Isolation and partial characterization of mutants of the trypanosomatid Crithidia fasciculata and their use in detecting genetic recombination

Crithidia fasciculata was used as a model trypanosomatid to study the possible existence of genetic recombination in this group of protozoa. The approach was based on the ability to select a variety of mutants on agar plates. Following mutagenesis of wild type cells by nitrosoguanidine or ethylmethanesulfonate, stable mutant phenotypes were obtained. These included mutants resistant to the drugs actinomycin D, 6-azauracil, 6-azauridine, and 5-fluorouracil, auxotrophs and colony morphology mutants. Following mixed growth of pairs of drug-resistant mutants on selective media, isolates exhibiting stable recombinant phenotypes were obtained. The data presented suggest that 1) Crithidia undergoes some type of genetic recombination and 2) Crithidia must be diploid at some time during this process.     

Genetic alleviation of the self-fertilization complication when hybridizing monoecious Gelidium vagum

Fundamental genetic studies were initiated for the monoecious red alga Gelidium vagum. Color and sterility mutants were isolated and characterized to provide genetic tools, initially to identify hybrid plants when they occurred in crosses, and secondarily to eliminate self-fertilization altogether. When fertility phenotypes were scored, rapid onset of reproduction in culture was favored by long day-length, moderately high irradiance levels from fluorescent lights, warm temperature and the addition of Tris buffer to the medium. A recessive green mutant (designated grn 1) was characterized and used in subsequent crosses to allow a clear distinction between non-hybrid (green) and hybrid (red) offspring. Additional color mutants and a variety of reproductive mutants were also isolated and characterized. Male-sterile mutants had phenotypes ranging from apparently normal plants to those that produced no spermatia. Female-sterile mutants also included a variety of phenotypes, some plants having post-fertilization malfunctions during the development of the carposporophyte. Only a fraction of the sterility mutations have been phenotypically or genetically characterized, but some are straightforwardly inherited as stable, nuclear, single-gene defects. From the genetic recombination pattern, one female-sterile mutant may be loosely linked (39 cMorgans) to the grn 1 marker gene. Male sterility very effectively eliminated selfing without affecting the production of carpospores in crosses, thereby overcoming one of the most serious genetic difficulties in working with this monoecious species.     

Meiotic recombination in Cochliobolus pallescens as a tool for strain improvement

Sterile morphological variants of the self-fertile Cochliobolus pallescens were obtained. Certain combinations of such strains formed fruit bodies. Selected strains were submitted to a genetic analysis, which resulted in the F2 to stable sterile mutants. These can be used to phenocopy mating types. Thus this fungus can be used for genetic experiments requiring meiotic recombination. This opens the possibility of using the mutants as recipients (zero strains) in strain improvement by genetic engineering for the anamorphic closely related Curvularia lunata, which is of great biotechnological value for the 11-OH hydroxylation of steroids. Correspondence to: K. Esser     

Different functional roles of RTR complex factors in DNA repair and meiosis in Arabidopsis and tomato

The RTR (R ecQ/Top 3/Rmi 1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyper-recombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: the top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom.     

A series of mutable alleles of the white locus in Drosophila melanogaster

Summary The origin and phenotypes of a number of zeste mutant stocks with mutable white loci are described. Each newly arising form was lighter in eye color than the mutant it originated from. In each case the lighter pigmentation is believed to be due to an increase in genetic material in the proximal region of the white locus, the increase supposedly being the result of unequal crossing over. Some of the mutations which arose in the mutable stocks are reversions. They occurred in males as well as in homo- and heterozygous females. The reversions are believed to be due to a decrease in genetic material in the proximal region of the white locus. The decrease is assumed to be the result of intrachromosomal recombination. At least some of these events took place premeiotically. New mutants which originate frequently from mutable stocks are stable. In addition to the structure of the mutable white locus there is probably at least one still unknown factor which affects its mutability since the frequency of mutations arising in the mutable stocks decreases over the years.     

Genetic nature, stability, and improved virulence of hybrids from protoplast fusion in Beauveria

Genetic improvement of two different strains of the entomopathogenic fungus Beauveria bassiana for more effective control of Ostrinia nubilalis and Leptinotarsa decemlineata was obtained by crosses with the insecticidal toxin-producing strain Beauveria sulfurescens. Protoplast fusion between diauxotrophic mutants resulted in the recovery of some stable prototrophic fusion products. The low levels of virulence of the wild type strain B. bassiana 28 isolated originally from L. decemlineata were enhanced both on L. decemlineata and O. nubilalis for one of the hybrids obtained (FP 8) from the cross B. bassiana 28 × B. sulfurescens 2. Fusion product 25 obtained from the cross between B. sulfurescens and the highly pathogenic strain B. bassiana 147 showed a three-day reduction in the LT₅₀ towards O. nubilalis. Southern blot hybridization with nine probe-enzyme combinations were conducted on genomic DNAs from the original wild strains, parental mutant strains, and fusion products. Additive banding patterns or unique banding pattern of either parental strain was observed in five hybrids, indicating their status as recombinant and/or partially diploïd. Combination of RFLP markers indicative of both parental genomes was never observed with fusion product FP 25. The stability of the virulence following passage through insect-host and stability of molecular structure for the fusion products FP 8 and FP 25 suggest that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi.     

A search for allelic recombination in Chinese hamster cell hybrids

Summary Mutants resistant to 6-thioguanine were selected from CHO cells which were either temperature sensitive or proline requiring. These mutants were stable and had low levels of hypoxanthine guanine phosphoribosyl transferase (HGPRT). Hybrids were selected which were heteroallelic at the hgprt locus and complementation between the mutants used was not observed. Interallelic recombination at this locus would generate hgprt ⁺ cells which could be selected in Littlefield's HAT medium. Selection experiments with hybrids containing three different pairs of mutants yielded no recombinants among populations of 4x10⁶-2x10⁷ cells. After treatment with the recombinagen mitomycin C, 3 putative recombinants were detected amongst 1.4x10⁷ surviving cells from one hybrid. One of these strains was examined and shown to have a normal level of HGPRT and its heterozygosity at this locus was demonstrated by the segregation of colonies resistant to 6-thioguanine. It cannot be excluded that the rare hgprt ⁺ colonies seen arose by mutation rather than by recombination. Mitotic allelic recombination therefore appears to be a much less frequent event in CHO cells than it is in lower eukaryotes. It is possible that mitotic recombination is effectively suppressed in mammalian cells to prevent the expression of deleterious recessive mutants.     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

Targeted deletion of the <Emphasis Type="Italic">uvrBA</Emphasis> operon and biological containment in the industrially important <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

From a Bacillus licheniformis wild type as well as a defined asporogenous derivative, stable UV hypersensitive mutants were generated by targeted deletion of the uvrBA operon, encoding highly conserved key components of the nucleotide excision repair. Comparative studies, which included the respective parental strains, revealed no negative side effects of the deletion, neither on enzyme secretion nor on vegetative propagation. Thus, the uvrBA locus proved to be a useful deletion target for achieving biological containment in this industrially exploited bacterium. In contrast to recA mutants, which also display UV hypersensitivity, further strain development via homologous recombination techniques will be still possible in such uvr mutants.     

Genetic Recombination in Colletotrichum sublineolum

Genetic recombination without typical parasexuality (parameiosis) was shown in the fungus Colletotrichum sublineolum, causal agent of anthracnose on sorghum. The cross among auxotrophic mutants and mutants resistant to benomyl and cycloheximide confirmed the occurrence of heterokaryosis in this species. Aneuploid and haploid recombinants were obtained from heterokaryons. Some segregants released sectors continually after several replication cycles, and were considered aneuploids in the process of haploidization. Heterokaryons derived from crosses among mutants that presented low pathogenicity produced more virulent recombinants. Parasexuality with parameiosis may represent a natural mechanism for genetic variability amplification explaining the rapid appearance of new C. sublineolum physiological races.     

Transformation and recombination in rad mutants of Saccharomyces cerevisiae

Summary Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single-and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trpl-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trpl-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura352 mutant locus where only integration events have been observed, and at the trpl-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.     

Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions

The RadA/Sms protein is a RecA‐related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double‐strand break repair for YejH.     

An isoenzyme study in the genus Lotus (Fabaceae). Experimental protocols and genetic basis of electrophoretic phenotype

Summary An isoenzyme survey of some taxa in the genus Lotus (Fabaceae) was undertaken to increase the number of genetic markers available to breeders and to students of Lotus phylogeny. Twenty-one enzymes were examined using starch gel electrophoresis and nine buffer systems. Clear, consistent banding patterns were obtained for PGI, TPI, MDH, IDH (NADP), PGM, 6-PGDH, and ME. Clear but inconsistent banding patterns were obtained for FDP, G₃PDH (NADP), -EST, LAP, MDH, DIA, and NADHDH. Phenotypes of the seven consistently resolved enzyme systems were obtained for different tissues for each of several genotypes at different stages of development. Variation in enzyme phenotypes of the same individuals under different growth conditions indicated the presence of different isozymic forms of these enzymes. Shoot tissue of plants over 6 weeks of age was found to be suitable material for further genetic studies, since phenotype for this tissue was constant despite changes in growing conditions. A formal genetic analysis of segregation and/or recombination of allozymes for the enzymes PGM, TPI, MDH, IDH, and 6-PGDH was undertaken. Isoenzyme phenotypes were examined for the diploids L. alpinus Schleich., L. burttii Sz. Borsos, L. conimbricensis Brot., L. ornithopodioides L., L. tennis Waldst. et Kit., and L. uliginosus Schkuhr; and for the diploid interspecific hybrids L. alpinus x L. conimbricensis, L. burttii x L. ornithopodioides, and L. japonicus x L. alpinus. Several new loci were identified for Lotus, namely, Idh1, Idh2, Mdh3, Pgi1, Pgi2, Tpi1, Tpi2, and 6-Pdgh1. Duplications of loci of IDH, MDH, PGI, and 6-PGDH were detected in the diploid (2n=12) interspecific hybrid L. japonicus x L. alpinus.     

Effect of Mutations for the ruvABC Genes on Recombination between Direct DNA Repeats in Escherichia coli Strains Carrying Extended Tandem Duplications

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants forruvABCgenes. Homologous recombination in duplications of rec ⁺ strains and in recBC sbcB, recQand recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.     

Isolation of Rec? mutants from an F-prime merodiploid strain of Escherichia coli K-12

Summary This paper describes a method of screening mutagenised populations of an E. coli gal _A ^– gal _B ^– F-prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal⁺ papillae. With a suitable choice of gal ^–mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.     

Genetic determination of ploidy level in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formely Phaffia rhodozyma) is a basidiomycetous yeast-like fungus that produces carotenoids useful for the food industry. Recently, its sexual cycle was reported but little is known about its genetic constitution. To inquire into the ploidy state of X. dendrorhous, biased mutant spectrum, genetic complementation and mitotic recombination analysis were used. A wild-type strain was subjected to N-methyl-N′-nitro-N-nitrosoguanidine mutagenic treatment. Auxotrophic and carotene mutants were forced to revert to the wild-type phenotype. Pigment producing and prototroph revertants behaved as diploid except for adenine less mutants. These results are in agreement with the limited spectrum of auxotrophs obtained in this strain for the ADE1 locus. To analyze the genetic characteristic of the adenine genetic marker of X. dendrorhous, protoplast fusion experiments with several adenine less mutants were performed. The experiments presented in this work suggest that the ATCC 2430 (UDC 67-385) strain of X. dendrorhous is diploid and a heterozygous constitution is proposed for the ADE1 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nuclear and extranuclear inheritance of oligomycin resistance in Aspergillus nidulans

Summary A number of spontaneously-occurring, stable oligomycin-resistant mutants have been isolated in Aspergillus nidulans. Genetic characterisation showed that while most of the mutants examined were nuclear, one mutant was extranuclear as judged by several criteria. While the nuclear mutants showed no abnormalities on drug-free medium, the extranuclear mutant exhibited impaired growth ability. This character never segregated from the oligomycin-resistance character in any of the genetic experiments carried out, and appeared to be a secondary effect of the same mutation. The extranuclear genetic element coding for the oligomycin-resistance character was unable to co-exist in a stable fashion within the same mycelium as the wild type element, and they tended to segregate into sectors consisting almost wholly of one type or the other. The nuclear mutants showed incomplete dominance in heterozygous diploids, segregating fully resistant homozygous areas. All nuclear mutants mapped on linkage group VII.     

Effects of short‐term exposure to naturally occurring thymol concentrations on transmission of a bumble bee parasite

1. Plants produce antimicrobial phytochemicals that can reduce growth and infectivity of parasites in animals. Pollinator parasites are transmitted between hosts that forage on shared flowers. Floral transmission directly exposes parasites to phytochemicals on floral surfaces and in nectar, both at flowers and, post‐ingestion, in the crop. This exposure could directly affect parasite transmission to new hosts. 2. Nectar chemical analyses were combined with field and cell culture experiments to test the effects of the floral phytochemical thymol on the transmission potential of the trypanosomatid gut parasite Crithidia in Bombus impatiens. First, thymol concentrations in Thymus vulgaris nectar were measured. Second, the effect of adding thymol to floral nectaries on parasite transmission to foraging bees was tested. Third, cell cultures were used to determine direct, dose‐dependent effects of short‐term thymol exposure on subsequent in vitro parasite growth. 3. A total of 26.1 ppm thymol was found in T. vulgaris nectar, five‐fold higher than previously documented in this species. However, addition of thymol to flowers of parasite‐inoculated inflorescences of four plant species did not affect acquisition of Crithidia infection during a foraging bout. Cell culture experiments showed that the thymol concentrations needed to reduce subsequent Crithidia growth by 50% (120 ppm) were 4.6‐fold higher than the highest detected nectar concentration. 4. Although thymol exposure can influence Crithidia viability, Crithidia are robust to the duration and magnitude of exposure encountered during floral foraging under natural conditions. These experiments suggest that any effects of thymol alone on Crithidia–host infection dynamics probably reflect indirect, possibly host‐mediated, effects of chronic thymol ingestion.     

Discovery of rice essential genes by characterizing a CRISPR‐edited mutation of closely related rice MAP kinase genes

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co‐expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA‐gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen‐activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR‐edited mutants. The true knock‐out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR‐induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45–86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene‐free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.