Gene. 134(1993)175~181 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 175 zyxwvutsr GENE 07423 A molecular and cytogenetic analysis of h20p7 fragment DNA from the proximal P-heterochromatin of Drosophila melanogaster (Recombinant RNA; genomic clone; polytene and metaphase chromosomes, in situ hybridization; pericentric heterochromatin; nuclear lamina) S.I. Baiborodin, E.M. Baricheva, S.S. Bogachev, I.V. Borisevich, O.V. Strotz, M-A. Filippova, 1-V. Sharakhova and A.C. Shilov Institute ~fCjto&y and Gwwtiw Siberian Branch of t/w Russiun Academy ofSciences. Nooosihirsk 63OWO. L~went’rv pr, 10, Rus,yi[l: und aReseurt,fi Institutr of’ Biology and Biophysics. Tomsk Stutr Unioersity, Tomsk, Russirr. Tel. ( 7-8382) 29091 I2 Received by G. Bernardi: 27 February 1993; Revised/Accepted: 15 May/23 May 1993; Received at publishers: I6 July 1993 SUMMARY A DNA fragment from the ~roso~~~l~ ~~~~~nogus~er genome, cloned in h20p7, was derived independently from clones h20 and hL [Baiborodin et al., Genetika 29 (1993) 403.---416; Sharakhov et al., Genetika 29 (1993) 392-4021. In situ hybridization of h20p7 DNA to the chromosomes of D. melanogaster demonstrated preferential hybridization of the fragment to the chromocenter of polytene chromosomes and to pericentric heterochromatin of chromosomes II, IV and X at the metaphase plate. Copy number per haploid genome for h20p7 was estimated as approximately 200. Based on Southern blotting, the major portion of this moderate repeat was localized in the region of a 5.5-kb Hind111 digest. In situ hybridization to polytene chromosomes from strainfi( 2 )B trophocytes revealed that repeats homologous to h20p7 are located in the proximal heterochromatin which undergoes structural reorganization during tissue differentiation. The nucleotide sequence of two segments of the clone h20p7, Dm0.9 and Dm270, was determined. Sequence analysis of the 300-bp Dm0.9 clone revealed that it contains Zl-bp and 30-bp d(GT\CA) sequences, a 12-bp AT box, recognition sites for nuclear factors NFI and SpI, and a set of inverted repeats. Clone Dm270 contains an open reading frame (ORF). The deduced amino acid (aa) sequence shares homology with the gag-like gene from type-1 (Rl) ribosomal DNA insertion and may code for a polypeptide of 10 kDa. The Dm270 sequence was found to contain two direct repeats showing homology to the human CENP-B box, INTRODUCTION The pericentromeric heterochromatin of Drosophiln rne~~~og~ster contains two large groups of repeats which form a- and ~-heterochromatin. Their general organiza- tion is thought to determine the different functional roles they play in the chromosome (Heitz, 1934). In D. melanogaster cl-heterochromatin blocks, which may be up to 1000 kb in length, contain satellite sequences re- peated in tandem arrays. These satellites are highly un- dcrreplicated in the polytene nuclei and they occur in the pericentric heterochromatin of mitotic chromosomes, similar to what has been found in other species (Pardue et al., 1971; Brutlag et al., 1978; Peacok et al., 1978; Lohe Correspondence to: Dr. S. Bogachev at his present address: School of Medicine, Department of Pharmacological Sciences, HSC BST 8-140. SUNY at Stony Brook, Stony Brook, NY 11794-8651, USA. Tel. (1-516) 444 3079; (l-516) 4443067; Fax (1-516) 444-3218; e-mail: SERGEI@pharm.som.sunysb.edu Abbreviations: aa, amino acid(s); bp, base pair(s); CENP, centromeric protein(s)-binding region; D., Drosophila; kb, kilobase or 1000 bp; LTR, Iong terminal repeat; NL, nuclear lamina; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylarnide gel eiectrophoresis; r, ribo- somal; SDS, sodium dodecyl sulfate: SSC, 0.15 M NaCl/0.015 M Na,eitrate pH 7.8.