Sequence and expression of NUC 1 , the gene encoding the mitochondria ] nuclease in

Abstract

The DNA sequence and studies on the expression of the NUC1 gene from Saecharomyces cerev is iae are presented. The NUC1 locus i s located in the d i s t a l por t ion of the l e f t arm of Chromosome X and encodes the major nuclease found 1n mitochondria. The i n fe r red amino a d d sequence of NUC1 predicts tha t the nuclease i s basic , r i c h in p ro l i nes , of average hydrophob id ty , and has a molecular weight f o r the primary t r a n s l a t i o n product o f 37,209 da l tons . NUC1 is very poorly expressed, consistent w i t h the codon usage bias determined from the DNA sequence and our previous determinat ion of the number of enzyme molecules per c e l l . Mapping of the 5' terminus of the NUC1 mRNA reveals tha t the mRNA has a long 400 base untranslated leader 1n which are found three open reading frames, each i n i t i a t e d by an AUG. The p o s s i b i l i t y tha t these upstream open reading frames con t r ibu te t o the poor expression of the NUC1 gene Is discussed. INTRODUCTION In yeast more than 50% of a l l c e l l u l a r nuclease a c t i v i t y i s due to a s ing le enzyme located in mitochondria ( 1 , 2 ) . This enzyme has both RNase and DNase a c t i v i t y , Is bound to the mitochondrial Inner membrane, and 1s encoded 1n the nucleus ( 3 ) . In v i t r o the enzyme lacks s i t e s p e c i f i c i t y fo r mitochondrial RNAs or DNA. In the accompanying repor t (4) we demonstrate tha t d i s rup t i on of NUC1, the gene encoding t h i s nuclease, produces a s t r a i n wi th no detectable mitochondrial DNase a c t i v i t y and l i t t l e nonspecif ic RNase a c t i v i t y . Despite the absence of nuclease a c t i v i t y 1n t h i s s t r a i n , mitochondrial func t ion appears to be normal s ince the s t r a i n 1s rho^. Moreover, a s t r a i n w i th 20-40 times the normal level of mitochondrial nuclease 1s also phenotypical ly rho+. Although the amount of enzymatic a c t i v i t y 1n the mitochondria of a w i ld t ype yeast s t r a i n i s prodig ious, the number of enzyme molecules 1n the mitochondria Is low, possibly as few as 100-300 per ce l l ( 3 ) . This level does not vary more than two f o l d comparing c e l l s grown i n fermentable versus © IRL Press Limited, Oxford, England. 3297 Nucleic Acids Research nonfermentable carbon sources, c e l l s i n logar i thmic versus s ta t ionary phase, or rho versus rhoc e l l s (3 ) . S imi la r potent DNase and RNase a c t i v i t i e s have been observed i n other eukaryotes ( 5 , 6 ) , although only an enzyme from Neurospora crassa has been completely p u r i f i e d ( 7 ) . A p a r t i a l l y p u r i f i e d enzyme preparat ion from beef heart mitochondria has also recen t l y been characterized ( 8 , 9 ) . In both species, 1t appears tha t the character ized enzymes are at least responsible fo r the ma jo r i t y of a l l mitochondrial nuclease a c t i v i t y and may possibly be the only nonspeci f ic nucleases present, as we have shown 1n yeast ( 3 , 4 ) . These observations suggest tha t the presence of a mitochondrial DNase/RNase may be widespread 1n eukaryotes. Although the func t ion of t h i s enzyme remains obscure, p a r t i c u l a r l y since our yeast mutant lack ing nuclease 1s phenotypical ly rho+ (3 ,4 ) , the mitochondrial locat ion and a c t i v i t y of t h i s class of enzymes appears to have been conserved during evo lu t ion . Here we present the nucleot ide sequence of the gene (NUC1) fo r mitochondrial DNase/RNase from yeast and an analys is of I t s expression. These data show t h a t the gene i s very poorly expressed, consis tent w i th I t s codon usage bias and our previous determinat ion of the amount of enzyme in mitochondr ia. MATERIALS AND METHODS DNA Sequencing DNA sequencing was performed by the chemical method of Maxam and G i l be r t (10) . Res t r i c t i on fragments were labeled (11) e i ther by a f i l l i n react ion fo r the 3' end or by end labe l ing the 5' end w i th T4 polynucleot ide kinase and [Y_32p]ATP a f t e r dephosphorylation wi th c a l f i n t e s t i n a l a l k a l i n e phosphatase. When necessary, labeled r e s t r i c t i o n fragments were Iso lated by gel e lectrophoresis and e lec t roe lu t i on (12) . A l l enzymes were purchased from New England Biolabs (Bever ly , MA) and Isotope from ICN ( I r v i n e , CA). Northern Blots RNA preparat ion. Total c e l l u l a r RNA was prepared from spheroplasts by the guanid1ne-HCl/CsCl method (13) . A l t e r n a t i v e l y , spheroplasts were lysed in lOOmM NaCl, lOmM Tr i s -HCl , pH 8 . 0 , lOmM EDTA, 0.5% SDS at 65°C, ext racted w i th phenol/chloroform ( 1 : 1 ) , and RNA p rec ip i ta ted from t o t a l nucleic a d d at 4°C fo r 24 hr a f t e r the addi t ion of L1C1 t o 2M (14) . Preparat ion of poly(A)-enr1ched RNA. A poly(A)-enr1ched RNA f r a c t i o n was prepared from t o t a l c e l l u l a r RNA ( I so la ted by the guanid1ne-HCl/CsCl method) by o l igo(dT) ce l l u l ose column chromatography as described by Mania t is , et a l . (11) w i th L1C1 subs t i tu ted f o r NaCl, except tha t the 0.1M s a l t wash was

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@inproceedings{Hofmann2005SequenceAE, title={Sequence and expression of NUC 1 , the gene encoding the mitochondria ] nuclease in}, author={Timothy J. Hofmann and Hans Peter Zassenhaus}, year={2005} }