Current Genetics

Current Genetics 3, 6 5 - 7 1 (1981)

© Springer-Verlag 1981

Physical Map of the COB Region in Mitochondrial DNA of Saccharomyces cerevisiae G. Grosch, C. Schmelzer and S. Mathews Genetisches Institut der Universit~it Miinchen, Maria-Ward-Str. la, D-8000 Mtinchen 19, Federal Republic of Germany

Summary. A physical map of the COB region in mtDNA of yeast has been established. This region harbours a split gene coding for apocytochrome b. It includes restriction sites of seven endonucleases (EcoRI, HindII, HindlII, HaeIII, HpaII, AluI and BamHI). Various mtDNA sequences of this region, retained in a series of genetically characterized rho- clones have been allocated to this map. The combination of this physical map with a genetic map of the r h o - clones revealed that 1) cob- mutational sites spread over 8,400 bp, 2) mutations in sequences coding for apocytochrome b map in five distinct segments which are separated by intervening sequences with minimum lengths from 350 to 1,900 bp. Key words: mtDNA of Saccharomyces cerevisiae - rhorestriction mapping - Intervening sequence mutations

in sequences coding for apocytochrome b; the others map in intervening sequences (Slonimski et al. 1978b; Haid et al. 1979; Kreike et al. 1979; Solioz and Schatz 1979). The number of coding sequences for apocytochrome b is five (Bechmann et al. in press). This split gene organization of the COB region is consistent with results obtained by electron microscopy of hybrids between the presumptive messenger RNA for apocytochrome b and DNA of the COB region (Grivell et al. 1979). In this report we establish a genetic/physical fine structure map of the COB region. Restriction analysis of mtDNA was performed with a collection of rhoclones which were known from genetic tests to retain various parts of the COB region marked by mutational sites. This led to the allocation of the genetically defined sequences coding for apocytochrome b to sectors within a physical map of the COB region.

Introduction Materials and Methods Cytochrome b apoprotein is coded for by a region on mtDNA called COB or BOX (Slonimski et al. 1978a, b; Haid et al. 1979; Solioz and Schatz 1979; Hanson et al. 1979). Physical mapping of mutations causing defective synthesis of apocytochrome b (cob-) has revealed that the COB region is of considerable length, exceeding severalfold the size necessary to code for apocytochrome b (Slonimski et at. 1978a; Hanson et al. 1979). By analysis of the effect of cob- mutations of the pattern of mitochondrial translation products it became clear that the COB region contains a split gene: only some of the c o b - mutations apparently are located Offprint requests to." C. Schmelzer Abbreviations: mtDNA - mitochondrial DNA; b -bases; bp -

basepairs

Yeast Strains. rho- clones were derived from wild-type strain Kl14-4A, except for rho- dD22, derived from strain RM 511-4A and rho- strain BD4, derived from wild-type strain RM 590-9D. Mit- mutants were isolated from wild-type strain 777-3A. Preparation of mtDNA. Mitochondrial DNA was extracted from

isolated mitochondria (Lang et al. 1977) and cleared of nuclear DNA contamination by CsC1/bisbenzimid density gradient centrifugation (Lang, personal communication; Miiller et al. 1975). Restriction Enzyme Digestion and Isolation of Restriction Fragments. The restriction endonucleasesEcoRI, HindII, HindIII,

BarnHI, HpaII, and AluI were puchased from Boehringer, Mannheim, and HaeIII from Biolabs, Beverly, Massachusetts. Electrophoresis of the digests was performed in 1.5% and 0.7% agarose slab gels, calibrated with HaeIII fragments of phage e~X174 and EeoRI fragments of phage P22, respectively, and containing 0172-8083/81/0003/0065/$ 01.40

G. Grosch et al. : Physical Map of the COB Region

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