Universal genetic code evidenced in mitochondria ofChlamydomonas reinhardii

Summary With the ultimate intent to establish a transformation system for eukaryotic organelles, the structure and organization of mitochondrial genes from the unicellular algaChlamydomonas reinhardii has been investigated. Using DNA hybridisation and DNA sequencing techniques, 3.9 kb of DNA, comprising about 25% of the mitochondrial genome, have been analysed in detail. By comparing the primary structure of homologous genes from other eukaryotic systems, we were able to identify the continuous genes coding for cytochrome oxidase subunit I (COI) and a NADH dehydrogenase subunit (ND5). The two genes are coded by opposite DNA strands and are not overlapping. The COI and the ND5 genes code for 505 and 567 amino acids, respectively. Interestingly, the comparative analysis with homologous genes from other eukaryotes shows that the universal genetic code is used in mitochondria ofC. reinhardii. This situation is different from all other mitochondrial systems studied so far. The results provide evidence thatC. reinhardii would be the appropriate organism for development of a transformation and expression system, where foreign genes, translated via the universal genetic code, are introduced into mitochondria.     

Time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas reinhardii

Contradictory data have been published concerning the time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas. In the present study, adjacent ultrathin sections of Chlamydomonas reinhardii zygotes of various ages were examined with the electron microscope. These sections clearly reveal that nuclear fusion precedes chloroplast fusion.     

The zygote cell wall of Chlamydomonas reinhardii: a structural,chemical and immunological approach

The zygote cell wall of Chlamydomonas reinhardii has been studied using structural, chemical and immunological methods. Monoclonal antibodies and polyclonal antisera that were originally raised to the major hydroxyproline-rich glycoproteins of the vegetative cell wall were used to probe the zygote wall for common antigenic components. These antibodies cross-reacted strongly and specifically with components of the zygote cell wall, and were used to show the origin, route of transport, and the location of these antigens within the zygote cell wall. The zygote cell wall contained about 10% protein, with hydroxyproline accounting for 22.5 mol % of the total amino acids present. Glucose was the most abundant sugar residue, and accounted for 56% of the total sugar present. Gas liquid chromatography-mass spectrometry showed the presence of a (1-3)-d-glucan as the major structural polysaccharide within the zygote cell wall. The (1-3)-d-glucan was detected and localised within the zygote cell wall by immunogold labelling of thin sections. Using an antiserum directed against (1-3)-d-linked glucose units, this polysaccharide was found to be consistently present within the non-staining layer of both young and mature zygote cell walls. (1-3)-d-Glucan was also detected in other wall layer using higher concentrations of antiserum. No intracellular labelling was found, indicating that the plasmamembrane is the site for the synthesis of this polysaccharide within the Chlamydomonas zygote.Abbreviations DGP antiserum to deglycosylated 2-BII glycoprotein - GLC-MS gas liquid chromatography-mass spectrometry - MAC monoclonal antibody centre     

The genetics ofChlamydomonas moewusii Gerloff

Journal of Genetics -     

The biological clock of Chlamydomonas reinhardii in space

The overt circadian rhythm in a wildtype (wt+) and a short period (s-) strain of Chlamydomonas reinhardii has been studied in space using the photoaccumulation behavior as the recorded parameter. The period of the wt+ was 29.6 h, of the s- 21.4 h and did not deviate significantly from ground controls performed exactly at the same time. The phase was delayed in space by 4.2 h in the wt+, but was not altered in the s-. In both strains the amplitudes were significantly higher in space than in the ground controls. During the recording period of 6.5 days the cell density increased in both strains. The survival rate, i.e. the ability to form colonies on agar petri dishes, was higher in space than on ground. The period was in both strains by 1.1 h longer in Florida (Kennedy Space Center) in both the flight and the control samples than in Europe. The significance of these results is discussed with respect to the endogenous nature of the biological clock and the role of the microgravity environment.     

The basal body-root complex ofChlamydomonas reinhardtii during mitosis

Summary The results of this work clarify several structural and temporal aspects of biogenesis of the basal body-root complex inChlamydomonas reinhardtii. The two phases of basal body development (probasal body assembly and conversion of probasal body into mature basal body) occur at identical mitotic stages in successive mitoses during multiple fission, which indicates a tight coupling between basal body development and the mitotic cycle. The two steps of basal body development are separated from one another in time,i.e. immature probasal bodies originate during an interval lasting ca. 5 min between mid-metaphase and early telophase, but mature after a quasi-dormant period only during early prophase of the next mitotic round. The duration of the dormant period depends on the interval between two mitoses: during synchronized vegetative growth there is an interval of ca. 20 h (interphase growth) between two rounds of multiple fissions, but only a maximum interval of 1.5 h between the successive mitoses of one round of multiple fissions.The microtubular root system, which is bisected at the same time as the basal body apparatus in a plane perpendicular to the distal connecting fiber during prophase, and whose roots seem to be reduced in length, starts duplication at early metaphase with the successive origin of two short bud-like partner roots just opposite the remnants. These initial roots elongate during subsequent phases by unilateral and radial growth from the basal bodies and along the cell's periphery, but exactly where they terminate is not known. The two-stranded roots opposite each other appear to be again connected as early as anaphase.The striation pattern of the distal connecting fiber is lost during early prophase thus indicating a partial breakdown of the fiber.Dedicated to Prof. Dr. C.-G. Arnold (Erlangen) on the occasion of his 60th birthday.     

The role of flagellar adhesion in sexual activation ofChlamydomonas eugametos

Summary Sex-specific agglutination in gametes ofChlamydomonas eugametos was carried out with dead partner cells (killed by glutaraldehyde, formaldehyde or OsO₄), with isolated flagella, flagellar appendages and isoagglutinins derived from cell-free culture medium. The activation of the plasma papilla in agglutinated cells was studied by scanning electron microscopy in relation to the agglutinative properties of the materials tested. The results reveal differences in agglutination and papilla activation between gametes of both mating types. They also indicate that papilla activation depends on the extent of agglutination, which is a function of sex-specific flagellar properties and the amount and nature of the agglutinative material used. A hypothesis is presented to explain the observed phenomena.     

The electrophoretic behaviour of mitochondria from kidney and liver of rats

1. The electrophoretic mobility values for mitochondria prepared from rat kidney and liver measured in 0.125m-potassium chloride-0.02m-tris, pH7.4, are: -0.78(s.e.m.+/-0.02)mu/sec./v/cm. (29 experiments) and -1.06(s.e.m.+/-0.01) mu/sec./v/cm. (21 experiments) respectively. 2. These mobility values are unaffected by washing and spontaneous swelling at 25 degrees , indicating a stable electrokinetic surface. 3. The mobility of rat-kidney mitochondria is unaffected by thyroxine-induced swelling, or by the state of hydration of the rat. 4. pH-mobility curves show similar surface ionogenic groups for kidney and liver mitochondria; their isoelectric points are pH3.9 and pH4.4 respectively.     

The nitrate-reducing enzyme system of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the reduction of nitrate to ammonia occurs in two independent enzymatic steps: 1. the two-electrons reduction of nitrate to nitrite catalyzed by NADH-nitrate reductase, and, 2. the six-electrons reduction of nitrite to ammonia catalyzed by ferredoxin-nitrite reductase. Both enzymes have been purified and characterized, and some of their properties have been studied.     

Unusual distribution of a glycolipid antigen in the flagella ofChlamydomonas

Summary Mouse hybridomas were obtained that secrete monoclonal antibodies recognizing glycolipid antigens located in the flagellar membrane of the biflagellate alga,Chlamydomonas reinhardtii. The antigen is an acidic lipid that migrates slightly slower than a GM1 ganglioside on thin layer chromotography. The binding of the antibodies to the thin layer plate was inhibited by periodate oxidation suggesting that the antibodies are recognizing a carbohydrate epitope. In a variety ofChlamydomonas strains, these antibodies were found to stain the flagella of only a sub-set of the cells in the population, generally varying from 50% to 75% of the cells. Even after cloning, the population of cells continued to express this variability in staining, and presumably, expression of the glycolipid epitope. Although most cells showed either strong staining of both flagella or no detectable staining of both flagella, a subset of the cells in the culture exhibited differential antibody labeling of the two flagella, suggesting that an individualChlamydomonas can exhibit a different glycolipid composition in each of its two flagellar membranes and even differential expression along the length of an individual flagellum.