A nuclease from Drosophila and rRNA processing in yeast: Part~I. Nuclease III from adult flies of Drosophila melanogaster. Part~II. Mutants involved in pre- rRNA processing in Saccharomyces cerevisiae

Abstract

Part I. An endo-exonuclease (designated nuclease III) has been purified to virtual homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a stokes radius of 27A. The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg{dollar}\sp{lcub}2+{rcub}{dollar} or Mn{dollar}\sp{lcub}2+{rcub}{dollar} but not Ca{dollar}\sp{lcub}2+{rcub}{dollar} or Co{dollar}\sp{lcub}2+{rcub}{dollar} for its activity. The enzyme activity on double-stranded DNA is inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA requires 100 mM NaCl for 50% inhibition. The enzyme can degrade DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5{dollar}\sp\prime{dollar}-P and 3{dollar}\sp\prime{dollar}-OH termini. Supercoiled DNA is converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion.;Part II. A temperature sensitive bank of 517 mutants has been screened for mutants involved in pre-rRNA processing by Northern analysis. Mutant 517, designated rrp2, has been found to accumulate three novel RNA species 23S{dollar}\sp\prime{dollar}, 18S{dollar}\sp\prime{dollar} and 7S{dollar}\sp\prime{dollar}.;The processing of 35S pre-rRNA is slowed and the first processing step which generates 20S and 27S rRNA is defective in the rrp2 mutant. Pre-mRNA splicing is not affected. At 37{dollar}\sp\circ{dollar}C, the rrp strain gradually stops growing after four to five generations. The rrp2 mutant is recessive. The 5{dollar}\sp\prime{dollar} terminus of 7S{dollar}\sp\prime{dollar} RNA was determined to be 6 nucleotides downstream of the normal cleavage site that produces 20S and 27S rRNA. 7S{dollar}\sp\prime{dollar} RNA can assemble into 60S ribosomal subunits, but such subunits are relatively ineffective in joining polyribosomes.;A cold sensitive mutant spb6 is found to be defective in both 27S and 20S rRNA processing. 20S rRNA is relatively stable and is very slowly processed to 18S rRNA. 27S rRNA is not efficiently processed to 25S rRNA and much of the 27S rRNA is degraded. mRNA splicing is normal in the spb6 mutant. 20S rRNA is found to be present in 40S subunits. 40S subunits containing 20S rRNA can not assemble with 60S subunits to form ribosomes.