The Genetics and Cytology of Chlamydomonas

Levine, R. P.; Ebersold, W. T.

doi:10.1146/annurev.mi.14.100160.001213

Cited by 246 publications

(106 citation statements)

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“…For long-term culture storage and mating, cultures were maintained on solid agar medium. Tetrad analysis was performed at 24°C using standard techniques (Levine and Ebersold, 1960). Diploid strains were constructed using the arg2 and arg7 mutations as described by James et al (1988).…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

Piasecki

LaVoie

Tam

et al. 2008

MBoC

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Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation. INTRODUCTIONCentrioles are evolutionarily ancient organelles, possibly extant in a common ancestor of all eukaryotic cells (CavalierSmith, 2002). They are required for the organization of interphase and mitotic microtubule arrays and as basal bodies they serve as templates for the growth of nine doublet microtubules in axonemes of cilia and flagella (for review see Marshall, 2007). Although flowering plants and more derived fungi have reduced or eliminated centrioles and cilia, metazoans have diversified their functions in numerous sensory and developmental processes (for reviews see Singla and Reiter, 2006;Dawe et al., 2007). Several mammalian genetic disorders have been linked to defects in centrioles or cilia (for review see Afzelius, 2004;Pan et al., 2005). For example, the proteins associated with Bardet-Biedl syndrome, a debilitating pleiotropic human disorder characterized by obesity, renal abnormalities, polydactyly, retinal degeneration, and genitourinary tract malformations have been shown to localize to or around centrioles and cilia (for review see Beales, 2005;Blacque and Leroux, 2006).Centrioles in actively dividing cells show conservative replication and semiconservative segregation during division (for review see Dawe et al., 2007). As a result, each pair of centrioles consists of an older and a younger centriole that differ in age by at least one cell generation. The "mother" centriole differs both in structure and function from the "daughter" centriole. For example, in the single pair of centrioles found in most types of mammalian cells, the mother centriole nucleates the growth of the primary cilium and contains proteins not present in the daughter centriole. The daughter centriole becomes competent for ciliary assembly only in the ensuing cell cycle (for review see Bornens, 2002). Extension of the pathway for sequential maturation of basal bodies has been ...

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Section: Strains and Culture Conditionsmentioning

confidence: 99%

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

Piasecki

LaVoie

Tam

et al. 2008

MBoC

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“…Before crossing to S1-D2, backcrosses to a wild-type laboratory strain were conducted to ensure that the mcd4 and mcd5 phenotypes were caused by single, nuclear mutations. Then, mcd4 and mcd5 were crossed to S1-D2, and progeny were dissected as described previously (Levine and Ebersold, 1960). Progeny were tested for photoautotrophic growth by plating on minimal medium lacking acetate (Harris, 1989) and otherwise maintained on Tris-acetate phosphate medium (TAP; Gorman and Levine, 1965) under 23 h light and 1 h dark at 25°C.…”

Section: Strains Media and Genetic Crossesmentioning

confidence: 99%

Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii

et al. 2005

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Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.Sometimes called green yeast (Goodenough, 1992), the unicellular, eukaryotic green alga Chlamydomonas reinhardtii (hereafter called Chlamydomonas) is a venerable model system for plant biology as well as for cell motility. The tag green yeast refers to its haploid vegetative state, the existence of two mating types, and the general similarity in applicable genetic techniques. These aspects of Chlamydomonas biology have been previously reviewed (Rochaix, 1995).Like many microorganisms, screening of Chlamydomonas strains for rare mutations is straightforward, since large numbers of cells can be plated on an appropriate selective medium, or nonswimmers, for example, can be selected from large numbers of swimming cells. At the same time, the ease of nuclear transformation in Chlamydomonas, coupled with the plant-like nonhomologous integration of transforming DNA, facilitates the creation of insertional mutant collections. Taken together, the assortment of techniques useable for Chlamydomonas indulges both the amateur and experienced geneticist, yielding sometimes overwhelming collections of mutant strains. In this report, we focus on mutants affecting photosynthesis, in keeping with the thrust of this journal, and the emphasis of the newly renewed and National Science Foundation-supported Chlamydomonas genome project (http://www.chlamy.org/). However, the map-based cloning tools described here are generally applicable to Chlamydomonas biology.The use of Chlamydomonas to study the elaboration and regulation of the photosynthetic apparatus is long established and was recently review...

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“…Genetic analysis was performed using standard techniques (Levine and Ebersold, 1960;Harris, 1989). To determine whether the motility phenotype of 5B9 was linked to the NIT1 plasmid used as a selectable marker, 5B9 was backcrossed to A54-B2 (nit1⌬ ac17 sr1), and the resulting progeny were analyzed for their motility phenotypes and their ability to grow on selective media.…”

Section: Genetic and Phenotypic Analysesmentioning

confidence: 99%

TheChlamydomonas PF6Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

Rupp

O’Toole

Porter

2001

MBoC

113

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Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized a Chlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection. INTRODUCTIONCilia and flagella are highly conserved structures found on diverse cell types, ranging from single-cell protozoa to multicellular tissues in humans, where they function to propel cells through a fluid environment or to transport fluid across a cell surface. Motile cilia are also found in the embryonic node (Sulik et al., 1994; Bellomo et al., 1996), where their motility appears to be critical for establishing the morphogenetic gradient that determines the left-right body axis in mammals (Nonaka et al., 1998). Defects in the assembly or activity of cilia and flagella result in a variety of abnormalities, including defects in left-right axis asymmetry, infertility, and respiratory disease (Afzelius et al., 1975; Afzelius, 1995;Supp et al., 1999). Most motile cilia and flagella contain an axoneme that consists of nine outer doublet microtubules surrounding two central singlet microtubules. The outer doublets contain binding sites for the inner and outer dynein arms, the molecular motors that power axoneme motility (reviewed by Porter, 1996;King, 2000). The dynein arms on one outer doublet interact transiently with the adjacent doublet to generate the force for interdoublet microtubule sliding. Other structures within the axoneme constrain and coordinate the activity of the multiple dynein motors and thereby convert microtubule sliding into flagellar bending.Both structural and genetic evidence indicate that the central pair microtubules and radial spokes play an important role in coordinating dynein activity. The two central pair microtubules are structurally asymmetric, and in several organisms, this apparatus has been shown to undergo clockwise rotation at a rate of approximately one turn per beat (reviewed by Omoto et al., 1999). Thes...

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The Genetics and Cytology of Chlamydomonas

Cited by 246 publications

References 0 publications

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii

TheChlamydomonas PF6Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

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