The repeating nucleotide sequence in the repetitive mitochondrial DNA from a “low-density” petite mutant of yeast

Nucleic Acids Research Volume 4 Number 7 July 1977 The repeating nucleotide sequence in the repetitive mitochondrial DNA from a "low-density" petite mutant of yeast C. F. Van Kreijl and J. L. Bos Section for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Eerste Constantijn Huygensstraat 20, Amsterdam, Netherlands Received 4 May 1977 The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic p e t i t e mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were used. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly a l l of the mutant mitochondrial genome. The sequence, moreover, also represents the f i r s t part of wild-type mtDNA sequenced so far. INTRODUCTION Growth of y e a s t in the presence of i n t e r c a l a t i n g dyes l i k e ethidiura leads to the induction of rho cytoplasmic p e t i t e mutants. These mutants are unable to make functional mitochond r i a and t h e i r mtDNA i s e i t h e r completely l o s t or replaced by an equivalent amount of grossly a l t e r e d mtDNA ( 1 ) . The mutagenic event includes massive d e l e t i o n s and compensatory amplif i c a t i o n s via mechanisms not yet understood ( 2 ) . In extreme cases more than 99% of the wild-type sequence can be d e l e t e d . The d e t a i l e d a n a l y s i s of one such mutant - RD1A - revealed t h a t i t s mtDNA c o n s i s t s for over 95% of a p e r f e c t l y - r e p e a t e d , very (AT)-rich segment of the wild-type mitochondrial genome ( 3 - 5 ) . I t s p r e c i s e GC-content (3%) and the approximate length of the r e p e a t i n g sequence ('WO base p a i r s ) have been r e c e n t l y d e t e r mined ( 6 ) . F u r t h e r , q u a l i t a t i v e as well as q u a n t i t a t i v e pyrimidine t r a c t information has been obtained for each of the complementary s t r a n d s (5,6). In t h i s paper we p r e s e n t the complete n u c l e o t i d e sequence of Abbreviations: H-strand, heavy strand; L-strand, light strand; cRNA, complementary RNA. 2369 © Information Retrieval Limited 1 Falconberg Court London W1V5FG England ABSTRACT Nucleic Acids Research the perfectly-repeated 68 base pairs in RD1A mtDNA. Sequence information is derived from specifically primed and/or terminated RNA copies which are obtained by in vitro transcription of the separated strands under conditions described before (7). Sixty-six consecutive AT base pairs are shown to be flanked by two GC base pairs. This sequence not only comprises nearly all of the RD1A mitochondrial genome but, moreover, represents the first part of wild-type yeast mtDNA sequenced. The possible biological function of this sequence will be discussed. Part of this work has been briefly summarized elsewhere (8,9) . RD1A mtDNA The isolation and purification of RD1A mtDNA and the separation of the complementary strands have been described previously (5,7) . Specific transcription of RD1A mtDNA The preparative synthesis of the oligonucleotide-primed H-strand and L-strand copies has been described (7). Synthesis was carried out with Escherichia coli RNA polymerase at elevated temperatures in the presence of excess primer and low ribo32 32 nucleotide concentration. Either (a- P)ATP, (a- P)UTP or both were used as labelled precursors (Radiochemical Centre, Amersham, UK; 5-10 Ci/mmol, in later experiments 50-100 Ci/mmol). Purification of the transcripts included phenol extraction, fractionation on Sephadex G25 or G50 and electrophoresis on 16% acrylamide gels under denaturing conditions. Electrophoresis and elution from the gels was done as described below. The synthesis of long L-strand complementary RNA (cRNA) for subsequent digestion with T. ribonuclease was carried out under standard reaction conditions as described previously (10) with minor modifications; addition of 150 mM KC1, 500 pM unlabelled 32 ATP and UTP and 50 yM (a- P)GTP (Radiochemical Centre, Amersham, UK; 10 Ci/mmol). Ten ug of RNA polymerase was used per 1.6 pg L-strand DNA (0.2 ml reaction volume) and synthesis was continued for 2 h at 40°C. The reaction was stopped by heating for 5 min at 65°C, addition of 30 pg carrier tRNA (E. coli; 2370 MATERIALS AND METHODS Nucleic Acids Research General Biochemicals, Ohio) and further incubation for 20 min at 37 C with deoxyribonuclease (25 ug/ml, ribonuclease free; Sigma). After addition of sodium dodecylsulphate to 0.1% the mixture was extracted with phenol (equilibrated with 5 mM TrisHC1 (pH 8.0)) and fractionated on a Sephadex G50 column in 5 mM Tris-HCl (pH 8.0). The excluded material was digested with T1 ribonuclease (Sankyo, Japan) for 60 min at 37°C in 10 mM TrisHCl, 1 mM EDTA (pH 7.5) (11). The enzyme to carrier tRNA weight ratio was 1:1500 to avoid possible over-digestion by contaminating endonucleases. After phenol extraction the T. fragment was purified by electrophoresis on 10% acrylamide gels under denat- Gel electrophoresis Gel electrophoresis at 60°C on either 16% or 10% acrylamide gels containing 8 M urea was performed as described (7). The relative positions of the markers were determined by scanning the gel at 260 nm before freezing and subsequent slicing. Specific RNA fragments were eluted from crushed gel slices at 4°C with 0.4 ml of 5 mM Tris-HCl, 1 M NaCl (pH 7.5) in the presence of carrier tRNA (12). Gel fragments were removed by repeated centrifugation at 10 000 x 2 for 10 min and finally an equal volume of isopropanol was added to precipitate the RNA. After standing overnight at -20 C the precipitate was spun down, washed with 70% ethanol (-20°C), dissolved in a small volume of 5 mM Tris-HCl (pH 7.5) and stored at -20°C until use. Enzymatic digestion and fingerprint analysis Transcription products were digested with either pancreatic ribonuclease A (Boehringer Mannheim, Germany), ribonuclease U_ (Sankyo, Japan) or spleen phosphodiesterase (Worthington, U S A ) . i) Total pancreatic ribonuclease digestion was carried out for 30 min at 37°C at an enzyme to carrier tRNA weight ratio of 1:20 in 10 mM Tris-HCl, 1 mM EDTA (pH 7.5) (11). The digestion products were separated by: a) two-dimensional fingerprinting as described by Sanger et al. (13): electrophoresis in the first dimension on cellulose acetate strips (pH 3.5) (Schleicher & Schull, Germany) and in 2371 uring conditions as described below. Nucleic Acids Research the second dimension on Whatman DE-81 paper, 7% formic acid; b) one-dimensional fingerprinting, using only the electrophoresis in the second dimension on DEAE-paper. Spots were located by autoradiography and the relative amounts determined by cutting out the paper and subsequent counting in a liquid scintillation counter. ii) Partial pancreatic ribonuclease digestion was performed as described above except th (...truncated)