A protocol for rapid DNA extraction from<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> for PCR analysis

We present a method for instant DNA extraction fromArabidopsis thaliana based on a simple DNA extraction method (Edwards et al., 1991). A piece of rosette leaf (typically 3–5 mg) was ground in a centrifuge tube in extraction solution. Extracted DNA was suitable for PCR analysis, without centrifugation. The feasibility of this method was confirmed by testing 24 primer sets. This method requires less than 1 mg of plant tissue and is useful for genetic mapping, transgene detection, and other experiments.     

A rapid procedure for the isolation of chloroplast DNA fromChlamydomonas using the TL-100 ultracentrifuge

We describe a rapid and efficient procedure for the isolation of chloroplast (and nuclear) DNA from walled cells ofChlamydomonas reinhardtii. Total nucleic acids are prepared and separated by equilibrium centrifugation in CsCl-bisbenzimide gradients using a high-speed table-top ultracentrifuge. Chloroplast DNA sufficient for several restriction analysis is obtained from 1 liter of cells in one to two days.     

Am improved procedure for the isolation of chloroplast DNA fromChlamydomonas reinhardtii

The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation. We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios. An erratum to this article is available at .     

A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants

Protocols for genomic DNA extraction from plants are generally lengthily, since they require that tissues be ground in liquid nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening projects.     

cDNA and deduced amino acid sequences of cytochrome c fromChlamydomonas reinhardtii: Unexpected functional and phylogenetic implications

Summary We have isolated complementary DNA (cDNA) clones for apocytochrome c from the green algaChlamydomonas reinhardtii and shown that they are encoded by a single nuclear gene termedcyc.Cyc mRNA levels are found to depend primarily on the presence of acetate as a reduced carbon source in the culture medium. The deduced amino acid sequence shows that, apart from the probable removal of the initiating methionine,C. reinhardtii apocytochrome c is syntheszed in its mature form. Its structure is generally similar to that of cytochromes c from higher plants. Several punctual deviations from the general pattern of cytochrome c sequences that is found in other organisms have interesting structural and functional implications. These include, in particular, valines 19 and 39, asparagine 78, and alanine 83. A phylogenetic tree was constructed by the matrix method from cytochrome c data for a representative range of species. The results suggest thatC. reinhardtii diverged from higher plants approximately 700–750 million years ago; they also are not easy to reconcile with the current attribution ofChlamydomonas reinhardtii andEnteromorpha intestinalis to a unique phylum, because these two species probably diverged from one another at about the same time as they diverged from the line leading to higher plants.     

Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci

The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles.     

Glutamate synthase from Chlamydomonas reinhardtii: Interaction studies with its substrate ferredoxin and molecular cloning

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able to form functional covalent complexes with its substrate ferredoxin (Fd), either wild-type (^WTFd) or recombinant form (^rFd). However, when Fd carboxyl groups were chemically modified (^mdFd), no complexes were detected and its ability to serve as electron donor for glutamate synthase activity was also decreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and a negative core in the helix α1 are critical for Fd interaction with this enzyme and its functionality as electron carrier for glutamate synthase. As a previous step to elucidate the specific positive charged residues involved in glutamate synthase interaction with Fd, we have isolated a cDNA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii. The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structurally more similar to higher plant enzymes than to the corresponding prokaryotic GOGAT. Two conserved domains were present in this protein fragment, the FMN-binding domain and the cysteines involved in the iron–sulfur cluster binding. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of a Chlamydomonas reinhardtii gene encoding a protein of the DNA photolyase/blue light photoreceptor family

The organization and nucleotide sequence of a gene from Chlamydomonas reinhardtii encoding a member of the DNA photolyase/blue light photoreceptor protein family is reported. A region of over 7 kb encompassing the gene was sequenced. Northern analysis detected a single 4.2 kb mRNA. The gene consists of eight exons and seven introns, and encodes a predicted protein of 867 amino acids. The first 500 amino acids exhibit significant homology with previously sequenced DNA photolyases, showing the closest relationship to mustard (Sinapis alba) photolyase (43% identity). An even higher identity, 49%, is obtained when the Chlamydomonas gene product is compared to the putative blue-light photoreceptor (HY4) from Arabidopsis thaliana. Both the Chlamydomonas and the Arabidopsis proteins differ from the well characterized DNA photolyases in that they contain a carboxyl terminal extension of 367 and 181 amino acids, respectively. However, there is very little homology between the carboxyl terminal domains of the two proteins. A previously isolated Chlamydomonas mutant, phrl, which is deficient in DNA photolyase activity, especially in the nucleus, was shown by RFLP analysis not to be linked to the gene we have isolated. We propose this gene encodes a candidate Chlamydomonas blue light photoreceptor.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii

The development of techniques allowing the unattended collection of RNA from cell samples at room temperature makes practical accurate and facile monitoring of circadian rhythms in Chlamydomonas reinhardtii. The utility of these methods was demonstrated by collecting RNA samples for three days from cells maintained in continuous darkness. Every hour, cells were automatically collected and lysed with buffer containing SDS and proteinase K. Samples were maintained at room temperature with little or no evidence of degradation of RNA. Strong, non-damping circadian rhythms of cab mRNA abundance were measured. Free-running rhythms of about 24 h were measured from cultures maintained at 16, 20, 25 and 30 °C, thus demonstrating temperature compensation of circadian period. Simultaneous collections from cultures previously entrained to 12 h light/12 h dark cycles of opposite phase displayed circadian rhythms of cab mRNA abundance that were in phase with their previous entraining light cycles. Thus, this result suggests that the measured circadian rhythms of cab mRNA abundance was not an artifact of the collection procedure.     

Chloroplast nucleoids in large number and large DNA amount with regard to maternal inheritance inChlamydomonas reinhardtii

Summary We studied the maternal chloroplast inheritance ofChlamydomonas reinhardtii by epifluorescence microscopy after staining with DNA specific fluorochrome DAPI and by genetic methods, using wild type cells and cells containing previously isolated mutation of cond-1 and cond-2. Wild type cells contained about 7 chloroplast (cp) nucleoids, while mutants, cond-1(+) and cond-2(+), contained about 14 and 23 cp nucleoids, respectively, after one week culture on agar plates. The total cpDNA contents were almost proportional to the numbers of cp nucleoids. When cells containing cond-1 or cond-2 mutation were used as a parental source to cross with wild type cells of the other parent, preferential digestion of cp nucleoids from male parent (mt^–) origin occurred in the zygotes, although the frequencies of the digestion were slightly lower than that in the zygotes from the cross between wild type cells. Western blot analysis of the protein ofzyslB gene, which has been found related to preferential digestion of mt^– origin cp-nucleoids DNA, showed that a high amount of this protein was detected with the initiation of preferential digestion of mt^– cp nucleoids and disappeared with the completion of the digestion. Cp genetic markers for antibiotic resistance were maternally inherited in all crosses. These results showed that although the preferential digestion of cp nucleoids consisting of large number and large cpDNA amount requires a slightly longer period to complete, this high ploidy of the cp nucleoids does not disturb maternal inheritance.     

Embryo sac isolation inArabidopsis thaliana: A simple and efficient technique for structure analysis and mutant selection

Arabidopsis thaliana has been widely used as a model plant in gene function analysis. However, its tiny flower and curved embryo sac make it difficult to study gene expression during megagametogenesis, fertilization, and early embryogenesis, especially in the screening of mutants from those developmental processes. The techniques currently available are sectioning and whole-mount clearing of ovules; however, sectioning is time consuming and laborious for quantitative analysis, and whole-mount clearing, makes clear cytological observation impossible. Reported here is a simple and efficient method based on enzymatic isolation of embryo sacs that enables both quantitative analysis and elaborate cytological observation for gene expression investigation and mutant screening.     

A colorimetric confirmation method for DNA amplification in PCR and its application to the detection of Giardia lamblia cysts

A simple and quick colorimetric method for confirming DNA amplification in polymerase chain reactions (PCR) is described and has been applied to the amplification of Giardia lamblia DNA. This method detects the release of pyrophosphate based on the competition between 1,10-phenanthroline and pyrophosphate complexing with ferrous ion. When 1,10-phenanthroline complexed with Fe²⁺ is added to the finished PCR solution, depending on whether or not the DNA was amplified, the mixture is, respectively, either bleached or red. The color changed optimally for 20–30 min at 60–80 °C, and the result could be determined by detecting an absorbancy change at 510 nm or a color change discernible to the naked eye. The extent of change in absorbance was proportional to the amount of pyrophosphate produced.     

Rapid isolation of fungal genomic DNA suitable for long distance PCR

A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications. We have compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. For Myrothecium gramineum, the highest DNA concentration was obtained with the procedure described by N. Vanittanakom et al. (J Clin Microbiol 2002, 40: 1739–1742). For A. nidulans, concentrations higher than 100 ng/μl were reached with the glass bead, the LiCl, the boiling, the liquid N₂ and the protoplast-based method. Samples of M. gramineum resulting from the boiling and the liquid N₂ procedure were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the reproducible amplification of fragments up to 1 kb. Amplification of fragments up to 4.3 kb requires the use of the Elongase Mix on samples extracted with the liquid N₂ procedure.     

Genes conferring late flowering inArabidopsis thaliana

Three naturally occurring late flowering, vernalization responsive ecotypes ofArabidopsis thaliana, Pitztal, Innsbruck and Kiruna-2, were each crossed with the early flowering ecotypes of Landsbergerecta, Columbia and Niederzenz. Analysis of the subsequent generations suggested that late flowering in Kiruna-2 is recessive and mainly determined by a single, late flowering gene. This late flowering gene is not, however, the same as that in any of the late flowering mutants generated in the Landsbergerecta background. Both Pitztal and Innsbruck appear to contain the same dominant gene which confers late flowering to these ecotypes. The early flowering parents Niederzenz and Landsberg both contain genes which modify the phenotype of this dominant late flowering locus, causing F₁ plants to flower either earlier (Landsberg) or later (Niederzenz) than the late parent. Mapping of the dominant late flowering locus from Pitztal demonstrated that late flowering co-segregated with an RFLP marker from one end of chromosome 4. This is a similar position to that ofFLA, the gene responsible for late flowering of theArabidopsis ecotypes Sf-2 and Le-O.     

The cytoplasmic ribosomes of Chlamydomonas reinhardtii: characterization of antibiotic sensitivity and cycloheximide-resistant mutants

Summary In vitro protein synthesis was used to characterize the antibiotic sensitivity of cytoplasmic ribosomes from wild-type and antibiotic-resistant strains of Chlamydomonas reinhardtii. Cytoplasmic ribosomes from two cycloheximide-resistant mutants, act-1 and act-2, were resistant to the antibiotic in vitro. The alteration effected by the act-1 mutation, which was dominant in diploids, was localized to the large subunit of the cytoplasmic ribosomes, but no ribosomal protein alterations were detected using two-dimensional gel electrophoresis. The act-2 mutation, which was semidominant in diploids, was frequently associated with a charge alteration in the large subunit ribosomal protein (r-protein) cyL38 that segregated independently from the antibiotic-resistant phenotype in crosses.     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.