A Drosophila ribosomal protein gene is located near repeated sequences including rDNA sequences

Volume 10 Number 19 1982 NucleJC A c i d s Research A DrosopMla nbosomal protein gene is located near repeated sequences including rDNA sequences Steven Fabijanski and Maria Pellegrini Molecular Biology Section, University of Southern California, Los Angeles, CA 90007, USA Received 17 June 1982; Revised and Accepted 23 August 1982 We have isolated a cloned segment of Drosophila genomic DNA containing a ribosomal protein gene. Hybridization analysis of the DNA in this clone indicates a complex organization of repeated elements within this cloned segment. At least one of these repeated elements is homologous to regions of rDNA. Restriction analysis of the clone shows that some of the repeated elements are present as tandem duplications and in scattered locations within the cloned DNA segment. There are also three non-ribosomal protein genes contained in this clone, each of which is expressed along with the ribosomal protein gene into RNA species present in Drosophila embryos . INTRODUCTION Recently, cloned DNA segments coding for ribosomal protein genes in various organisms have been described (1-6). These include mouse, yeast, Xenopus and Drosophila• We have recently reported the isolation of a ribosomal protein gene-containing clone from a Charon 4 (Ch4) library of Drosophila genomic DNA. This cloned DNA contains a single ribosomal protein gene and at least one sequence present in 5 other ribosomal protein mRNAs (7). This paper details the organization and presence of a variety of repeated elements in this cloned DNA segment. MATERIALS AND METHODS DNA Isolation, Restriction Analysis and Hybridization Reactions. DNA from clone 2 (C2) was isolated as previously described (7). The 17 Kb insertion of pDmlO3 (Dml03'), containing a ribosomal RNA repeat unit with a Type I insertion in the 28S gene (8), was cloned in pACYC184 and was generously provided by Drs. D. Fouts and J. Manning. © I R L Pre» Limited, Oxford, England. 0305-1048/82/1019-597982.00/0 5979 ABSTRACT Nucleic Acids Research Restriction enzymes were obtained from Bethesda Research Labs, Inc. Restriction digests were separated on agarose gels in 40 mM Tris base, pH 8.0, 5 raM sodium acetate, 1 mM EDTA. The buffer was recirculated during electrophoresis. DNA was transferred to nitrocellulose using the procedure of Southern (9). DNA fragments were isolated from gels using the Nal procedure of Vogelstien and Gillespie (10). DNA was labelled with [32P]a-dCTP using the nick translation kit available from New England Nuclear . Electron Microscopy. For R-loop analysis, cloned DNA was incubated with cytoplasmic RNA or poly(A)+ RNA from Drosophila embryos (7) under conditions described by Woolford and Rosbash (12). The DNA concentration was 20 - 30 iig/mL and the RNA concentration was 500 ng/mL. The R-looped DNA was separated from the non-hybridized RNA by Sepharose column chromatography and directly spread for electron microscopy (12). The plasraid pBR322 was used as an internal length standard. RESULTS Repeated Elements in Clone 2 DNA Preliminary restriction analysis of C2 by hybridization of nick-translated restriction fragments to blots of various restriction enzyme digests of C2 indicated a complex arrangement of homologous sequences were present in this cloned DNA. 5980 Therefore, Hybridizations were carried out according to the procedure of Wahl et al. (11), with the following modifications. The prehybridization mixture contained 50% formamide, 0.8 M NaCl, 3X Denhardt's solution, 0.1 M Pipes, pH 6.4, 0.01% N-laurylsarcosine, and 500 iig/mL denatured, sonicated calf thymus DNA. The hybridization mixture was the same as the prehybridization mixture, except that it contained, in addition to probe, 10% dextran sulfate. Following hybridization, DNA blots were washed in 2 changes of 2X SSC, 0.01% NaPPi, 0.01% N-lauryl-sarcosine, each of 500 ml, at room temperature for 10 minutes, then in 0.2X SSC, 0.01% NaPPi, 0.01% N-lauryl-sarcosine at 60°C, 4 changes each of 1 liter for 2 hours. The blots were exposed to XAR-5 Xray film at -70*C with an intensifying screen. Nucleic Acids Research Kb ^ ^ 10 $EcoRl 1 20 ' 22 PBomHI r 24 •Hindm 26 28 $SstI 30 32 J Pstl 34 40 45 ABflHI it was necessary to determine the pattern of restriction sites by both single and double digestions of isolated restriction fragments . These data were used to construct the restriction map shown in Figure 1. Not all the Pstl sites were localized, only those within the 2.6 and 4.1 Kb BamHl fragments. There is a region of DNA in the middle of this clone which contains a series of Hindlll and Pstl sites separated by spacings of approximately 250 and 500 bp, respectively. These repeats are not exact, however, since the lengths of these units show some heterogeneity as judged by their gel patterns and the appearance of other non-repeated restrictions sites in this area of the DNA. The exact amount of this heterogeneity was not determined in these experiments. In order to further investigate the amount of repeated DNA in C2, nick translated C2 DNA was used to probe an EcoRl blot of total Drosophila DNA. Large amounts of hybridization were seen to several regions of the blot (data not shown), showing that sequences in C2 are repeated in the Drosophila genome. To determine if these repeated sequences were localized to any one region of C2 DNA, restriction digests of C2 DNA were blotted to nitrocellulose and probed with nick translated Drosophila total 5981 Figure 1. Restriction Analysis of Clone 2 DNA. Only those restriction sites within the 14.4 Kb inserted DNA are shown. Not all Pstl sites are shown, only those within the 4.1 Kb and 2.6 Kb BamHl fragments. Position of the EcoRl sites was determined by EcoRl digestion of isolated BamHl fragments. Positions of the Sstl and BglII sites were determined by measuring fragment lengths of digested C2 DNA, and positioning these fragments using the known sites in Ch 4. Positions of some of the Hindlll and BamHl sites were determined in the same way. Other sites were located by secondary digestion of isolated restriction fragments with the appropriate enzymes. The bars below the restriction may represent regions at least partially homologous to Dm 103'. Nucleic Acids Research genomic DNA. In this type of experiment, only those C2 DNA segments which contain an element repeated in the Drosophila genome will show significant hybridization (see legend to Fig. 2 ) . When single restriction digestions of C2 are analyzed using this technique, all inserted DNA fragments show hybridization, indicating that repeated elements are present throughout the inserted DNA. Scattered locations of homologous repeat units in C2 DNA. When nick translated individual restriction fragments of C2 DNA are used to probe total restriction digests of C2 DNA, hybridization of a single C2 fragment was observed to many other C2 fragments which are widely separated in the clone . For example, when the 4.4 Kb (...truncated)