA from the and Emmons 1988). Some of the C.elegans strains used in this genomic repetitive fraction give much stronger hybridisation experiment have a high copy number ofthe transposable element sigals (7). The repetitive portion of the original cosmid was then Tcl and among these some contain a mutator activity that results subcloned and used as probe on two tpes of indexed library grids in an increase in the rate of transposition ('hopper' genetic on nylon membrane (12, 7). The first kind of grid represents background, 21). We observe that the RcS5 probe generally a subset of cosmids already positioned on the map, the 1934 recognises the same number ofbands of similar size, suggesting Nucleic Acids Research, 1993, Vol. 21, No. 5 1135

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Figure 2. Physical distribution of RcS5 and Cerep3 elements along the physical map of the five autosomes and of the chromosome X. The distribution has been obtained by hybridising two types of indexed library grids on nylon membrane: a subset of 1934 cosmids already positioned on the map covering about 80% of the genome in a non-overlapping manner and a set of overlapping YAC clones representing almost 95% of the genome (12, 7). The probes used in the screening are: a HindE fragment containing the entire RcS5 # 1 element and a 728bp flanking DNA region (on the left side of the element illustrated in figure 5, ac. no.: X68553), an EcoRI-BamnHI fragment from pCel7(Cerep3. 1) containing a Cerep3 entire element and about 200bp of flanking sequences on both sides (8), and a synthetic oligonucleotide 48bp in length containing 8 repeats of the putative telomeric hexamer (TTAGGC). Hybridisations of libraries with RcS5 and Cerep3 probes were performed in 6XSPC, 0.9% Sarkosyl, 10% dextran sulphate, 100ltg/ml single strand DNA carrier at 65°C, hybridization with the single stranded telomeric probe was performed in the same mix at 60°C. In all cases, filters were washed four times in 1 XSPC, 1 %SDS at 50°C. The RcS5 elements and the Cerep3 elements are reported as small vertical rods in the second and third row respectively. Interstitial telomeric repeats are represented by round bold dots under the physical map. (Physical map version 920628) an average constant copy number, although RFLPs are detectable We have also tested for possible cross-hybridisations between especially in CB4854 and CB4856 strains. These are not 'hopper' our repetitive probes and DNAs from other free living nematode strains, but show a low Tcl copy number and mobility. Those species (figure 4, upper panels). We also have on the same gel RFLPs are therefore not likely to be due to accidental three C.elegans N2 derived lines. No detectable RFLPs were transposition of an active Tcl next to a RcS5 element. The observed among N2 derived lines. Using an internal fragment patterns obtained on the same strains with the Cerep3 probe of the RcS5(ZK1091) element, common to all the members of appear more variable, though not so well resolved due to the the family, we did not detect any cross-species hybridisation under high number of Cerep3 loci in genome. relaxed stringency conditions (hybridisation at 65°C in 6 x SPC, 1136 Nucleic Acids Research, 1993, Vol. 21, No. S

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Figure 4. Southern transfer and hybridisation with RcS5 and Cerep3 specific probes ofdifferent closely related nematode species. DNA in all cases was digested Figure 3. Southern transfer and hybridisation with RcS5 and Cerep3 specific with EcoRI and run on a 1% agarose gel in Tris-Acetate buffer, following the wild type DNA was probes ofindependendy isolated C.elegans strains. digested hybridisation in 6xSPC, 0.9% Sarkosyl, 10% dextran sulphate at 650C, filters with EcoRI and run on a 1% agarose gel in Tris-Acetate buffer, following the were washed four times in 2xSPC, 1% SDS at 420C.The gel in the upper panel hybridisation in 6XSPC, 0.9% Sarkosyl, 10% dextran sulphate at 65°C, filters contains, left to right, C.remanei, PG-3,C.bniggsae, PG-2, C.marpasi , followed were washed four times in 1 at From left to W- xSPC, 1%SDS 50°C. right by three N2 derived C.elegans strains A, B, C, an empty lane and P. redivivus DH424 RW7000* PA-lH(Ca,USA), W-PA-2H(Ca,USA), (Ca,USA), (France), DNA. The gel was hybridised with an RcS5 specific probe, (Eco RI-Sac I CB4507 CB4555* RC301* (Germany), TR403* (Wi, USA), (Ca,USA), fragment from the RcS5(ZK1091) element containing only RcS5 sequences, see (Ca,USA), CB4853 (Ca,USA), CB4854 (Ca,USA), CB4855 (Ca,USA), CB4856 figure 5 for details) left, and a Cerep3 specific probe (EcoRI-BamnHI from CB4857 Strains with an asterisks have a copy number (Haway), (Ca,USA). high pCel7(Cerep3. 1) conaining a Cerep3 enire element, 8) right. In the lower panel, of Tcl elements. of the strains is indicated in Origin parenthesis. Upper panel: we have a different gel hybridised with two subclones of an RcS5 element. The the filter was with an RcS5 Lower a second hybridised specific probe. panel: gel contains, left to right, C.remanei, PG-3, P.redivivus, C.briggsae, PG-2, copy of the same gel transferred on filter was hybridised with a Cerep3 specific C.marpasi cerevisiae DNA as negative control, two empty lanes probe. ,Saccharomzyces and CB1392. The filter on the left was hybridised with the RcS5 internal firgment described above, the one on the right with a SacI-HindM fiagment spanning the junction between the RcS5(ZK1091) element and its rigth flanking sequence (accession number: X69082). 0.9% Sarkosyl and washes at 420C in 2xSPC, 1 %SDS), even after a week exposure (not shown). Similarly, we confirmed the observation of Felsenstein and Emmons 1988 that there is no elements, 1.3kb in length and composed oftwo terminal inverted cross-species hybridisation with the Cerep3 probe. However a repeats of about 250bp separated by an intervening sequence fragment spanning the RcS5(ZK1091) element and its flanking around 800bp in length. Within the intervening sequence of the sequence gave multiple bands with all nematode species tested, two complete elements we find a duplication of over 150 bp. but not with the yeast DNA used as control, raising the question RcS5(ZK1091) has also a duplication of 69bp within the inverted of what kind of repetitive sequence, so widespread among other repeat at the 3' end. The other two, RcS5 # 1, unmapped, and nematdes, lies next to this RcS5 element (figure 4, lower panels). RcS5(T17H5) on chromosome II, are truncated elements missing the 3' inverted repeat and part of the intervening sequence (see Comparison within RcS5 elements and within their flanking figure 5) . Sequence identity between elements ranges between sequences 86% and 93%. We did not find any open reading frame inside We have determined the DNA sequence of four independent the elements maldng it unlikely that any of these could be an members ofthe RcS5 family.Two, RcS5(T25H8) on chromosome active member of an unhindered transposon family such as Tcl. I and RcS5(ZK1091) on chromosome V, represent complete An interesting feature of the flanking regions of the RcS5 Nucleic Acids Research, 1993, Vol. 21, No. 5 1137

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Figure 5. A graphic representation of four repetitive elements of the RcS5 family. The complete sequence data obtained (including flanking sequences) have been submitted to EMBL Data Library (accession numbers are: X69085, X69082, X68553, X68498 respectively). Arrows indicate duplications, white boxes the inverted terminal repeats, the bold boxes tandem repetitions of telomeric hexamers. On the right, physical locations of the sequenced elements are shown when known.

presence 10 20 30 40 50 elements is the oftelomeric repeats. The inverted repeats 3' RcS5 AGGCTTAGGCMTAGACATAAGCTGAGGCTCTGGTTCAGGCMTGGCMTAA of each element ends with the sequence AGGGTT (in opposite 111111111111 I11111 11111 11 1 11111 AscTel CGAAAGTTrATGCAAGGAATTGGMTTAGGCTTAGGCMTAGGCTTAGGCM1AGGCM1AGGMTrAGGCMTAGGMTIAG orientations), and adjacent to each element either at the 5' end 10 20 30 40 50 60 70

(in RcS5(T17H5)) or at the 3' end (in RcS5(T25H8), 60 70 80 90 3' RcS5 TCTTAGACTTAAGTAGGCTTAAGTrIAGGCTTA-ACTMAAGCTT RcS5(ZK1091) and RcS5 # 1) we find a number of repetitions 1IIIIIIII111111111 1iii III of the AscTel GCTTAGGCMTAGGCMTAGGCTTAGGCTrAGGCTTAGGCTTAGGCTTAGGCTTAGGCTTAGGCTTAGGCTTAGGCMr putative telomeric hexamer of C.elegans TTAGGC (as s0 90 100 120 130 140 150 defined by C. Wicky by means of Bal3 1 experiments, personal communication). This finding accounts for the results obtained 10 20 30 40 in the Southern experiments with the panel of the free living 3'RcS5 AGGCTTAGGCTTAGACATAAGCTGAGG---CTCTGG1TCAGGCTTTG 11111 1111111H111M11 IIIIII III II 1111 1 nematode species: in fact, the probe that only recognises Loarep TTATGCMTAGGCTTAGGCM1AGGTTCTTCGGGTTTATGCTTAGGCTTAGGCTTAGGMTTCTCGGGTTTATGM1TAG C.elegans DNA was an Eco RI-Sac I fragment from the 220 230 240 250 260 270 280 RcS5(ZK1091) element containing only RcS5 sequence, while 50 60 70 80 90 3' RcS5 GCTTAATCMTAGACTTAAGTTTAGGCMlAAGMT1AGGCMlA-AMTrAAGCTT the other probe, detecting sequences in all the nematode species, I I 11111 1111 1 11111111 111111111 li1 li1 Loarep GTrCATGCTTAGGCMTAGGCTTAGGCMTGGGCTMAGGCMrAGGCMFAGGCMTAGGTITCGGGTTrATGCTTAGG was a SacI-HindI fragment encompassing the 3' end of 290 300 310 320 330 340 350 360 RcS5(ZK1091) and entering the flanking region for 98 bp. We also the filter shown in hybridised figure 4, bottom pannel, with Figure 6. Sequence similarity between RcS5 flanking region and nematode a oligomer containing 8 tandem repetitions of the TTAGGC telomeres. The figure shows the alignment between the 98bp laying next to hexamer, and we detected the same pattern obtained with the RcS5(ZK1091) element and theAscatis lwnbricoides telomeric sequence, flanking SacI-HindI probe including the flanking sequence of this and the other RcS5 elements we sequenced we found a number of perfect RcS5(ZK1091) (data not shown). Evidence for the occurrence telomeric hexamers interspersed with degenerated copies. In the bottom part of the figure sequence alignment between the same 98bp sequence and a Loa Loa of intrachromosomal telomeric repeats in C.elegans has been repetitive DNA sequence is also shown (from 30). obtained in other laboratories (Donna Albertson, Yuko Kozono, and Chantal Wicky, personal communication). Sequence comparison between this 98bp fragment flanking RcS5(ZK1091) and the sequence of the cloned Ascaris telomere (17) is shown hybridised with a terminal labelled 48mer containing eight tandem in fig. 6 (top). repeats of the TTAGGC hexamer. Interstitial telomeric repeats once again turned out to be clustered in the terminal 30% of the Distribution of interstitial telomeric repeats in the C.elegans chromosomes (see bold dots in figure 2). They appear to be more genome abundant than the repetitive elements belonging to RcS5 and Transposable elements flanked by telomere-like repeats have been Cerep3 families, although they are often found associated either isolated in several protozoa (22, 23). Could the occurrence of with Cerep3 or with RcS5 or with both, in fact cosmids containing intrachromosomal telomeric hexamers be closely linked to RcS5 RcS5 and/or Cerep3 often hybridise also with the telomeric probe. elements? We mapped the interstitial telomeric repeats making There is a positive correlation between enhancement of crossing use of the same indexed filters already used for the physical overs in this part of the genome and frequency of interstitial mapping of the RcS5 and Cerep3 families. The filters were telomeric repeats (figure 7). 1138 Nucleic Acids Research, 1993, Vol. 21, No. S III 1 111111111111111111 JA r X 5^,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~[UJJ tss~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~lt-6 Cerep3. *- . * @ * * - * - *- - *...... dpy . 01020304050 60 70 80 90 100 0 10 20 30 40 S0 60 70gOI10SLI80

Figure 7. Recombination frequencies and repetitive sequences distribution along the chromosomes. In this figure we compare the genetic map, the physical map and the RcS5 and Cerep3 element distribution of chromosomes II and chromosome IV. As in figure 1, the scale used for both maps is expressed in percent of the chromosome length. On the genetic map, top, all the genetic loci (unspecified) are shown as vertical bars. The bolded box represents the region named 'gene cluster' and the bare terminal portions of the chromosomes are evident. Slanted lines point at corresponding loci in the genetic map and physical map underneath. Variations in the frequency of recombination account for the discrepancies between genetic and physical distances. The RcS5 elements and the Cerep3 elements are reported as small vertical rods in the second and third row respectively, pseudo-telomeric repeats (TTAGGC) are represented by round bold dots under the physical map. (Physical map version 920628.) DISCUSSION lumbricoides, where these DNA elements are completely eliminated from the somatic genome during the process of The work reported in this paper is directed at defining the chromatin elimination (17), and in the filarial species Loa Loa distribution of repetitive elements in the terminal portions of the (30). Klion et al. (30) claim that such repeated DNA is a species- chromosomes of C. elegans. We were particularly interested in specific diagnostic probe. Under our stringency conditions as litde these regions because of their involvement in recombination and as 8 tandem duplications of the TTAGGC repeat cross-hybridise chromosome stability. In fact, recombination frequencies vary with all nematode species analysed. Sequence similarity between along these chromosomes being higher in the proterninal C.elegans interstitial telomeric repeats and Loa Loa repetitive portions. C.elegans chromosomes are holocentric (also found in DNA sequence is shown in figure 6 (bottom). other nematodes, in some insects and plants). Kinetocores are We also observed that interstitial telomeric repeats are not visible during meiosis I, the two main functions of the associated in C.elegans with bonafide repetitive DNA elements. centromeres (attachment to the spindle and holding the bivalent The RcS5 and Cerep3 families are also preferencially located together) being adopted by the chromosome termini (Albertson in the proterminal last 30% of the chromosomes. Elements personal communication). One end of the chromosome is oriented belonging to the Cerep3 family have been shown to direct towards the spindle pole while the other guarantees the replication and correct segregation in Saccharomyces cerevisiae maintenance of the bivalent (probably by terminalization of the , stabilising yeast plasmids during mitosis and meiosis and also chiasmata). Notably, it is not always the same end to be used lowering the plasmid copy number (8). Taking into account the for each function (Albertson personal communication). Some non-uniform distribution of Cerep3 elements in C.elegans , it mutations, that affect meiotic disjunction, have been shown to is suggestive to imagne that they might play a role in segregation have recombination abnormalities suggesting that exchange may in this organism. be necessary for proper disjunction in C.elegans as well as in Internally located telomeric sequences in the germ-line other organisms (24, 25). We observe a positive correlation chromosomes of Tetrahymena mark the ends of transposon-like between abundance of interstitial telomeric repeats, and elements (31). The authors suggest that transposition of these enhancement of recombination along the chromosomes (see figure elements involves a linear form and that telomeric repeats are 7). Telomeric repeats have been shown to be highly added to the ends of these free linear elements. Transposons recombinogenic in other organisms (26, 27, 28, 29). In bounded by C4A4 telomeric repeats were also found in

Saccharomyces cerevisiae , for example, telomeric repeats moved Oxytricha fallax (22) and shown to excise precisely during inside the chromosome create a hot-spot of recombination (Petes, macronuclear development (23). RcS5 might be a vestigial mobile personal communication). This observation suggests that the element of the kind observed in protozoa and the cause of occurrance of stretches of degenerate telomeric hexamers, interspersion oftelomeric repeats in the C. elegans 'chromosome

invading the last 30% of the chromosomes in C elegans , might arms'. Once moved inside the chromosome, telomeric repeats account for the increase in the recombination frequency in the might have been furtherly amplified by gene conversion, unal- 'chromosome arms'. In this perspective these DNA elements crossing over or DNA polymerase slippage (32, 33, 34). might play an important functional role in chromosome Alternatively, the amplification of telomeric repeats in the disjunction favouring the formation of sub-terminal chiasmata chromosome arms has independently occurred and the putative during meiosis. Middle repetitive DNA families, containing transposon has acquired the hexamer TTAGGC as preferential several TTAGGC repeating units, have been detected in Ascaris target site of integration. Nucleic Acids Research, 1993, Vol. 21, No. 5 1139 ACKNOWLEDGEMENTS We are grateful to Clara Frontali, Alan Coulson and John Pulitzer for stimulating discussions and helpful suggestions concerning the manuscript. We thank Donna Albertson, and Chantal Wicky for suggestions and for sharing informations prior publication. We thank Scott Emmons for the Cerep3 plasmid, and L.W.Nawrocki for non-C. elegans nematode DNAs. Some nematode strains used in this work were 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 Contract SC 1 *-CT9 1-0633, CNR Progetto Speciale Bioinformatica and CNR Target Project Biotechnology and Bioinstrumentation.

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