Volume 1 1 Number 5 1983 Nucleic Acids Research Volume 11 Number 5 1983 Nucleic Acids Research Repetitive satellite-ike sequences are present within or upstream from 3 avian protein-coding genes L.Maroteaux, R. Heilig, D. Dupret and J.L. Mandel Laboratoire de Genetique Mol&culaire des Eucaryotes du CNRS, Unite 184 de Biologie Moleculaire et de Genie Genetique de l'INSERM, Faculte de M&decine, 11 Rue Humann, 67085 Strasbourg Cedex, France Received 17 January 1983; Accepted 9 February 1983 ABSTRACT. Peculiar DNA sequences made up by the tandem repetition of a 5 bp unit have been identified within or upstream from three avian protein-coding genes. One sequence is located within an intron of the chicken "ovalbumin-X" gene with 5'-TCTCC-3' as basic repeat unit (36 repeats). Another sequence made of 27 repeats of a 5'-GGAAG-3' basic unit is found 2500 base pairs upstream from the promoter of the chicken ovotransferrin (conalbumin) gene. A related but different sequence is present in the corresponding region of the ovotransferrin gene in the pheasant, with 5'-GGAAA-3' as the basic unit (55 repeats). These three satellite-like elements are thus characterized by a total assymetry in base distribution, with purines restricted to one strand, and pyrimidines to the other. Two of the basic repeat units can be derived from the third one (GGAAA) by a single base pair change. These rela- ted sequences are found repeated in three avian genomes, at degrees which vary both with the sequence type and the genome type. Evolution of tandemly repeated sequences (including satellites) is in general studied by analysing randomly picked elements. The presence of conserved protein-coding regions neighbouring satellite-like sequences allow to follow their evolution at a single locus, as examplified by the striking comparison of the pheasant and chicken sequences upstream from the ovotransferrin gene. INTRODUCTION Families of middle or highly repetitive sequences have been found in all eucaryotic genomes [and even in archaebacteria, (1)]. Some of these sequences are interspersed with unique sequences ; the Alu type sequences for instance can be found in the vicinity of expressed genes, or even within a transcription unit (2, 3, 4). Other families appear in the genome as long tandem repeats which can confer characteristic physical properties to the DNA, allowing their isolation as satellite components by buoyant density centrifugation. Some of these satellite DNAs are characterized by very short repeat units (5-10 bp) while others have a much longer basic element which in some cases contain hidden internal homologies (see ref. 5 for a review). Satellite sequences have been localised, by in situ hybridization, to hete- rochromatic regions (as in Drosophila) including centromeres and telomeres, © I R L Press Limited, Oxford, England. 1 227 Nucleic Acids Research and it is generally assumed that these sequences are not transcribed. It has been proposed that interspersed repetitive sequences play a role in specific gene regulation (6) and more recently attention has been focused on their possible mobility within the genome (7). Satellite sequences on the contrary have been thought to play a structural role in chromosome organization and in chromosome mechanic [pairing, recombination, etc. (see ref. 8)]. The presence of repetitive sequences has been demonstrated in the vici- nity or within some of the genes coding for egg white proteins in the chi- cken : ovotransferrin (9), lysozyme (10), ovalbumin X and Y genes (11, 12), ovomucoid (Gerlinger,personal communication). All these genes are expressed in the oviduct under similar steroid hormone control. It was thus interes- ting to look for possible similarities in the organization of such repeti- tive sequences, and in their localization with respect to functional parts of the genes or of the active chromatin domain (13, 14). We report here the presence of sequences constituted by tandem repeats of a 5 bp unit, 2.5 kb upstream from the chicken and pheasant ovotransferrin genes and within an intron of the ovalbumin-X gene, which corresponds to some of the repetitive sequences previously identified. Each of these satellite-like elements has a characteristic repeat unit: two of these units (GGAAG and GGAGA) can be derived from the third one (GGAAA) by a single nucleotide change. MATERIALS AND METHODS. Clones and DNA sequencing. For analysis of sequences upstream from the chicken ovotransferrin gene, we subcloned a 1.5 kb EcoRI-PstI fragment derived from the 4 kb "Ecob" fragment (9, see Fig. 1A, line a). A 6 kb EcoRI fragment containing the first exon and upstream sequences from the pheasant ovotransferrin gene was isolated after screening, with a chicken ovotransferrin cDNA probe, of a pheasant erythrocyte DNA library in bacteriophage x (Dupret et al., in pre- paration). A 950 bp PstI fragment containing the repetitive sequence was subcloned in pBR322 and used for sequence determination (see Fig. 1B, line b). The 1.7 kb and the 4.4 kb EcoRI subclones of the ovalbumin-X gene have been described previously (11, see Fig. 1C, line a). DNA sequencing was performed according to the Maxam and Gilbert proce- dure (15) using 5' end-labelled restriction fragments which had been in general submitted to strand separation by polyacrylamide gel electrophoresis as described previously (16). 1228 Nucleic Acids Research Hybridization experiments. Fragments of cloned DNA were immobilized on DBM paper according to a modification (17) of the method of Alwine et al. (18) and hybridized to 150 ng of total nick-translated cellular DNA (specific activity 1.4 108 cpm/pg) for 18 h at 42°C in 6.4 ml of 40 % deionized formamide, 40 mM phosphate buffer (pH 6,5), 0.7 M NaCl, 2 mM EDTA, 1 x Denhardt Solution, 8 % dextran sulphate (19), containing 50 jg/ml of sonicated heat-denatured salmon sperm DNA. Filters were washed stepwise under three conditions of increasing stringency 200C 0.5 x SSC - 0.1 % SDS ; 68°C 2 x SSC - 0.1 % SDS and 68°C 0.5 x SSC - 0.1 % SDS. After each washing step the filters were exposed to Fuji RX films at -80°C, with a Philips Ultra-S intensifying screen. RESULTS Location of repetitive sequences upstream chicken and pheasant ovotransfer- rin genes. Previous study of the cloned EcoRI "b" fragment containing the first exon from the chicken ovotransferrin gene (and upstream sequences) had de- monstrated the presence of a sequence highly repeated in the chicken genome, in a region located 2000 to 3200 bp upstream from the 5' end of the gene, corresponding to the fragment "Mbo a" (9, see Fig. 1A, line d). In the nei- ghbouring "Mbo b" fragment a sequence is located which cross-hybridizes to intron B of the same gene and which shows a lower degree of repetition in the chicken genome (9). We have performed a more precise localization of the two repetitive sequences by blotting onto DBM paper of restriction digests of a plasmid containing the 1.5 kb EcoRI-PstI fragment and hybridizing them to nick-translated total cellular chicken DNA. This allowed to map the high- ly repetitive sequence (labelled S in Fig. 1A and 5A1) within a 190 bp MspI-MboII fragment, and the other repetitive sequence to the adjacent 180 bp MboII-KpnI fragment (labelled R in Fig. 1A and in Fig. 5A1, lane 3). The ovotransferrin gene from pheasant was cloned in order to study its structure and compare it to that of the chicken gene (Dupret et al. in pre- paration). The first exon of the gene is contained within a 6 kb EcoRI frag- ment. Using the experimental design outlined above, we found repetitive sequences within the 620 bp PstI-SphI fragment located 2300 bp upstream from exon 1 (Fig. 1B and Fig. 5B, lanes 5 to 7). Location of repetitive sequences within introns of the chicken "ovalbumin-X" gene. We have previously shown that introns A and C of the chicken "oval- 1229 Nucleic Acids Research 0 1kb transcription El EJa S.~~~~~~R PI A t_ b c t za t b d 111j e L.~ E 1 E . ' H ii I .1 1? n | I II ITn.c IIIe E E E|tRA i B | C UH D | E Ia C * r '~ ~~ 11 1I . 6t' ? . c II1 1I 1111i1 I .d IJ f Figure 1 Maps of the three genomic regions. Satellite-like sequences (S) are ind cated by the hatched areas. The dotted lines correspond to other repetitive sequences (R). Exons (numbered 1, 2, etc.) are represented by black boxes. The arrows above the S regions indicate the direction and ex- tent of sequence determinations. The thick arrows correspond to the auto- radiograms presented in Fig. 2. A) Chicken ovotransferrin gene. Line a: map of the 4 kb EcoRI fragment (fl, E2) and of the 1.5 kb EcoRI (El, P1) fragment used in this study. Lines b to d: location of repetitive sequences by blot-hybridization with nick-translated chicken DNA (lines b to d corres- pond to lanes 1 to 3 in Fig. 5). The plasmid containing the Ecol-Pstl frag- ment was digested by EcoRI + PstI + MspI (?) (b), by KpnI + AvaI (c) and by EcoRI + PstI + MboII (t) + HhaI (d). The MboII fragments "a" and "b" contain the S and the R elements respectively (see text). Strongly hybridizing frag- ments are indicated by heavy lines, weaker signals by thin lines. Line e resulting location of S and R elements. B) Pheasant ovotransferrin gene line a : Map of the 6 kb EcoRI fragment cToned from pheasant DNA (D. D. et al., in preparation). Lines b to d : location of the repetitive element by blot hybridization with nick-translated pheasant DNA, (lines b to d corres- pond to lanes 5 to 7 in Fig.