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A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.

The molecular composition and organization of the row of axonemal inner dynein arms were investigated by biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axonemes. Three inner arm structures could be distinguished on the basis of their molecular composition and position in the axoneme as determined by analysis of pf30 and pf23 mutants. The three inner arm structures repeat every 96 nm and are referred to here as inner arms I1, I2, and I3. I1 is proximal to the radial spoke S1, whereas I2 and I3 are distal to spokes S1 and S2, respectively. The mutant pf30 lacks I1 whereas the mutant pf23 lacks both I1 and I2 but has a normal inner arm I3. Each of the six heavy chains that was identified as an inner dynein arm subunit has a site for ATP binding and hydrolysis. Two of the heavy chains together with a polypeptide of 140,000 molecular weight form the inner arm I1 and were extracted from the axoneme as a complex that had a sedimentation coefficient close to 21S at high ionic strength. Different subsets of two of the remaining four heavy chains form the inner arms I2 and I3. These arms at high ionic strength are dissociated as 11S particles that include one heavy chain, one intermediate chain, two light chains, and actin. These and other lines of evidence indicate that the inner arm I1 is different in structure and function from the inner arms I2 and I3.

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S-containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8-7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild-type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.

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.

This paper presents segregation and cosegregation data for a set of 15 chloroplast genes of Chlamydomonas, and uses these data to generate a linear map of the chloroplast genome. The data were derived from pedigree analysis of a total of 1596 zoospore clones resulting from 12 crosses in each of which 4 to 7 pairs of chloroplast alleles were segregating. The crosses are a subset of those previously described (Sager and Ramanis 1976). By means of pedigree analysis, Type II segregations (nonreciprocal conversion-like events) were distinguished from Type III segregations (reciprocal events). The average frequency of Type II segregation was found to be the same for all 15 genes, indicating randomness of this event with respect to map location (Figure 1). Type III segregations occurred with a different and characteristic frequency for each gene, and were interpreted as a measure of the distance of each gene from the postulated centromere-like attachment point.Cosegregations, involving two or more genes, occurred with frequencies characteristic of the particular genes and much lower than expected for the product of single-gene events, indicating strong positive interference. Pairwise cosegregation frequencies provided unambiguous data for the gene order, confirmed by cosegregation runs of three or more genes. Apparent lengths of cosegregation runs, as fractions of the total map, indicate much longer stretches of gene conversion-like events than have been reported for other genetic systems. Comparisons of cosegregation frequencies in cross 20 after 15'', 30'' and 15'' UV irradiation of the mt(+) before mating, indicate little if any consistent effect of this irradiation on segregation events.

A novel mapping procedure is presented for organelle genes or any other genetic system exhibiting a measurable frequency of exchanges occurring at a constant rate over a measurable time interval. For a set of markers in a multiply-marked cross, the exchange rates measure relative map distances from a centromere-like attachment point.With this method, we present mapping data and a linear map of genes in the chlcroplast genome of Chlamydomonas. The data are plotted as log (percent remaining heterozygotes) against time and map distances are taken as proportional to slope.A statistical method which is an adaptation of jackknife methodology to a regression problem was developed to estimate slope values. A single line is fitted to pooled data for each marker from several crosses, and then lines are re-fit to a series of pooled data sets in each of which the observations from a single cross have been omitted. From these data sets a final summary slope is computed as well as a statement of its variability. The relative positions of new markers present in single crosses can then be estimated utilizing data from many crosses. The method does not distinguish between one-armed and two-armed linear or circular maps. However, evaluation of this map in conjunction with cosegregation frequency data (Sager and Ramanis 1976b) provides unambiguous evidence of the genetic circularity of the Chlamydomonas chloroplast genome.

The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its (35)S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with (35)S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes (pf-17, pf-24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes. Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology. Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci (pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components. In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants. In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides.

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.

This paper presents allelic segregation data from a series of 16 crosses segregated for nuclear and chloroplast genes. By means of pedigree analysis, segregants of chloroplast markers occurring in the zygote have been distinguished from those occurring in zoospore clones. The genes ac1, ac2, and tm1 showed little if any deviation from 1:1 either in zygotic segregation or in zoospore clones. The genes sm2, ery, and spc showed a significant excess of the allele from the mt (+) parent in zygotes. However, in zoospores, mt( +) excess was seen only when that allele was the mutant (resistant) form but not when it was wild type (sensitive).These results show that the extent of preferential segregation differs in zygotes and in zoospores, and that preferential segregation is influenced by map location and by allele specificity. A comparison of progeny from zygotes mated after 0, 15'', 30'', and 50'' UV irradiation of the mt(+) gametes demonstrated the lack of an effect of UV upon allelic segregation ratios. In total, these results exclude the multi-copy model of chloroplast genome segregation suggested by Gillham, Boynton and Lee (1974) and support the diploid model we have previously proposed (Sager and Ramanis 1968, 1970; Sager 1972).

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak-like projections found within the B-tubules of outer doublets 5 and 6.

The uni1 mutant of Chlamydomonas reinhardtii lacks one of the paired flagella seen in wild-type cells. The missing flagellum is cis to the eye spot and at this site the basal body is incomplete, lacking a transition zone. Together with nine other loci affecting flagellar assembly and two loci affecting flagellar function uni1 defines an approximately equal to 100-centimorgan linkage group with a circular map. Measurements of gene-centromere distances on the uni linkage group are consistent with the ordering of loci determined by recombination analysis and place the centromere near pf29, a motility-defective mutant. In well-synchronized meiotic cycles, recombination frequencies between loci on the uni linkage group but not loci on other linkage groups show striking temperature sensitivity during a 1- to 2-hr period 5 days before meiosis.

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.

Two mutations are described that alter the pattern of inheritance of chloroplast genes in Chlamydomonas. The mutant gene mat-1 linked to the mating type allele mt(-) greatly increases the frequency of exceptional zygotes, i.e., zygotes that transmit chloroplast genes from the mt(-) (male) parent. In some crosses, 80-90% of the zygotes are biparental, transmitting chloroplast genes from both parents. The mat-2 mutation, linked to mt(+), acts to decrease the frequency of exceptional zygotes below the spontaneous level. The mutant effects are discussed in terms of a DNA modification-restriction system, postulated to regulate the transmission of chloroplast DNA in zygotes.

The cytoplasmic linkage group of Chlamydomonas shows maternal inheritance, i.e. preferential transmission of cytogenes from the female (m t (+)) parent and loss of the corresponding male (m t (-)) genome in sexual crosses. The mechanism of this process is postulated to be enzymatic modification of chloroplast DNA of the female to protect it from a restriction enzyme which degrades the chloroplast DNA of the male parent in the zygote soon after fusion. Genetic, biochemical and physical data bearing on this hypothesis are summarized and discussed.

A mutation, car, determining resistance to several macrolide antibiotics, including carbomycin, has been identified in the alga Chlamydomonas as cytoplasmic, and mapped in the known cytoplasmic linkage group close to genes determining resistance to other antibiotics, including streptomycin, erythromycin, and spectinomycin. The effect of the car mutation on chloroplast ribosome function was demonstrated with an in vitro system incorporating amino acids especially developed to assess activity of 70S chloroplast ribosomes. In an S-30 extract containing both 70S chloroplast and 80S cytoplasmic ribosomes, low concentrations of Mg(++) and spermidine favored 80S ribosome activity, and high concentrations activated 70S ribosomes and reversibly inactivated the 80S component. Under conditions favoring chloroplast ribosome activity, carbomycin inhibited incorporation by an S-30 extract, and by purified 70S ribosomes from wild-type but not from car cells. These results show that cytoplasmic genes are directly involved in chloroplast ribosome function and they suggest that the car gene product is a ribosomal protein; the results further strengthen the evidence that the cytoplasmic linkage group is located in chloroplast DNA.

A group of eight non-Mendelian genes have been shown by recombination analysis to be linked into a linear structure or chromosome. Similar genetic maps of gene order and relative distances between genes have been constructed by two methods, one based on additivity of recombination frequencies, the other on frequency of reciprocal recombination with a postulated attachment point. The data indicate that the progeny are diploid for this linkage group, and that the strands are distributed in a precisely oriented manner at mitosis. Evidence is discussed in support of the view that this linkage group is located in chloroplast DNA.