David J.L. Luck

Suppressor mutations in chlamydomonas reveal a regulatory mechanism for flagellar function

Reversion analysis of flagellar-motility mutants of Chlamydomonas reinhardtii yields an unusual class of intergenic suppressor mutations that restore flagellar activity to paralyzed radial-spoke or central-pair mutants without altering the structural or molecular defects associated with the original mutations. Four suppressors representing independent genetic loci were studied in detail. Two of the mutations, suppf1 and suppf2, restore flagellar motility to either radial-spoke or central-pair mutants of different genes. The mutants suppf3 and suppf 4 suppress flagellar paralysis associated only with mutants defective for the radial spokes. Analyses of the axonemal polypeptides of suppf1, suppf3 and suppf4 mutants indicate that the mutations restore flagellar activity to paralyzed radial-spoke or central-pair mutants by altering other components of the flagellar axoneme. suppf1 shows an altered electrophoretic migration for a 325,000 molecular weight polypeptide known to be a subunit of an outer-arm dynein. suppf3 and suppf4 are missing different axonemal polypeptides with molecular weights of 60,000 (in the case of suppf3), and 40,000 and 29,000 (in the case of suppf4). Genetic evidence has been obtained indicating that the polypeptides affected in suppf3 and suppf4 are components of a newly identified functional and/or structural compartment of the flagellar axoneme. The suppressor mutations described here reveal the operation of a control mechanism that inhibits the operations of flagellar movements in the presence of radial-spoke or central-pair defects. Suppressor mutations release the inhibition. The molecular defects of suppf1, suppf3 and suppf4 provide evidence that the inhibitory mechanism can be interrupted at two different levels of axonemal function.

Uniflagellar mutants of chlamydomonas: Evidence for the role of basal bodies in transmission of positional information

A series of uniflagellar mutants isolated following mutagenesis of Chlamydomonas reinhardtii (strain 137c) with ICR-191 show a remarkable positional phenotype. The flagellum that fails to develop is cis to the eyespot in more than 95% of the cells examined. Both the positional and the uniflagellar phenotypes are transmitted stably through mitotic and meiotic divisions, and in backcrosses the meiotic segregation is two mutant to two wild-type progeny. Four of the mutants, uni1, uni2, uni3 and uni4, have been studied extensively. They appear to be alleles of a single gene locus or to be closely linked (less than or equal to 0.06 map units). The characteristic expression of the uniflagellar defect in cells under different growth conditions or in stable diploids indicates that the mutations alter the rate of development of the flagellum in the cis-eyespot flagellum. Electron microscopic studies suggest that the developmental defect resides in the basal body. Extensive recombination analysis to 33 nuclear markers representing the 16 linkage groups failed to establish linkage. The uni mutants, however, showed linkage to four unmapped mutant loci. Mutations for each of these loci also affect flagellar assembly.

Basal body/centriolar DNA: Molecular genetic studies in chlamydomonas

In Chlamydomonas reinhardtii, mutations on an unusual linkage group, the uni linkage group (ULG), affect structure and function of basal bodies. The ULG shows Mendelian segregation, but its genetic map is circular. Molecular cloning of fragments of the ULG was accomplished by taking advantage of restriction fragment length polymorphisms generated by crosses to Chlamydomonas smithii. These clones were used as probes to determine the size and form of the ULG chromosome; it is a 6-9 megabase linear molecule. Use of the probes for in situ DNA hybridization in cells localized the ULG chromosome to basal bodies.

Inner arm dyneins from flagella of chlamydomonas reinhardtii

Two dyneins have been isolated from axonemes of Chlamydomonas flagella by a three step procedure consisting of extraction in a high salt containing buffer, hydroxyapatite chromatography and sedimentation in sucrose gradient. A dynein with Mg+2- dependent ATPase activity 6.0 mumole Pi/min/mg, sedimenting at 12.5S was found associated with a polypeptide of molecular weight 310,000. A second dynein with specific activity of 3.7, sedimenting at 10-11S was found associated with a polypeptide of molecular weight 315,000. In their most purified forms, the two dyneins are complexed with nonstoichiometric amounts of four polypeptides ranging in molecular weight between 42,000 and 19,000. The 42,000 component has been identified previously as an actin-like protein. The high molecular weight subunits of both dyneins and two polypeptides of 28,000 and 19,000 molecular weight were found to be phosphorylated by in vivo pulse-labeling with 32P-phosphoric acid. All components of the 12.5S and 10-11S dynein complexes, with the exception of the 19,000 polypeptide, form a subset of polypeptides found to be deficient in pf-23, a chlamydomonas mutant, which is defective for inner arms.

Hybridization of mitochondrial ribosomal RNA

Abstract Mitochondrial and cytoplasmic ribosomal RNAs of Neurospora crassa were hybridized with mitochondrial or nuclear DNA which was immobilized on nitrocellulose filters. The 25 s and 19 s mitochondrial RNAs were complementary to 6.1% and 2.8% of the mitochondrial DNA, respectively. The 28 s and 18 s cytoplasmic RNAs were complementary to 0.67 and 0.33% of the nuclear DNA. Renaturation kinetics showed that the sequence length molecular weight of Neurospora mitochondrial DNA is more than 66 × 106. Therefore, the genes for 25 s and 19 s RNA are repeated at least four times in the mitochondrial DNA.

Mitochondrial ribosome assembly in Neurospora. Preparation of mitochondrial ribosomal precursor particles, site of synthesis of mitochondrial ribosomal proteins and studies on the poky mutant

Mitochondrial ribosomal RNAs in Neurospora are transcribed in tandem as a32 S precursor molecule which is subsequently cleaved and trimmed to yieldthe mature 19 S and 25 S RNA species ( Kuriyama & Luck,1973 a ). As part of a study of mitochondrial ribosome assembly and the poky (mi-1) mutant, procedures were developed for isolating mitochondrial ribosomal precursor particles which consist of 32 S RNA and 15 or more newly synthesized large and small subunit protiens.The precursor particles comprise less than 5% of the total mitochondrial ribonucleprotein and were initially identified in pulse-labeling experiments by cosedimentation (approx. 30 S)of peaks containing newly synthesized RNA and protein.Although not yet completely free of mature ribosomal subunits, the precursor particles could be studied in pulse and pulse-chase experiments combined with electrophoretic analysis of RNA and protein. Such experiments provided strong evidence that the precursor particles are formed in vivo by specific association of the RNA and protein species. The pulse-labeling experiments led to additional information about mitochondrial ribosome processing. Several small subunit proteins were identified which have exceptionally small free pools (as judged by high specific activity of pulse-label in mature small subunits), and which may thus control the rate of mitochondrial ribosome assembly. Experiments using inhibitors of protein synthesis (anisomycin and chloramphenicol) showed that one of these protiens, S-4a (apparent M r =52,000), is synthesized inside the mitochondria, whereas all other mitochondrial ribosomal proteins are synthesized in the cytosol. Finally, studies on poky , a non-Mendelian mutant with defective mitochondrial ribosome assembly( Rifkin & Luck, 1971 ) suggested that poky small subunits are deficient in several proteins, possibly including S-4a. These several lines of evidence are discussed in light of the possiblity that S-4a is the site of the primary defect in poky .

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-3H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNAs. Both mature ribosomal RNAs compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNAs. The large molecular weight precursor (32 S) of both ribosomal RNAs appears to be 2.4 × 106 daltons in size. Maturation to large subunit RNA (1.28 × 106 daltons) is assumed to involve an intermediate ~1.6 × 106 daltons in size, while cleavage to form small subunit RNA (0.72 × 106 daltons) presumably involves a 0.9 × 106 dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNAs, the mitochondrial precursor RNA has a strikingly low G + C content.

Methylation and processing of mitochondrial ribosomal RNAs in poky and wild-type Neurospora crassa.

Abstract Mitochondrial ribosomal RNAs (25 S and 19 S) are produced by cleavage of a common high molecular weight precursor (32 S), which is a transcript of mitochondrial DNA. The poky strain in Neurospora crassa is characterized by a deficiency of mitochondrial small-subunit ribosomes. Pulse-labeling studies were carried out in poky indicating that the maturation and cleavage steps, which lead to the production of mitochondrial ribosomal RNAs, are abnormal. Studies of RNA methylation in methionine-requiring strains were also made. In control cells, the time course of incorporation of [C3H3]methionine label into mitochondrial RNAs, and the contents of methyl groups in the RNAs suggest that 32 S RNA is the major site for methylation. In poky, mitochondrial 25 S and 19 S RNAs have methyl contents of approximately 70% and 55%, respectively, of control values. The cytoplasmic ribosomal RNAs and mitochondrial 4 S RNAs, by contrast, show no differences between poky and control cultures. It is concluded that in poky, most of the putative 19 S RNA is degraded before it is incorporated into small ribosomal subunits. This defect could be a consequence of undermethylation, or both defects could result from a primary abnormality yet undefined.

Methylation of mitochondrial RNA species in the wild-type and poky strains of Neurospora crassa☆

Abstract Methylation of mitochondrial RNAs in the me-3 and poky f+ me-3 strains of Neurospora crassa has been re-examined using procedures based on steadystate labeling with [methyl-3H]methionine and taking the precaution of adding sodium formate to suppress randomization of the methyl-3H label. Under these conditions, the values measured for the mitochondrial ribosomal RNAs are 0·05 to 0·16 methyl group per 100 nucleotides, much lower than had been reported previously and, in addition, there is no longer a significant difference between the me-3 and poky f+ me-3 strains (contrast Kuriyama & Luck, 1974). Control experiments rule out the possibility that the much lower values result from grossly inefficient labeling of intra-mitochondrial methyl groups.