Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Abstract

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt- parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COX1 have been isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of Dark Respiration and Reduction of Phototrophic Growth in a Mitochondrial DNA Deletion Mutant of Chlamydomonas Lacking cob, nd4, and the 3′ End of nd5

“…In this case, all of the meiotic progeny were phenotypically wild type. This exclusive transmission of the phenotype from the mt Ϫ parent is typical of the transmission pattern observed for mitochondrial mutations (Matagne et al, 1989;Bennoun et al, 1991;Dorthu et al, 1992;Colin et al, 1995); thus, it indicates that the dum24 mutation resides in the genome of this organelle. The new mutant was named dum24 .…”

“…Compared with both the wild-type strain and the dum19 mutant, which lacks cytochrome c oxidase activity (Colin et al, 1995), the dum24 strain produced very small colonies on Tris-minimal-phosphate (TMP) and Tris-acetate-phosphate (TAP) agar plates (Table 1). The slow growth of the dum24 mutant under photoautotrophic (TMP) conditions was confirmed in aerated liquid culture (data not shown).…”

“…In contrast to wild-type cells, which contain a single type of mitochondrial genome, the deletion mutants are heteroplasmons in which deleted monomers and dimers resulting from the fusion of two monomers by their deleted ends coexist (Dorthu et al, 1992;Colin et al, 1995). The deletion mutants also share the property of mitotically segregating a majority of viable cells and a few cells that divide eight or nine times to produce "minute" lethal colonies (Dorthu et al, 1992;Randolph-Anderson et al, 1993).…”

“…dum24 cells were grown under mixotrophic conditions (light, plus acetate as a carbon source), and their dark respiration activity was measured in the absence or presence of respiratory inhibitors. Cells from the wild type and from the dum19 mutant deprived of cytochrome c oxidase activity (Colin et al, 1995) were used as controls. The total respiratory rate of dum24 cells was low and represented only 22 and 42% of that found with wild-type and dum19 cells, respectively (Table 3).…”

“…Its small 15.8-kb linear mitochondrial genome has been totally sequenced, and all of the genes residing in the organelle have been identified (Vahrenholz et al, 1993; Figure 1). Moreover, several mutations altering the mitochondrial genes encoding apocytochrome b ( cob gene) or subunit 1 of cytochrome c oxidase ( cox1 gene) have been characterized (Matagne et al, 1989;Dorthu et al, 1992;Randolph-Anderson et al, 1993;Colin et al, 1995). Mutant cells are homoplasmic for the mutations and do not retain any DNA copies carrying the corresponding functional gene.…”