Abstract. We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the Pb16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XnI. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pfl) strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pfl) mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flageUar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.T HE dynein ATPases are large and complex multisubunit enzymes that provide the force for a variety ofmicrotubule based movements inside cells (reviewed in Gibbons, 1988;Porter and Johnson, 1989). In cilia and flagella, at least nine different dynein heavy chains have been described; these and other polypeptides are arranged into at least four different dynein arm subunits (Piperno et al., 1990). The presence of these multiple dynein isoforms within the axoneme provides the cell with several different potential mechanisms for affecting the flagellar waveform, but how the activity of the different dyneins is coordinated is not well understood. Both the inner and outer dynein arms can drive the sliding movements between adjacent outer doublet microtubules that serve as the basis for ciliary movement. However, study of flagellar mutations in Chlamydomonas has revealed that the different dynein arms have different functions in the generation of flagellar motility. For instance, mutant strains that lack outer arm polypeptides swim with a reduced flagellar beat frequency, whereas mutant strains that lack subsets of inner arm polypeptides swim with an altered flagellar waveform (Brokaw and Kamiya, 1987).The flagellar dyneins also play an important but poorly understood role in modifying the flagellar waveform in response to signals f...

Abstract. Strains of Chlamydomonas reinhardtii with a mutant allele at the BOP2 locus swim slowly and have an abnormal flagellar waveform similar to previously identified strains with defects in the inner arm region. Double mutant strains with the bop2-1 allele and any of 17 different mutations that affect the dynein arm region swim more slowly than either parent, which suggests that the bop2-1 mutation does not affect solely the outer dynein arms, the I1 or ida4 inner dynein arms, or the dynein regulatory complex. Electron microscopic analysis shows that bop2-1 axonemes are missing density in the inner dynein arm region. Surprisingly, two populations of images were observed in longitudinal sections of axonemes from the bop2-1 strain. In the 10 longitudinal axonemes examined, a portion of the dynein regulatory complex and a newly identified structure, the projection, are affected. In five of these 10 longitudinal axonemes exarnined, two lobes of the ida4 inner arm are also missing. By examining the cross-sectional images of wild-type and bop2-1 axonemes at each outer doublet position around the axoneme, we have determined that the bop2-1 mutation affects the assembly of inner arm region components in a doublet specific manner. Doublets 5, 6, and 8 have the most severe deficiency, doublet 9 has an intermediate phenotype, and doublets 2, 3, 4, and 7 have the least severe phenotype. The bop2-1 mutation provides the first evidence of radial asymmetry in the inner dynein arm region. IN the unicellular green alga Chlamydomonas reinhardtii, two flagella generate force to move the cell. The beat stroke is highly asymmetric and consists of two stages. In the effective stage, a power stroke, which travels almost entirely in two dimensions, is initiated by a single bend that forms near the base of the flagellum while the remainder of the flageHurn is straight. In the recovery stage, a threedimensional stroke is initiated near the base of the flagellum and propagates distally, leaving the flagellum in the starting position (Brokaw et al., 1982). An asymmetric beat stroke suggests that the flagella are structurally asymmetric. The focus of much research in recent years has been the regulation of dynein activity, but the relationship between asymmetry of the beat stroke and dynein regulation is not well understood.Three a B-tubule projection, which extends partially across the lumen of the microtubule (Hoops and Witman, 1983). These three doublets are located in the plane of the power stroke; doublets in these positions are predicted to exhibit the least microtubule sliding relative to their neighbors during the power stroke (Hoops and Witman, 1983). Several mutant strains (mbol,2,3), which are missing the B-tubule projections from doublets 5 and 6, are able to swim only backwards and the flagella utilize a sinusoidal waveform that is similar to sperm flagella waveforms (Segal et al., 1984). A twomembered cross-bridge that extends from doublet 1 towards doublet 2 and the absence of an outer dynein arm from over 90% of the len...

Well-designed "controls" distinguish experimental from non-experimental studies. Surprisingly, we found that a high percentage of students had difficulty identifying control experiments even after completing three university-level laboratory courses. To address this issue, we designed and ran a revised cell biology lab course in which students participated in weekly "experimental control exercises." To measure student understanding of control experiments, we developed a set of assessment questions; these were given to students prior to and following completion of either a standard cell biology lab course or the revised cell biology lab course. Not unexpectedly, the results indicate that the revised course led to greater improvements in students' ability to identify and explain the purpose of control experiments. Based on these observations, we recommend that explicit and detailed discussions designed to identify the design and purpose behind control experiments become a standard component of all laboratory courses.

In Chlamydomonas reinhardtii, we have found that linkage groups XII and XIII define only a single linkage group and that linkage groups XVI and XVII also define a single linkage group. The interdigitation of the genetic maps of linkage groups XII and XIII and of XVI and XVII is presented. At present, 17 linkage groups that display Mendelian segregation have been identified in C. reinhardtii.