A single message of 1 1
A single message of 1 1.3 kb is greatly up-regulated after flagellar excision. two or more intermediate chains (ICs) (usually WD-repeat proteins) and dimers of three different classes of light chains (LCs) (the LC8, Tctex1/Tctex2, and LC7/Roadblock families). These distinct dyneins also contain additional components specific to a particular TIE1 enzyme class, such as the light intermediate chains of cytoplasmic dynein and the regulatory light chains directly associated with the motor units of the outer arm. The Tctex1 and Tctex2 proteins were first identified as candidates for distorter/sterility factors involved in the non-Mendelian transmission of mouse capsid protein VP26 [Douglas are viable, although these mutants are male sterile. Phenotypically, these strains exhibit defects in spermiogenesis where a nuclear cap of cytoplasmic dynein is incorrectly localized and a connection between the nucleus and basal body is not formed appropriately; they also have immotile sperm possibly due to the lack of Tctex1 in axonemal dyneins (Caggese outer dynein arm (Piperno and Luck, 1979 ; Pfister inner dynein arm I1 (Harrison genome and demonstrate that it is an integral component of the outer dynein arm associated with both intermediate chains. Thus, in this organism distinct Tctex1-related proteins are used within the flagellar inner and outer arm systems. Furthermore, we find that this LC9 protein together with BT-13 LC2 (a Tctex2) and LC6 (related to the highly conserved LC8 protein; King and Patel-King, 1995 ) are missing in a mutant strain (double mutant provides further support for this model and yields intriguing insight into the mechanisms of outer arm dynein assembly and the molecular architecture of the IC/LC complex. MATERIALS AND METHODS Chlamydomonas Strains The strains used in this study are indicated in Table 1. The and pseudorevertants were generously provided by Dr. David Mitchell (SUNY Upstate Medical Center, Syracuse, NY). The double BT-13 mutant was constructed using standard methods (Harris, 1989 ). The genotype of the tetrad progeny from this cross was determined using the PCR to obtain appropriate regions from both genes. The frame-shift mutation forms a novel allele is a null and thus only the wild-type LC2 gene was detected by the PCR. Table 1. Strains used in this study Strain Description Reference cc124 Wild type g1 Used as parent for generation of null mutants; derived from a cc124 cross Pazour (1995 ) IC2 frame-shift resulting in a premature stop; cannot assemble outer arms; slow swimming Kamiya (1988 ); Mitchell and Kang (1993 ) intragenic pseudorevertant; assembles outer arms and restores wild-type beat frequency Mitchell and Kang (1993 ) intragenic BT-13 pseudorevertant; assembles outer arms, but exhibits reduced beat frequency Mitchell and Kang (1993 ) Defective for 1 HC; lacks inner arm I1 Kamiya (1991 ); Porter (1992 ) Defective for p28; lacks a subset of inner arms Kamiya (1991 ); LeDizet and Piperno (1995 ) Defective for DC1; lacks outer arms and the outer arm docking complex Kamiya (1988 ); Koutoulis (1997 ) Defective for IC1; lacks outer arms, but assembles the docking complex Kamiya (1988 BT-13 ); Wilkerson (1995 ) Null mutant for LC2; lacks functional outer arms Pazour (1999 ) Lacks 3 end of LC2 gene; defective outer arm assembly Pazour (1999 ) Null mutant for LC2, rescued with a 3.1-kb genomic region containing the wild-type LC2 gene Pazour (1999 ) Null mutant for LC6; assembles outer arms and exhibits a minor swimming defect Pazour and Witman (2000 ) Null mutant for LC7a; assembles reduced levels of outer arms and inner arm I1 Pazour and Witman, (2000 ); DiBella (2004a ) Double mutant with altered IC2 and null for LC2 This study; Mitchell and Kang (1993 ); Pazour (1999 ) Defective for RSP3; lacks radial spokes; paralyzed flagella Huang (1981 ) Lacks central pair microtubule complex; paralyzed flagella Adams.