Analyses of 22S Dynein Binding to Tetrahymena Axonemes Lacking Outer Dynein Arms

ABSTRACT. Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature of 39° C. Axonemes isolated from nonmotile oad mutants ( oad 39° C axonemes) lack approximately 90% of their outer dynein arms and are deficient in 22S dynein. Here we report that oad 39° C axonemes contain 40% of the 22S dynein heavy chains that wild-type axonemes contain and that oad axonemes do not undergo ATP-induced microtubule sliding in vitro. Wild-type 22S dynein will bind to the outer arm position in oad axonemes and restore ATP-induced microtubule sliding in those axonemes. Unlike wild-type 22S dynein, oad 22S dynein does not bind to the outer arm position in oad axonemes. These data indicate that the oad mutation affects some component of the outer arm dynein itself rather than the outer arm dynein binding site. These data also indicate that oad axonemes can be used to assay outer dynein arm function.     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Effects of sulfhydryl reagents on the ATPase activity of solubilized 14S and 30S dyneins and on whole ciliary axonemes as a function of pH

The effects of five sulfhydryl (SH) reagents – N-ethylmaleimide (NEM), a spin-labeled maleimide (SLM), N-N′-phenylenedimaleimide (PPDM), bis(4-fluoro-3-nitrophenyl)sulfone (FNS), and carboxypyridine disulfide (CPDS) – on glycerol-treated, Triton X-100-demembranated ciliary axonemes of Tetrahymena, on the 30S and 14S dyneins extracted from such axonemes, and on the residual ATPase activity remaining associated with axonemes that have been extracted twice with Tris-EDTA have been examined as a function of pH in the range 6.9–8.6. Preincubation of axonemes and of solubilized 30S dynein with low concentrations of each of the five SH reagents, at 0°C and at 25°C, caused enhancement of the latent ATPase activity. PPDM was the most effective reagent, causing half-maximal enhancement (after 18 h at 0°C) at ～ 0.5 μM, corresponding to 0.19 moles/10⁵ g axonemal protein. The rate constants, k_a, for the enhancement reaction at 0°C depended on whether the 30S dynein was in situ or solubilized; the ratio k_a (in situ) /k_a (solubilized) was > 1 for NEM, ～ 1 for PPDM, and < 1 for FNS. For each SH reagent except CPDS, k_a (at 0°C) increased markedly with increasing pH in the range pH 6.9–8.6; for CPDS k_a increased only about fourfold. At long times of preincubation and high concentrations of NEM, SLM, PPDM, and CPDS, the enhancement of ATPase activity was followed by a loss of activity. The values of k_L, the rate constants for loss of ATPase activity from the peak enhanced level, were much lower than the corresponding values for k_a, and increased with increasing pH. With SLM and PPDM, inhibition continued until the ATPase activity was almost completely inhibited. With NEM, however, the initial rate of loss from the peak enhanced value decreased as the ATPase activity returned toward the control (unmodified) level, and further inhibition was very slow. The differences in degree of inhibition obtained with SLM as compared to NEM suggest that there are at least two classes of inhibitory SH groups on 30S dynein. The ATPase activity of 14S dynein was only inhibited by preincubation with NEM, SLM, PPDM, and, to a lesser extent, CPDS; k_L increased with increasing pH. Preincubation of 14S dynein with FNS yielded conflicting results when the reaction was “stopped” by adding dithiothreitol. When 14S dynein was preincubated at 0 C with FNS and the ATPase activity was then assayed at 25°C, a biphasic pattern of enhancement followed by inhibition was obtained. The residual ATPase activity of twice-extracted axomenes was relatively insensitive to each of the SH reagents studied; an initial rapid loss of some 20–40% of the ATPase activity occurred, followed by a very slow further loss of activity. Increasing the pH increased this slow rate of inhibition. The residual ATPase activity of unmodified twice-extracted axonemes decreased slightly with increasing pH, in contrast to the slight increase observed with increasing pH for the ATPase activity of axonemes and of solubilized 30S and 14S dyneins. The presence of ATP during preincubation of axonemes with PPDM at O°C prevented the enhancement of ATPase activity; only a slow loss of ATPase activity was observed. This rate of loss of ATPase activity was slower than the rate of loss observed (after peak enhancement of activity was reached) when PPDM reacted with axonemes in the absence of ATP. In these properties the SH groups of 30s dynein responsible for the enhancement of latent ATPase activity and for the inhibition of ATPase activity do not resemble the SH1 and SH2 groups of myosin, respectively, since the presence of ATP increases the rates of reaction of SH1 and SH₂ of myosin with SH reagents.     

小麦体细胞无性系Glu-1基因突变体的遗传分析

对 Glu-1基因的Glu-Al和Glu-Bl位点都发生突变的1个小麦体细胞无性系后代的高分子量谷蛋白亚基组成进行的分析结果,进一步证实了体细胞无性系发生了基因突变, 且发现同源染色体的等位基因易发生相同突变,突变体多是纯合基因型。认为这是体细胞无性系变异稳定快的遗传根源。 Abstract:Evidence for specific gene mutation has been obtained in our previous research by analyzing high-molecular-weight glutenin subunits(HMW-GS)controlled by Glu-1 gene in wheat somaclons.On the basis of the result,the present study detected in the offspring of a HMW-GS somaclone the mutants at both Glu-A1 and Glu-B1 loci.The occurrence of gene mutation in wheat somaclones was further proved.The results showed that a allele on homologous chromosomes often mutated identically.Genotype of this kind mutation migh be pure.Thie is considered to be genetic reason for quickly stabilizing of somaclonal variation.     

Biochemical Analysis of Myelin Proteins in a Novel Neurological Mutant: The Taiep Rat

Abstract: Hemispheres, spinal cords, and sciatic nerves were taken from taiep, carrier, and control rats at ages ranging from 1 day to 16 months. Absolute myelin yields from CNS taiep tissues peaked at ～2 months and then decreased until they reached a low but stable level. Myelin yield from the affected hemispheres expressed as a percentage of age-matched controls decreased continuously from 2 weeks until it reached a stable level of ～10–15%. The same was true for the spinal cords, but here the myelin yield reached a plateau at a slightly higher percentage of 20–25%. In comparison with control rats, isolated CNS myelin fractions from the affected rats had a greater content of high molecular weight proteins. Western blot analyses of CNS homogenates revealed that myelin basic protein (MBP), proteolipid protein, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase were all present but decreased to levels generally consistent with the deficiencies of myelin. However myelin-associated glycoprotein (MAG) levels always were reduced much more than those of the other three myelin proteins, and at younger ages the apparent molecular weight for MAG was increased in the mutants. Western blot analyses of sciatic nerve homogenates showed that the levels of MBP, MAG, and P₀ were not significantly different in control and mutant animals. These results suggested an early hypomyelination of the CNS, with peak levels of myelin at 2 months, followed by a prolonged period of myelin loss, until a very low but stable myelin level was reached. The consistently greater loss of MAG, in comparison with other CNS myelin proteins, is different from most other hypomyelinating mutants in which MAG is relatively preserved in comparison with the proteins of compact myelin. This might be due to microtubular abnormalities in the taiep mutant interfering with transport of myelin proteins and having the greatest effect on MAG because of its most distal location in the periaxonal oligodendroglial membranes.