Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5814-5818, July 1991

Sequence conservation in avian CR1: An interspersed repetitive DNA family evolving under functional constraints (molecular evolution/avian genome organization) Z.-Q. CHEN*, R. G. RITZEL*, C. C. LIN*t, AND R. B. HODGETTS* *Department of , University of Alberta, Edmonton, AB, T6G 2E9, Canada; and tDepartment of Pathology, University of Alberta, Edmonton, AB, T6G 2R7, Canada Communicated by Roy J. Britten, April 3, 1991 (receivedfor review September 11, 1990)

ABSTRACT CR1 is a short interspersed repetitive DNA various positions, reminiscent of the Li family in mammals element originally identified in the domestic chicken (Galus (11). Also reminiscent of L1, the CR1 unit characterized in gallus). However, unlike virtually all other such sequences ref. 12 has an open reading frame. However, unlike the Li described to date, CR1 is not confined to one or a few closely elements, whose 3' ends usually terminate with a poly(A) related species. It is probably a ubiquitous component of the tract, the 3' end of most CR1 units is a tandemly duplicated avian genome, having been detected in representatives of nine octamer. Recently, it was found that a CR1 member present orders encompassing a wide spectrum of the class Aves. This in the chicken vitellogenin (qiVTIII) was absent identification was made possible by using the polymerase chain from the ancestral VTIII . This fact, taken together with reaction (PCR), which revealed interspecific similarities not the observed duplication of target sequences, has prompted detected by conventional Southern analysis. DNA sequence the speculation that CR1 is a novel family (12). comparisons between a CR1 element isolated from a sarus crane Additional functional properties have been ascribed to (Grus antigone) and those isolated from an emu (Dromaius CR1. Chromatographic columns containing CR1 DNA were novaehollandiae) showed that two short highly conserved regions used to isolate specific nuclear from chicken oviduct are present. These are included within two regions previously tissue, and the fractionated proteins were found to bind at a characterized in the CR1 units of domestic fowl. One of these 3' region of the CR1 sequence (13). Second, in dissecting the behaves as a transcriptional silencer and the other is a binding upstream region of the chicken lysozyme gene, Baniahamad site for a nuclear . Our observations suggest that CR1 has et al. (14) used a reporter gene to define a silencer element in evolved under functional constraints and that interspersed re- the central portion of a CR1 member found here. petitive sequences as a class may constitute a more significant Preliminary evidence has shown that the CR1 family exists component of the eukaryotic genome than is generally acknowl- in duck and peacock (15), and in this paper we provide edged. evidence that the CR1 DNA family exists in a wide range of avian genomes, including species as diverse as emu and Repetitive DNA families have proved to be useful for studies black-billed magpie. The persistence of CR1 throughout on molecular evolution (1). In the pursuit ofour interest in the avian evolution argues strongly for a functional role, and this repetitive component of the avian genome, we have de- is supported by our finding that two regions are highly scribed and speculated about the function of a tandemly conserved: one known to interact with a nuclear protein(s) repeated, centromeric DNA family in cranes (Gruidae) (2). In and the other known to contribute to the regulation of gene the present study, we explore the characteristics of an expression. interspersed repetitive DNA family. The eukaryotic inter- spersed DNA families studied to date appear, with only one exception [the L1 family found in a wide range of mammalian MATERIALS AND METHODS species (3)], to be confined to closely related species. This Specimens. Avian species used in this study included emu narrow distribution of interspersed DNA families has led (Dromaius novaehollandiae), southern cassowary (Casuar- some to speculate that they make up a nonfunctional ius casuarius), American white pelican (Pelecanus erythro- ("junk") component of the genome (4, 5). However, since rhynchos), cormorant (Phalacrocorax auritus), white stork their discovery by Britten and Kohne (6), many functional (Ciconia ciconia), Andean condor (Vultur gryphus), Austra- roles have been suggested, most notable of which are those lian shelduck (Tadorna tadornoides), Japanese quail involved in gene regulation (7, 8). If any functionally signif- (Coturnix coturnix japonica), sarus crane (Grus antigone), icant, interspersed DNA families exist in birds, we hypoth- long-eared owl (Asio otus), and black-billed magpie (Pica esized that they should be highly conserved. Therefore, we pica). Blood samples from these birds were kindly provided undertook a screen of the crane genome, hoping to identify by R. M. Cooper and M. Mainka (Calgary City Zoo, Alberta, repetitive sequence elements that were present in many other Canada). Genomic from alpine newt (Triturus alpes- avian species. The first clone we identified: turned out to be tris), African clawed frog (Xenopus laevis), goldfish (Caras- a member of the interspersed repetitive DNA family CR1, sius auratus), lobster (Homarus vulgaris), mouse (Mus mus- discovered by Stumph et al. (9) in domestic fowl (chickens, culus), and normal human leukocytes were kindly provided Gallus gallus). by R. Sasi (Department ofPathology, University ofAlberta). The chicken CR1 family has been the subject of extensive DNA Extraction and Southern and Dot-Blot Hybridization. study. It is composed of approximately 7000-20,000 repeats The protocol for extracting genomic DNA from avian blood per haploid genome, the lengths of individual members samples and the DNA hybridization conditions have been varying from 160 to 850 base pairs (bp) (9, 10). Interestingly, described (2). the 5' ends of the known CR1 members are all truncated at Isolation ofConserved Repetitive DNA Families. A library of EcoRI restriction fragments from genomic DNA of the sarus The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" fThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M68957 and M68958).

5814 Downloaded by guest on September 25, 2021 Evolution: Chen et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5815 crane was constructed in the vector pUC19. The bacterial a E P P V H V E strain JM83 was transformed with a sample ofthe library, and sc1o L1. I transformants that contained repetitive DNA sequences were I identified by the intensity of their signals after colony hy- /f 1 kb bridizations with crane genomic DNA. The repetitive nature V I N S X V of these clones was confirmed by isolating and labeling the m. %a plasmids and hybridizing them to Southern blots of restric- SC105

tion endonuclease-digested crane genomic DNA. The extent W.. of sequence conservation was determined by dot hybridiza- tion of labeled clones to a test' panel of DNA samples from a pelican, condor, crane, chicken, owl, magpie, mouse, and human. b 1 2 3 PCR and Subsequent Cloning. The primers used for the PCR 'were 5'-CAGGACAAGGGGTAATGGGT-3' and 5'- kb CATAGAATGGTTTGGGTTGG-3'. Reaction mixtures con- 4.8- 43 tained 100 ng of each primer, 30 ng ofthe template DNA, and 1 unit of Taq DNA polymerase (Bio/Can Scientific, Missis- 2.8-_ saugua, ON, Canada) in a buffer containing 50 mM Tris HCl at pH 9.0, 10 mM MgCl2, 15 mM (NH4)2SO4, 0.1 ofbovine FIG. 1. (a) Restriction maps of Itg SC10 and SC105. The restriction serum albumin, and 2 mM each of the four deoxyribonucle- enzymes used were EcoRI (E), oside triphosphates. The reactions were carried out in an . Pst I (P), Pvu 11 (V), HindIII (H), automated thermocycler (Tyler Research, Edmonton, AB, Sau3a (S), and Nco I (N). The Canada). Initial incubation was at 950C for 4 min, followed by shaded box indicates the CR1 el- 16-30 cycles at 60'C for 1 min, at 730C for 3 min, and at 91'C ement. Lines beneath the SC105 for 5 sec. Aliquots of the amplification reaction products map show the sequencing strategy were mixed with 50o (vol/vol) glycerol and loaded onto 2% employed. (b) Autoradiographs of agarose gels. After electrophoresis, the products were Southern hybridizations of Hin- stained and visualized under UV light and photographed. dIII-digested crane genomic DNA an of the (5 ,g) probed with 32P-labeled Pst When subsequent cloning was undertaken, aliquot I fragment (lane 1), Pst I/Pvu II PCR mixture was incubated briefly with T4 DNA polymerase fragment (lane 2), and Pvu II frag- and additional nucleotides to blunt the ends of the fragments ment (lane 3). (16). Afterwards, the fragments were purified and concen- trated with glass powder (Geneclean; Bio/Can Scientific) and quences stored in the MicroGenie (Beckman) data bank, then ligated into blunt-ended pUC19 vector DNA and used to which revealed substantial relatedness between this DNA transform DH5a competent cells (GIBCO/ sequence and members of the CR1 family. A comparison of BRL). the consensus DNA sequence of the chicken CR1 family DNA Sequencing. Cloned plasmids were isolated from 1-ml members (18) with the analogous sequence of the crane cultures of their bacterial hosts (17), and a 17 DNA poly- repetitive element is shown in Fig. 2a. There is =80% identity merase sequencing kit (Pharmacia) was employed for double- between these two sequences, over about 300 bp. The stranded DNA sequencing. The 35S-labeled reaction products differences between the two' sequences were primarily small were fractionated on 6% acrylamide gels under denaturing deletions and base substitutions. conditions and visualized by autoradiography of the dried Conservation of the CR1 Homologous Sequence in Other gels. Avian Species. To determine the extent of conservation ofthe CR1 sequence in birds, SC105 was labeled and hybridized to RESULTS a Southern blot containing EcoRI-digested genomic DNAs from emu, cassowary, pelican, stork, condor, quail, crane, Isolation and Restriction Mapping of SC10. From the plas- detected mid library of crane DNA, a repetitive clone designated as owl, and magpie. As shown in Fig. 3a, the probe SC10 was selected for study because it showed DNA se- smears of different intensity in each of the samples, except quence similarity to all six avian DNA samples but did not for the emu and cassowary, where hybridization was barely hybridize to any of the mammalian samples used in the test detected. A very pronounced band, about 2.8 kb, was de- pane! (data not shown). SC10 is a 4.6-kilobase (kb) fragment tected in stork DNA, suggesting that a significant number of of crane DNA, the repetitive component of which was the CR1 units are clustered in a tandem array in the genome determined from the restriction analysis shown in Fig. la. of this species. The same blot was stripped and rehybridized Individual restriction fragments isolated from SC10 were with the HindIII/EcoRI fragment of SC10, which does not used as probes for Southern hybridizations with HindIII- include any CR1 sequence (see Fig. la). The discrete bands digested crane genomic DNA. As shown in Fig. lb, the Pst observed (Fig. 3b) prove that the smears seen in Fig. 3a were I and Pvu II fragments hybridized to a corresponding band or not due to incomplete digestion or degradation of the.DNA bands on the Southern blots (lanes 1 and 3, respectively), samples. When the SC105 probe was hybridized to a South- indicating their single-copy nature. By contrast, the Pst ern blot containing human, mouse, newt, frog, and fish DNA I/Pvu II fragment hybridized to similar sequences throughout samples, no related DNA sequence was detected in any of the genome (lane 2). These results demonstrated the inter- these cases (data not shown). spersed nature of the repetitive DNA in clone SC10 and The failure to detect cross-hybridization between the crane suggested that an entire repeat unit was confined to the Pst CR1 repeat and a comparable sequence in either the emu or I/Pvu II fragment. This fragment, SC105, was isolated and cassowary might simply reflect the high stringency of the hy- ligated into Pst I/HincII-digested pUC19 for subsequent bridization conditions (2). Since low-stringency conditions using sequencing. repetitive DNA probes are liable to give false positives, we felt DNA Sequencing of the Repetitive Unit in SC10. The se- that the application ofPCR might reveal the presence ofthe CR1 quencing strategy carried out on SC105 is summarized in Fig. family in these distantly related birds. Two oligonucleotide la. The DNA sequence obtained was compared with se- primers identical to sequences near each end of the crane CR1 Downloaded by guest on September 25, 2021 5816 Evolution: Chen et al. Proc. Natl. Acad. Sci. USA 88 (1991) a 200 210 220 230 240 250 the amplification products were examined on 2% agarose gels. As in CHICK TTGAAGTTGAAGGAGGGAAGATTNAGGTTGGATATCAGGGGGAAGTTCTTTACTATGA shown Fig. 4, all the avian samples (lanes 2-11), including CRANE ..-.....C. TC.. . .T..A.....G.T . AA .. .A...CA. ..G the emu and cassowary, had a predominant band ofabout 250 bp EMU ACA. .-C...... AC.CA.G.A ... CCATC . A.Y G. .AAA.C.. G... and several other discrete bands. The four nonavian samples, 260 270 280 290 300 310 three vertebrates and an invertebrate, all lacked the major band GAGTGGTGAGGTGCTGGAACAGGCTGCCCAGAG^AGTGTGGATGCCGTCCATCCCTGGAGGTG in .G . CA . CAC ..T .-.G.G ...... A... (lanes 12-15). Larger amplification products addition to the .G.. TGAACA . T.GT.--.... T.A.A major band were produced in the avian samples as well as in the 320 330 340 350 360 370 380 nonavian samples. For the emu, three clones from this popula- TTCAARGCCAGGYTGGATGGGGCCCTGGGCAGCCTGGTCTAGTAYTGTGTGGAGGYTGATGGCC tion were analyzed and shown to contain CR1 (see Fig. 5b). For .....G ...... T ...... A... TT ...... A....A...... AA. .CAAC.. ..CAC...... A...... GTGA.------the nonavian species, several explanations for the longer prod- ucts exist and further 390 400 410 420 430 analysis is required to distinguish among 1 them. CTGCCYATAGCAGGGGGGGGGGTTGAAGCTTGGTGATCC TTGAGGTCCCTT .....C..-... C.C .------. . .G.A..-.A ...... Conclusive evidence that the major bands seen in the avian .... .-TTG ...... G..Y. .A-A TCCA ------PCR smaples were genuine CR1 amplification products comes from Southern and DNA b hybridizations sequencing. 200 210 220 230 240 250 1 l When the DNA fragments in the emu's major band (as shown EMU C ACAAACTGAAACACAGGAAATTCCATCTGAAYATGAGG AAAAA CTTCTTT ACTGTGAG in Fig. 4) were labeled and used to reprobe the Southern blot 1 .. ... C.G ...T...... C. 2TG..T T...A. G...CT.A..G..C..A.A3.GT. presented in Fig. 3 a and b, a strong hybridization signal was 3 C.GG G. C.GA .....C T.-.

4 .G. GT ... T. ....C ...... seen in both the emu and cassowary lanes (Fig. 3c). Sequence 5 C..-G C...C..C..A ...C. .C.A...C. 6 G. T. I-.C.C T..A.C similarity was also detected between this emu probe and the 7 G.. A.. T. I-.C.C T..A..G... 8. other avian ... ----T .C. GT. species, with the exception of the magpie. The 9.... ----T .C. GT. CR1 similarity between the emu and the crane, shown in 10 GGCTTG.T.A .C..A..A ... C. A. C Fig. 11 GG. .CTTG.T.A .C..A..A ... C. A. C 3c, was not apparent in Fig. 3a. Thus, the CR1 element in SC10 has apparently diverged significantly from emu ele- 260 270 280 290 300 310 320 ments, although among the emu elements present in the GGTGGTTGAACACTGGAA CAGGTTGCCCAGAGAGGTTGTGGAGTCTCCATCCTTGGAGATATTCAA

.ACA. C.. ... A. G.T TC. PCR-generated probe there exist some with detectable sim- A.. .A.CT ...... A...... CA.CAC.A ...... TGA ...... C ilarity to some crane CR1 members. Since the emu ACA ...... A.C...... C .. .A.TC .--.. .A...... C... probe .AC-AG-. ..AA. T. represents only a subset of CR1 family members in the emu A..A. .TA.G A.. A. A CA.A. C.. .A.A- .T...C.. .C. T-C. C.... genome, the implication of its failure to cross-hybridize with CA.A. C.. .A.A- .T.. .C..C T-C. C...... T.GC. .T. G ..A.AA T. C.T.. sequences in the magpie DNA requires further study. .T.GC.T..G. A.TA T. C.T..

T.C. . A.A T-- T.C..TG ...A. ------.... TG. The mixture ofPCR-amplified fragments from the emu was T.C A.A ...A. T------.... TG. cloned in pUC19 and DNA sequences were obtained from 11 330 340 350 360 390 400 clones in the 250-bp range and 3 clones in the 600-bp range. AA CCCAACTGGACACGGTCCTGGGCAACCTGCTCTAGGTGA CCC TGCTTGAGCA GGGGGG Sequences of the crane and 250-bp emu clones were con- .... TG..A..A..T....TG. C.A. firmed on both strands, while sequences of the longer emu A.ACGA.CC-TGA------.C ...... ------GCTGT....A...TGC. .. T A. clones were obtained on one strand only. Fig. 2a compares .. G.GT G.AA. .TA ...... G ......

.AT.. .. A2 the A.A..A ---...... A2...... consensus of the 250-bp emu clones (1-11) with both the

.TTAC . T..A ...-. ...G. A..T A ..... crane and ..T chicken CR1. The emu consensus was derived .AC-. T..A ...G A ...... T...... G...... C...... T... A. from the individual sequences (1-11) shown in Fig. 2b...... 0...... G.C C...... T...... A. TGTG. T. G ... C-AGA2 ...... T. Among the emu clones, there exist three pairs of very closely TGTG.. C.. .AC.AGA2 T ...... T. related sequences: 6-7, 8-9, and 10-11. These may represent 410 420 430 pairs of very similar template elements. On the other hand, TTGGAYC AGATGATCTC CAGAGGTCCCTT we cannot rule out that disproportionate amplification of CT.. .T...... certain templates occurred. The slight differences between C...T ...... T...... pairs could then be ascribed to replication errors of the Taq ...... T. .G...... CGTA... . .A...... DNA polymerase (19)...... AT.C Fig. 5a illustrates schematically the longer emu clones C...... AT.C T. --...... T.C.. (clones 12-14), and their partial DNA sequence is presented :: C. C. T.. AT... in Fig. 5b, again in comparison with the crane and chicken C...... T... CR1. These clones are each about 600 bp in size. Though they all have a region of about 250 bp similar to clones FIG. 2. (a) Nucleotide sequences of CR1 elements. The CR1 1-11, consensus sequence of chickens (CHICK) is taken from ref. 18; N is priming has occurred within the CR1 region at only one of the any nucleotide, R is a purine nucleotide, and Y is a pyrimidine two sites. The cloned DNA fragments extend beyond the nucleotide. Differences from this consensus sequence as they exist mismatched sites to a suitable template in flanking DNA with in SC105 (CRANE) and in the consensus of 11 emu clones (EMU) are which the primer could form a stable hybrid. The regions in shown below; dots indicate identical residues. A gap has been these clones beyond the CR1 exhibit no similarity to each introduced into the chicken sequence to account for an extra other or to the crane or chicken CR1, indicating that the emu nucleotide present in the emu sequence, and nucleotides not present CR1 units were resident at different sites in the genome. in chicken are shown as -. Nucleotides are numbered according to In comparing DNA sequences of the emu CR1 elements ref. 18, and the DNA sequence shown lies between the primers used (clones 1-11) with the crane and chicken CRls (Fig. 2a), two to amplify the emu DNA (see Fig. 5b). The silencer consensus (284-292) and part of the putative binding domain of the nuclear highly conserved regions are evident: the first between protein (424-433) are underlined. (b) The nucleotide sequence of the positions 279 and 297 and the second between 424 and 433. 11 emu CR1 elements (1-11) used to derive the consensus (EMU C) Because the emu clones 12 and 13 were primed outside the 3' shown on the top lines. I represents an insertion of the sequence end of the CR1 element (Fig. 5a), the latter region of 5'-GGGAGGAAAAAAACAAACCCAAAACTGAGAAGCAC-3' conservation appears to extend to position 439 (Fig. Sb). after position 235. A3 and A2 represent AAA and AA. Elsewhere, substantial divergence has occurred in the form of base substitutions and, from about position 360 to 404, as sequence (see Fig. 5b) were synthesized. PCRs were performed deletions of nucleotides in the emu and crane elements on DNA samples of 10 avian species and 4 nonavian species, and relative to the chicken consensus. Downloaded by guest on September 25, 2021 Evolution: Chen et aL Proc. Natl. Acad. Sci. USA 88 (1991) 5817

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

FIG. 3. (a) Autoradiograph of a kb Southern hybridization of 32P-labeled SC105 with EcoRI digests of 5-pg ge- 4-- nomic DNA samples isolated from emu 3- (lane 1), cassowary (lane 2), pelican (lane 3), stork (lane 4), condor (lane 5), quail 2- (lane 6), crane (lane 7), owl (lane 8), and magpie (lane 9). (b) The blot in a was stripped and reprobed with the Hindlll! EcoRI fragment of SC1O, which does not I1- include the CR1 element. (c) The blot in .bS b was then stripped and probed with labeled DNA fragments isolated from the major band of the emu PCR product (see a c Fig. 4). DISCUSSION fiable template among the collection of CR1 units in the emu is very high, given the length of the two crane primers that We have applied PCR to reveal the interspecific conservation were used. By contrast, the average composition of the ofthe repetitive DNA family CR1. Our results, some ofwhich family members diverges so substantially between the crane were unexpected, point to PCR's potential use in the study of and the emu that no hybridization was observed in a Southern we have genome organization. For each ofthe avian samples blot using a single CR1 unit as a probe. These results lead us examined, the amplification products formed a pronounced to suggest that the interspecific conservation of interspersed 4, lanes 2-11). By contrast, this band 200-300 bp long (Fig. repetitive DNA families may be more widespread than pres- band was absent from the amplification products of the samples (lanes 12-15). The CR1-like templates in ently believed. nonavian A comparison ofthe CR1 sequences obtained from the emu the emu and cassowary DNA were not predicted from the genome with the ones obtained from the genomes of the Southern hybridization experiment (Fig. 3a), which failed to chicken and crane revealed two highly conserved regions detect cross-hybridization between these species and the within the CR1: a region between positions 279 and 297 and crane CR1 probe. Because of the discrepancy between the another between positions 424 and 439 (Figs. 2 and 5). By was to results from these two experiments, it essential contrast, the remaining part ofthe CR1 diverged substantially confirm that the amplification band from the emu and cas- between these species. It is unlikely that this high degree of sowary samples was indeed CR1 related. When the DNA sequence conservation occurred by chance alone in such in the major emu amplification band were labeled fragments distantly related species as the emu and chicken. In fact, it to a Southern blot that had previously been and used reprobe has already been pointed out that these regions were also well with the crane CR1 probe (Fig. 3a), both emu and hybridized conserved among the chicken CR1 members (9, 18). The cassowary lanes 1 and 2) showed a strong signal. (Fig. 3c, region between 284 and 292 matches a consensus silencer and This contrasts with the weak signal seen in these samples is included in a 172-bp restriction fragment upstream of the when the crane CR1 repeat was used as a probe. Second, the chicken lysozyme gene with demonstrable effects on tran- cloning and sequencing of individual amplified DNA se- scription: it exerts a strong repressing effect on weak pro- quences from the emu sample provide direct evidence that moters but has a weak effect on strong transcription units belong to the CR1 family (Figs. 2 these amplified products (14). The region between 424 and 439 is included in a and 5). somewhat larger domain that has been shown to bind a The failure of the Southern analysis (Fig. 3a) to detect nuclear protein of unknown identity (13). sequences in the emu sample is likely due to the CR1-related The presence of islands of conserved sequence within limited sequence identity between emu CR1 elements and the otherwise divergent sequences raises interesting questions crane CR1 probe. PCR, when compared with DNADNA about the function of this interspersed DNA family. Our data is clearly a more senstive method for detecting hybridization, show that the sequence conservation actually extends 5 bases interspecific sequence similarities. We think the reason for beyond either end of the silencer domain. This region bears this is that the probability of there being at least one ampli- no significant similarity to the binding domains of any eu- 1 2 3 4 S A 7 A Q In 11 4Kdo n 1A karyotic transcription factors characterized by Wingender (20). However, in searching the human DNA data base, a perfect match to the sequence 5'-GCCCAGAGAGG-3' was found within or near five . In two of these genes, encoding dopamine p-hydroxylase and prothrombin, the sequence occurs in the 5' untranslated leader; in the trans- forming growth factor-,8 gene, it lies in the 3' untranslated region; in the estradiol 17,8-dehydrogenase gene, it occurs in an intron; and in c-myc, it is located about 1.9 kb upstream ofthe gene. Whether or not these sequences actually serve as silencers remains to be established. In the light of current notions regarding silencers (21), we FIG. 4. An ethidium bromide-stained agarose gel (2%) on which feel that the likelihood of there being a protein that binds to aliquots of PCR products using various templates were loaded. Lane between the 1 contains 1-kb ladder DNA (GIBCO/BRL) as a molecular weight this region of CR1 is high. The slight differences marker. Lanes 2-15 contain samples obtained from, in order, quail, emu elements between 279 and 297 do raise the possibility shelduck, cormorant, cassowary, emu, condor, magpie, owl, peli- that the silencer may not be equally active at all chromosomal can, stork, newt, frog, goldfish, and lobster. locations of CR1. Indeed, if CR1 units are serving to regulate Downloaded by guest on September 25, 2021 5818 Evolution: Chen et al. Proc. Natl. Acad. Sci. USA 88 (1991)

a CHICKEN CRANE EMU 12 -C3...... = EMU 13 FIG. 5. (a) Schematic comparison of EMU 14 the chicken and crane CR1, with three emu clones (12-14) representative of the b 57 3' longer emu PCR products. The solid lines 178 262 301 delineate CR1 DNA. The broken lines indicate non-CR1 sequences. A hatched CHICK CAGGACAAGGGGGAATGG-T*****GAGGTGCTGGAACAGGCTGCCCAGAGAGGTT.GTGGATGCC box indicates sequences matching per-

. T CA CAC. .T CRANE G.*****...... G.-- fectly with one of the primers used for EMU12 . T G.*****TGAACA ...... T. ....GT.- whereas an box shows some EMU13 . T G.*****AGA.CA ...... T. .GT.PCR, open EMU14 A.C. .C. .G.*****AG ..CA.C T A...... GT.- divergence from the primer. (b) Partial DNA sequence of the CR1 elements, 417 5a showing the conserved domains between nucleotides 279 and 297 (which contain CHICK *****GATCC-TTGAGGTCCCTTCCAACCCAGGCCATTCTATG the underlined silencer consensus) and CRANE ***** ...... AA. between nucleotides 424 and 439. The EMU12 ***** .... TCCA TCA-.T.C-. .G.. extent of the primers is shown above the EMU13 ***** ...... -A TCA.A. G..

EMU14 ***** .... TCCA AA. sequence.

a battery of genes as suggested in an early model (8), it will Natural Sciences and Engineering Research Council ofCanada Grant be important to ascertain if the set of genes so regulated is (A6477) to R.B.H. even conserved between species. If this is so, interspecific 1. Britten, R. J. (1986) Science 231, 1393-1398. differences in the silencers at a given chromosomal location 2. Chen, Z.-Q., Lin, C. C. & Hodgetts, R. B. (1989) Genome 32, should be minimal compared to those seen when comparing 646-654. silencers at different sites. The association of transcriptional 3. Voliva, C. F., Jahn, C. L., Comer, M. B., Edgell, M. H. & silencers with an interspersed repetitive element has been Hutchison, C. H., III (1983) Nucleic Acids Res. 11, 8847-8859. reported for the LINE family in rats (22). The distribution of 4. Doolittle, W. F. & Sapienza, C. (1980) Nature (London) 284, transcriptional silencers throughout the genome on the ele- 601-603. 5. Orgel, L. E. & Crick, F. H. C. (1980) Nature (London) 284, ments of a repeated gene family may play a role in estab- 604-607. lishing the default mode ofchromatin, namely transcriptional 6. Britten, R. J. & Kohne, D. E. (1968) Science 161, 529-540. inactivity. 7. Britten, R. J. & Davidson, E. H. (1969) Science 165, 349-357. The second conserved region, located near the 3' end of 8. Davidson, E. H. & Britten, R. J. (1979) Science 204, 1052- CR1, is included in a region that binds a nuclear protein that 1059. W. may in If this is true, the 9. Stumph, W. E., Kristo, P., Tsai, M. J. & O'Malley, B. be involved transposition (12). (1981) Nucleic Acids Res. 9, 5383-5397. protein might be encoded by the open reading frame on the 10. Hache, R. J. G. & Deeley, R. G. (1988) Nucleic Acids Res. 16, putative master CR1 unit. Alternatively, a nucleoprotein 97-113. complex in the region could facilitate the down-regulation of 11. Singer, M. F. & Skowronski, J. (1985) Trends Biochem. Sci. 10, transcription in concert with the silencer. Or, finally, since 119-122. the nuclear protein that binds to this region has not been 12. Silva, R. & Burch, J. B. (1989) Mol. Cell. Biol. 9, 3563-3566. 13. Sanzo, M., Stevens, B., Tsai, M. J. & O'Malley, B. W. (1984) characterized, this DNA sequence could play a structural Biochemistry 23, 6491-6498. role in the organization of chromatin within the nucleus, 14. Baniahamad, A., Muller, M., Steiner, C. & Renkawitz, R. although we would exclude at present the matrix attachment (1987) EMBO J. 6, 2297-2303. sites because those described to date appear to be A+T-rich 15. Schip, F. V. H., Samallo, J., Meijilink, F., Gruber, M. & regions of DNA (23). Geert, A. B. (1987) Nucleic Acids Res. 15, 4193-4202. We propose that the conserved domains of each CR1 16. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular A Manual Harbor Cold member are due to functional constraints and that sequences Cloning: Laboratory (Cold Spring Lab., Spring Harbor, NY), p. 395. elsewhere diverge in the absence of selective pressures. We 17. Qi, W. (1990) MSc Thesis (University of Alberta, Edmonton, are encouraged to think that the ubiquity ofCR1 units in birds AB, Canada). signifies an important functional role and that a character- 18. Stumph, W. E., Hodgson, C. P., Tsai, M. J. & O'Malley, ization ofthe proteins that may bind to the conserved regions B. W. (1984) Proc. Natl. Acad. Sci. USA 81, 6667-6671. could reveal a more general role for the interspersed repet- 19. Dunning, A. M., Talmud, P. & Humphries, S. E. (1988) Nu- cleic Acids Res. 16, 10393-10394. itive component of eukaryotic genomes. 20. Wingender, E. (1988) Nucleic Acids Res. 16, 1879-1902. 21. Renkawitz, R. (1990) Trends Genet. 6, 192-197. We thank Drs. Curtis Strobeck, David Boag, and Bill Addison for 22. Laimins, L., Holmgren-Konig, M. & Khoury, G. (1986) Proc. their comments on this manuscript, Dr. Ken Roy for synthesis of the NatI. Acad. Sci. USA 83, 3151-3155. primers, and Dr. Frank Nargang and Laura Querengesser for assis- 23. Gasser, S. M., Amati, B. B., Cardenas, M. E. & Hoffmann, tance with the DNA searches. This study was supported by a Medical J. F. X. (1989) in IRC Reviews in Cytology, ed. Jeon, K. Research Council of Canada Grant (MA-5488) to C.C.L. and by a (Academic, New York), pp. 57-96. Downloaded by guest on September 25, 2021