Anita Johnson

Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation.

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

SummaryThe respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 × 10−7) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10−7). No colonies were seen on control plates (frequency < 0.96 × 10−9). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.

A Standardized TLC Analysis of f3-Orcinol Depsidones

Many p-orcinol depsidones with low Rf values in the three stan- dardized TLC solvents commonly used in lichenology are better resolved by a solvent system of toluenelethyl acetate/formic acid. Standardized chromatographic data and examples of specific problems resolved by one- and two-dimensional TLC with this solvent system are given. Chemical analyses are reported for 25 lichen species from 17 genera. A vexing problem in the identification of lichen products by thin-layer chromatography (TLC) has been the separation of some 35 low-Rf, p-orcinol compounds many of which are still unknowns. In the three standardized solvent systems (Culberson & Kristinsson 1970; Culberson 1972, 1974; Culberson & Ammann 1979), most of these compounds run below the control substance norstictic acid, which has Rf values between 28 and 40. Even if the spots for these substances were evenly spaced, the Rf values would differ by only about one millimeter. In addition, we now know that these compounds occur in complicated mixtures and consequently accurate identifications will require more sophisticated chro- matographic techniques than those routinely used. Reports in the recent literature of low- Rf compounds (even common ones like salazinic, constictic and connorstictic acids) may be misidentifications based upon insufficient TLC evidence. The purpose of the present paper is to give standardized TLC data for a fourth solvent system (Solvent G) to assist the resolution of difficult mixtures of low-Rf, p-orcinol depsidones by one- and two-dimen- sional TLC. The chromatographic procedure followed the standardized method (Culberson 1972) except that toluene (dried over CaCl2 and redistilled) was used in place of benzene. Solvent G, toluene/ethyl acetate/formic acid (139:83:8, v/v/v; 230 ml), is modified from previously suggested systems (Stahl & Schorn 1961; Hirayama et al. 1976). Formic acid (95-97%) and ethyl acetate were not purified or redistilled; it is not necessary to purify a good grade of toluene for satisfactory results with Solvent G. As with Solvent B, however, a grade of formic acid with low water content is essential. TLC plates were Merck Silica Gel 60 F254 (0.25-mm layer; No. 5765). Chromatographic tanks (Desaga- Heidelberg) were insulated from external temperature fluctuations but were not lined with filter paper. Pre-equilibration of the plates in 60% formic acid (Culberson 1974) did not improve the quality of chromatograms in Solvent G. Unless noted otherwise, the standardized data in Table 1 were from shortened plates (12.5 x 20 cm) developed to 10 cm. Initial spots were 2 cm from the bottom of the plate and 0.5 cm above the solvent. Controls for standardization were included at three positions on every plate. The Rf data in Table 1 are standardized with respect to fumarprotocetraric acid in addition to the usual control substances, atranorin and norstictic acid. Two-dimensional chromatograms were prepared by the standardized method (Culberson & John- son 1976) except that full plates (20 x 20 cm) were used with seven one-dimensional comparisons (including two atranorin-norstictic acid controls) in each direction. In addition, for the second direc- tion, an atranorin-norstictic acid control was added just to the left of the origin on the two-dimensional portion of the plate. This control helps to align spots and to monitor effects of residual first-direction solvent on R, values in the second direction. (In the present report, first- and second-direction solvents

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii

SummaryWe have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.

Xanthones and depsidones of the lichen Lecanora dispersa in nature and of its mycobiont in culture

A chemotype ofthe natural lichen Lecanora dispersa contains 2,7-dichlorolichexanthone as the major secondary compound, but cultured spore isolates, growing without the alga, produced pannarin and related depsidones (HPLC, TLC and MS) instead. HPLC data suggested that traces of the xanthone characteristic of the source lichen might also be in some of the pannarin-producing sporelings, but concentrations were not sufficient for TLC or MS confirmation. Surprisingly, pannarin could not be confirmed in voucher material of the natural source lichen, but the biosynthetic potential for this depsidone was proven for the species by a herbarium survey. When museum samples larger than single apothecia were analyzed, trace to moderate proportions of pannarin were confirmed in some specimens. That the source lichen produced primarily xanthones and the mycobiont produced primarily depsidones is surprising because xanthones are a class of compounds also common in non-lichen fungi whereas depsidones like pannarin are nearly restricted to lichens. Pannarin, which has previously not been reported from an isolated lichen mycobiont, was produced copiously by some cultures.

A standardized two-dimensional thin-layer chromatographic method for lichen products

Abstract Two-dimensional thin-layer chromatography is useful for microchemical studies on mixtures difficult to resolve by the standardized one-dimensional thin-layer chromatographic method now commonly used for lichen products. A modified two-dimensional technique uses the large body of standardized RF data already accumulated for these compounds. In addition, correlations of RF values with chemical structures permit tentative identifications of many trace constituents, including new natural products, resolved from microextracts by the two-dimensional method. The standardized two-dimensional procedure also allows more reliable comparisons of chromatograms and the determination of RF classes of components of complex mixtures. The method is illustrated for the orcinol-type depsides of two closely related species, Parmelia loxodes and P. verruculifera.

Characteristic lichen products in cultures of chemotypes of the Ramalina siliquosa complex

Sixteen characteristic aromatic lichen products were identified in alga-free fungal cultures derived from single-spore isolates of four chemotypes of the Ramalina siliquosa species complex. The chemistries of many cultures are more complex than those of natural thalli. The compounds identified include the major f-orcinol depsidones that characterize the chemotypes as well as biogenetically reasonable precursors not yet proved in natural thalli. Single-spore progeny from natural thalli can be assigned to chemotype without being lichenized with an alga. This result simplifies the use of secondary-product chemistry as a genetic marker to assess the limits of gene flow between chemotypes in natural populations.

Manipulating the Chloroplast Genome of Chlamydomonas — Molecular Genetics and Transformation

In photosynthetic eukaryotes, genes encoding chloroplast proteins of multimeric complexes involved in photosynthesis and protein synthesis are partitioned between the cell’s nuclear and chloroplast genomes. The chloroplast genome’s relatively small size, high copy number and great evolutionary conservation have led to rapid progress in identifying and characterizing chloroplast genes. In fact, the entire chloroplast genomes of tobacco, liverwort and rice have been sequenced within the last three years (1–3). Somewhat less progress has been made in identifying the nuclear genes for the remainder of the chloroplast polypeptides, due to the larger number of genes and the much larger size and evolutionary diversity of the nuclear genomes themselves. However in many plant species, nuclear genes can be engineered in vitro, the constructs introduced by transformation, and their function studied in vivo.