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1.
Marieke R. Samson Frans M. Klis Wieger L. Homan Piet van Egmond Alan Musgrave Herman van den Ende 《Planta》1987,170(3):314-321
Sexual interaction between gametes of opposite mating type (mt) of the unicellular green alga Chlamydomonas eugametos starts with agglutination of the cells via particular glycoproteins on the flagellar surface. Purification of these socalled agglutinins was achieved by a three-step procedure consisting of, successively, gel filtration, anion-exchange chromatography, and high-performance gel filtration. The amino-acid and sugar compositions of both agglutinins showed a high degree of similarity; the most prominent amino acids were hydroxyproline, serine and glycine, and the main sugars were arabinose and galactose. The carbohydrate portions represented about half of the molecular mass of both agglutinins. Using high-performance gel filtration, a calibration curve was constructed for high-molecular-mass compounds from which the Stokes' radius of the sexual agglutinins could be estimated. The mt
+ agglutinin had a Stokes' radius of 39 nm and a sedimentation coefficient of 9.3 S. From these data its molecular mass was estimated to be 1.2·106. The corresponding data for the mt
- agglutinin were 38 nm, 9.7 S and 1.3·106, respectively. The biological activity of both agglutinins was destroyed by mild periodate treatment. Treatment with specific glycosidases had a differential effect on the biological activity of the agglutinins. These observations indicate that carbohydrate side-chains are needed for biological activity and perhaps are responsible for the specifity of the sexual agglutinins. A comparison of both agglutinins is given and their possible structure is discussed in relation to their amino-acid and sugar compositions.Abbreviations HP
high performance
-
mt
mating type
- SDS
sodium dodecyl sulfate 相似文献
2.
Robert W. Lee Carole Dumas Claude Lemieux Monique Turmel 《Molecular & general genetics : MGG》1991,231(1):53-58
Summary We report that the mitochondrial genome of Chlamydomonas moewusii has a 22 kb circular map and thus contrasts with the mitochondrial genome of Chlamydomonas reinhardtii, which is linear and about 6 kb shorter. Overlapping restriction fragments spanning over 90% of the C. moewusii mitochondrial DNA (mtDNA) were identified in a clone bank constructed using a Sau3AI partial digest of a C. moewusii DNA fraction enriched for mtDNA by preparative CsCI density gradient centrifugation. Overlapping Sau3AI clones were identified by a chromosome walk initiated with a clone of C. moewusii mtDNA. The mtDNA map was completed by Southern blot analysis of the C. moewusii mtDNA fraction using isolated mtDNA clones. Regions that hybridized to C. reinhardtii or wheat mitochondrial gene probes for subunit I of cytochrome oxidase (cox1), apocytochrome b (cob), three subunits of NADH dehydrogenase (nadl, nad2 and nad5) and the small and the large ribosomal RNAs (rrnS and rrnL, respectively) were localized on the C. moewusii mtDNA map by Southern blot analysis. The results show that the order of genes in the mitochondrial genome of C. moewusii is completely rearranged relative to that of C. reinhardtii. 相似文献
3.
H. van den Ende 《Sexual plant reproduction》1995,8(3):139-142
In Chlamydomonas monoica, cell division and mating are interdependent processes, since under gametogenic conditions only newly born cells are mating competent. By refeeding nitrogen-starved cells with nitrate or ammonium ions, cell division and mating were synchronized. The mating competence of the progeny cells was dependent on the amount of the nitrogen source parent cells were refed, with an optimum around 0.1 mol·105 cells. A second treatment with nitrate inhibited gametogenesis, but only when applied during the first part of the cell cycle, suggesting that an essential part of sexual development takes place during this period. During the latter part of the cell cycle, cells required light to acquire mating competence. 相似文献
4.
Monique Turmel Guy Bellemare Robert W. Lee Claude Lemieux 《Plant molecular biology》1986,6(5):313-319
Summary The unicellular green alga Chlamydomonas moewusii contains small DNA species of unknown cellular location. We report that the most abundant of these DNAs, here designated low-molecular-weight DNA (LMW DNA), is a linear molecule of 5.9 kilobase pairs (kbp). Southern blot hybridization and restriction enzyme analysis revealed that the LMW DNA sequence also exists as an integrated sequence in a discrete region of the chloroplast genome. We have confirmed earlier reports that small DNA species related to the LMW DNA are absent from Chlamydomonas eugametos, an alga which is interfertile with C. moewusii. In the C. eugametos chloroplast genome we found only remnants of the LMW DNA sequence. 相似文献
5.
Marinus Versluis Frans Schuring Frans M. Klis Piet van Egmond Herman van den Ende 《FEMS microbiology letters》1992,97(1-2):101-105
Abstract The sexual mating reaction between gametes of the green alga Chlamydomonas eugametos starts by cell-cell contacts involving sex-specific cell-adhesion molecules (agglutinins) at the flagellar membrane. An in vitro adhesion assay is described using glutaraldehyde-fixed gametes. In vitro adhesion was fully comparable to in vivo adhesion, making it a reliable assay to study the initial recognition step of sexual adhesion in vivo. It was shown that both agglutinins are capable of inhibiting sexual adhesion at similar concentrations (1−2×10−10 M), indicating that mt+ and mt− agglutinins interact with each other during binding. This was confirmed by demonstrating that charcoal particles adsorbed with purified agglutinins of the opposite mating type aggregate with each other. 相似文献
6.
A. M. Tomson R. Demets E. A. van Spronsen G. J. Brakenhoff D. Stegwee H. van den Ende 《Protoplasma》1990,155(1-3):200-209
Summary During gamete-gamete adhesion in the unicellular green algaChlamydomonas eugametos, the sexual adhesion molecules or agglutinins that are located on the flagella are subject to tip-oriented migration and rapid inactivation. It is demonstrated that sexual adhesiveness is maintained by incorporation of additional agglutinins, recruited from a cellular pool. The location of this reservoir is unknown but, as indicated by its insensitivity to the chaotropic agent guanidine thiocyanate, it appears to be distinct from the large amount of agglutinins on the plasma membrane of the cell body. By viewing flagella of conjugating gametes in a confocal scanning laser microscope after immuno-labelling of the agglutinins, evidence was obtained for a linear arrangement of the agglutinins in two rows on the flagellar surface. This suggests that after insertion at the base of the flagellum, the agglutinins follow linear tracks to the tip and that the transport system is confined to two longitudinal domains. It is estimated that the half-life of flagellar agglutinins drops from 1–2 h in nonconjugating gametes to 1 min during conjugation, which suggests that after incorporation at the flagellar base, the agglutinins migrate to the tip with a velocity of 100 nm/s. Presumably after arrival at the tip, the molecules are inactivated. It is postulated that rapid turnover and transport of agglutinins are required for optimal signalling between partner gametes.Abbreviations BSA
bovine serum albumine
- CHI
cycloheximide
- CSLM
confocal scanning laser microscope
- GA
glutaraldehyde
- GTC
guanidine thiocyanate
- GAM-IgG
goat-anti-mouse immuno-globuline
- mAb
monoclonal antibody
- mt
mating type
- PBS
phosphate-buffered saline
- SDS
sodiumdodecyl sulphate
- TRIS
tris-(hydroxymethyl)-aminomethane 相似文献
7.
8.
9.
K. J. Crabbendam N. Nanninga A. Musgrave H. van den Ende 《Archives of microbiology》1984,138(3):220-223
An alteration of the form and ultrastructure of the tips of the flagella of Chlamydomonas eugametos, occurring during sexual agglutination, is shown to be persistent in the mt
- flagella of the resulting vis-à-vis pairs. It is argued that this phenomenon is related to the lack of motility of mt
- flagella in vis-à-vis pairs of this species. 相似文献
10.
In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)+ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40 000 clones in each library revealed 44 clones which hybridize preferentially with a [32P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [32P] cDNA probe synthesized from poly(A)+ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis. 相似文献
11.
Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells 总被引:4,自引:0,他引:4
P. C. Sijmons A. J. A. Nederbragt F. M. Klis H. Van Den Ende 《Archives of microbiology》1987,148(3):208-212
Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mta and mt) were purified and analysed. The constitutive agglutinin from mta cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mta cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt cells only doubled the apparent agglutinin activity.Abbreviations mt
mating type
- DTT
dithiothreitol
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- YPG
yeast-peptone-glucose
- PAS
periodic-acid-Schiff reagent 相似文献
12.
The gamete activity of compatible mating strains of the isogamous, heterothallic species Chlamydomonas eugametos was investigated. Gamete activity was optimum within 4 h after flooding of agar slants and was maintained over a 24-h period. When male and female mating strains were mixed in proportions of 1:4, 2:3, 1:1, 3:2, and 4:1, the results based on zygote yield, indicated the strains exhibited different degrees of gamete activity. The male strain consistently showed less gamete activity than the female strain in a variety of culture conditions. 相似文献
13.
14.
Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels. 相似文献
15.
Ligation-mediated suppression-PCR as a powerful tool to analyse nuclear gene sequences in the green alga Chlamydomonas reinhardtii 总被引:1,自引:0,他引:1
To improve the analysis of unknown flanking DNA sequences adjacent to known sequences in nuclear genomes of photoautotrophic
eukaryotic organisms, we established the technique of ligation-mediated suppression-PCR (LMS-PCR) in the green alga Chlamydomonas reinhardtii for (1) walking from a specific nuclear insertion fragment of random knockout mutants into the unknown flanking DNA sequence to identify and analyse disrupted genomic DNA regions and for (2) walking
from highly conserved DNA regions derived from known gene iso-forms into flanking DNA sequences to identify new members of
protein families. The feasibility of LMS-PCR for these applications was successfully demonstrated in two different approaches.
The first resulted in the identification of a genomic DNA fragment flanking a nuclear insertion vector in a random knockout mutant whose phenotype was characterised by its inability to perform functional LHC state transitions. The second approach
targeted the cab gene family. An oligonucleotide of a cabII gene, derived from a highly conserved region, was used to identify
potential cab gene regions in the nuclear genome of Chlamydomonas. LMS-PCR combined with 3′ rapid amplification of cDNA ends (3′ RACE) and a PCR-based screening of a cDNA library resulted in the identification of the new cabII gene lhcb4. Both results
clearly indicate that LMS-PCR is a powerful tool for the identification of flanking DNA sequences in the nuclear genome of
Chlamydomonas reinhardtii.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
16.
Marie Hugosson Ghasem Nurani Elzbieta Glaser Lars-Gunnar Franzén 《Plant molecular biology》1995,28(3):525-535
It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F1 subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP
chloroplast transit peptide
- mTP
mitochondrial targeting peptide
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- pF1(1,25)
a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F1 subunit
- PsaF(2–30) and PsaF(2–61)
mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively 相似文献
17.
The snow alga Chlamydomonas nivalis was collected from the Sierra Nevada, California, USA, and examined for its ability to produce phenolic compounds, free proline,
and provide antioxidant protection factor in response to UV-A and UV-C light. Exposure of C. nivalis cells to UV-A light (365nm) for 5 days resulted in a 5–12% increase in total phenolics, where as exposure to UV-C light (254
nm) resulted in a 12–24% increase in phenolics after 7 days of exposure. Free proline was not affected by UV-A, but increased
markedly after UV-C exposure. A three-fold increase in free proline occurred within two days after exposure to UV-C, but then
dropped as cells became bleached. Antioxidant protection factor (PF) increased after treatment of cells with UV-A and remained
constant throughout UV-C exposure. Spectral analysis of algal extracts revealed a decrease in absorption in the 215–225 nm
region, short-term (2day) stimulation of pigment at 280 nm, and an increase in carotenoids (473 nm), after exposure to UV-A.
Snow alga exposed to UV-C light had a different spectrum from that of UV-A exposed cells, i.e. an enhancement of three major
peaks at 220, 260, and 280 nm, and loss of absorption in the carotenoid region.We report that UV light exposure, especially
in the UV-C range, can stimulate phenolic-antioxidant production in aplanospores of C. nivalis effecting biochemical pathways related to proline metabolism.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
18.
A set of chlorophyll fluorescence methods, including PEA (Plant Efficiency Analyser), PAM (Pulse Amplitude Modulated fluorometer),
and picosecond fluorometer, was employed to study PS 2 heterogeneity in sulfur deprived green algae Chlamydomonas reinhardtii. The regression method and JIP test were applied to analyze chlorophyll fluorescence kinetics. The fractions of PS 2 characterized
by the energetic disconnection, smaller antenna size, elevated constant rate of primary photochemistry, and inability to maintain
ΔpH-dependent energy dissipation increased essentially already after 12 h of incubation in sulfur depleted medium. The amount
of PS 2 centers with reduced QA (closed state), QB-non-reducing centers with impaired water splitting function, and centers coupled to the plastoquinone pool with the slow
cycle rate increased dramatically after 24 h period of deprivation. The mechanisms of PS 2 inactivation under sulfur deprivation
are discussed. 相似文献
19.
Heather C. Huppe Frédéric de Lamotte-Guéry Jean-Pierre Jacquot Bob B. Buchanan 《Planta》1990,180(3):341-351
The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE
diethylaminoethyl
- ELISA
enzyme-linked immunosorption assay
- FBPase
fructose 1,6-bisphosphatase
- Fd
ferredoxin
- FPLC
fast protein liquid chromatography
- FTR
ferredoxin-thioredoxin reductase
- FT system
ferredoxin-thioredoxin system
- kDa
kilodaltons
- Mr
relative molecular mass
- NADP-MDH
NADP-malate dehydrogenase
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures. 相似文献
20.
Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K
m values for the oxidized form of glutathione (approx. 50 M). However, the K
m values for NADPH were different, being 7 M and 28 M for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.Abbreviations GSSG
glutathione, oxidized form
- GR
NAD-PH-glutathione reductase (EC 1.6.4.2)
- G3P
glyceraldehyde-3-phosphate
- pI
isoelectric point
- SDS-PAGE
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
This work was supported in part by grants NO. PB 87–401, PB 90–99 and BIO 91–1078 of the DGICYT (Ministerio de Educatión y Ciencia, Spain) and the Autonomous Government of Andalusia (Spain). Postdoctoral aid from the Alexander von Humboldt Foundation (Bonn, FRG) to A.S. is also acknowledged. 相似文献