Molecular Analysis of Diepoxybutane-Induced Mutations at the rosy Locus of Drosophila melanogaster

We have analyzed at the molecular level diepoxybutane-induced mutants determined to have lesions affecting expression of the ry locus. Of the 21 mutants analyzed here, genetic analysis suggested that five were putative deficiencies involving ry and adjacent lethal loci. However, molecular analysis confirmed that only two of these five putative deficiencies were in fact deletions detectable by the methods used in the analysis. The remaining 16 mutants were viable as homozygotes, suggesting that their lesions were confined to the ry locus. Seven of these 16 intragenic mutants were determined to be deletions of genetic material as evidenced by altered restriction patterns relative to the wild type patterns. Thus, nine of 21 (43%) diepoxybutane-induced mutants are due to deletions ranging in size from approximately 50 base pairs to more than 8 kilobase pairs. Most of the deletions (seven of nine or 78%) are intragenic and less than 250 base pairs in size; it seems that most, if not all, affect coding rather than regulatory sequences.     

Comparative genomic hybridization detects secondary chromosomal deletions in Escherichia coli K-12 MG1655 mutants and highlights instability in the flhDC region

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655, we identified unintended secondary genomic deletions in the flhDC region in Δfnr, Δcrp, and ΔcreB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.     

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N₂ fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261–357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.Subject terms: Soil microbiology, Molecular evolution     

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region (“NB loop”). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called “I-loop” region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K_m with PPi (up to 33 mM), whereas Ncut deletions had greatly increased K_m with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K_m values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.     

An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.     

Apparent Mutagenic Effect of Induction of Lambda Prophage Inserted Between lysA and thyA

Induction of a heat-inducible abnormal lambda prophage inserted between lysA and thyA in Escherichia coli resulted in a number of auxotrophic mutants in the surviving cured-cell populations. These mutants could not be accounted for by deletions arising on formation of lambda hybrid particles carrying regions adjacent to the insertion site. The properties of these mutants, which were almost all spontaneously revertable, have been described and mapped by F′ episome complementation. Tentatively, it was suggested that induction of the lambda lysogen leads to a mutagenic state.     

Molecular analysis of mutation at the hprt locus of Chinese hamster V79 cells induced by ethyl methanesulphonate and mitomycin C

Mutation at the hprt locus of Chinese hamster V79 cells were induced by treatment with ethyl methanesulphonate (EMS), considered primarily a point mutagen and mitomycin C (MMC), a potent clastogen. EMS gave a dose-dependent induction of mutants while MMC induced a poor mutagenic response. Mutations were analysed using Southern and Northern blotting.Analysis of 9 EMS-induced and 4 spontaneous mutants yielded no detectable alterations in the hprt locus after digestion of DNA with 6 restriction enzymes. Mutants without detectable changes carried presumptive point mutations. In contrast, 4 out of 12 MMC-induced mutants had detectable alterations. 2 of these appeared to have lost the entire hprt gene while the other 2had prodable partial deletions. For these 4 deletion mutants no hprt mRNA was detected. 3 MMC-induced and 1 EMS-induced mutants had reduced levels of hprt mRNA. All the other mutants showed normal levels of hprt mRNA and the message detected was always of the correct size.It is suggested that the poor mutagenic response induced by MMC may be due to the lethal nature of large deletions involving both the hemizygous hprt locus and adjacent essential genes. This may lead to an underestimate of the mutagenicity of clastogenic agents such as MMC in the V79 HPRT mutation assay.     

Genetic evidence of mutator-induced deletions in the short arm of chromosome 9 of maize

Evidence is presented that at least 12 of the Mu-induced yg2 mutants found in an extensive mutation analysis of this locus are the result of deletions in the region of the yg2 locus on the short arm of chromosome 9. Twelve of these putative deletions were characterized genetically, and in every instance, they were confirmed to be deletions involving chromosomal segments that include the yg2 and wd loci as well as additional portions of the short arm of chromosome 9.     

Isolation of plasmid deletion mutants and study of their instability

We describe a method which allows isolation of deletions within hybrid plasmids. It is based on the fact that the tetracycline resistance (Tc^R) gene of pBR322 can be inactivated by inserting foreign DNA into its HindIII site, and that the easily selectable Tc^R mutants of such plasmids are generally (>90%) due to deletions of certain hybrid plasmid sequences. We have found that Tc^R mutants are usually maintained within the cell recombined with the parental Tc^S plasmids. Such heterodimers dissociate in both Rec⁺ and in recA hosts. Parental rather than mutant plasmids are then retained by the host cell.     

Genetic and Physiological Characterization of met15 Mutants of SACCHAROMYCES CEREVISIAE: a Selective System for Forward and Reverse Mutations

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus.—It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.     

Fine Structure Mapping of the am (Gdh) Locus of Neurospora

Utilizing a combination of flanking marker analysis and deletion mapping we have constructed a fine structure map of the am locus which includes 63 point mutants and ten unique deletions. Positions of point mutants can be rapidly assigned to one of 13 segments within the gene on the basis of crosses to nine deletion strains.     

Genetic analysis of non-essential bacteriophage T7 genes

Isolation and genetic characterization of a series of deletions and point mutants affecting two non-essential genes of bacteriophage T7 is described. The T7 ligase gene falls between genes 1 and 2, and is designated gene 1.3. Another non-essential gene, designated gene 0.7, has been mapped to the left of gene 1. In order to facilitate isolation and characterization of these mutants, host strains were found in which one or both of these T7 genes is required for growth.     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ^? mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.     

Genetic map of an octopine TI-plasmid.

Several deletion mutants of an octopine TI-plasmid were mapped by digestion with the restriction enzyme Sma I. The T region, as it is defined on the B6-806 plasmid, does not appear to be an essential area for tumour induction on the plasmid of Ach5. The genes for octopine breakdown, plasmid transfer, and the replicator were roughly localized. The possibility of using mutants with large deletions as a cloning vehicle in Agrobacterium tumefaciens is discussed.     

Metabolic modeling for multi-objective optimization of ethanol production in a Synechocystis mutant

Cyanobacteria have potential to produce drop-in bio-fuels such as ethanol via photoautotrophic metabolism. Although model cyanobacterial strains have been engineered to produce such products, systematic metabolic engineering studies to identify optimal strains for the same have not been performed. In this work, we identify optimal ethanol producing mutants corresponding to appropriate gene deletions that result in a suitable redirection in the carbon flux. In particular, we systematically simulate exhaustive single and double gene deletions considering a genome scale metabolic model of a mutant strain of the unicellular cyanobacterium Synechocystis species strain PCC 6803. Various optimization based metabolic modeling techniques, such as flux balance analysis (FBA), method of minimization of metabolic adjustment (MOMA) and regulatory on/off minimization (ROOM) were used for this analysis. For single gene deletion MOMA simulations, the Pareto front with biomass and ethanol fluxes as the two objectives to be maximized was obtained and analyzed. Points on the Pareto front represent maximal utilization of resources constrained by substrate uptake thereby representing an optimal trade-off between the two fluxes. Pareto analysis was also performed for double gene deletion MOMA and single and double gene deletion ROOM simulations. Based on these analyses, two mutants, with combined gene deletions in ethanol and purine metabolism pathways, were identified as promising candidates for ethanol production. The relevant genes were adk, pta and ackA. An ethanol productivity of approximately 0.15 mmol/(gDW h) was predicted for these mutants which appears to be reasonable based on experimentally reported values in literature for other strains.     

Octopine Ti-plasmid deletion mutants of agrobacterium tumefaciens with emphasis on the right side of the T-region

One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ^? phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the T_L + T_R region. Most of the T_R region, present as T_R-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the T_R region together with a small part of the adjacent T_L region, which is always present as T_L-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoë and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.     

Efficient isolation of targeted Caenorhabditis elegans deletion strains using highly thermostable restriction endonucleases and PCR

Reverse genetic approaches to understanding gene function would be greatly facilitated by increasing the efficiency of methods for isolating mutants without the reliance on a predicted phenotype. Established PCR-based methods of isolating deletion mutants are widely used for this purpose in Caenorhabditis elegans. However, these methods are inefficient at isolating small deletions. We report here a novel modification of PCR-based methods, employing thermostable restriction enzymes to block the synthesis of wild-type PCR product, so that only the deletion PCR product is amplified. This modification greatly increases the efficiency of isolating small targeted deletions in C.elegans. Using this method six new deletion strains were isolated from a small screen of approximately 400000 haploid genomes, most with deletions <1.0 kb. Greater PCR detection sensitivity by this modification permitted ~10-fold greater pooling of DNA samples, reducing the effort and reagents required for screens. In addition, effective suppression of non-specific amplification allowed multiplexing with several independent primer pairs. The increased efficiency of this technique makes it more practical for small laboratories to undertake gene knock-out screens.     

Fine Structure Analysis of the ade3 Locus in SACCHAROMYCES CEREVISIAE

Twenty-six spontaneous mutants at the ade3 locus of Saccharomyces cerevisiae have been mapped and characterized with respect to revertibility, osmotic remediability and temperature sensitivity. Twelve of the twenty-six are temperature sensitive, 25 of 26 are osmotic remedial and 21 of 26 revert. Two of the mutants map as deletions. At least five of the 26 are nonsense mutations but are also, unexpectedly, osmotic remedial. Three nonsense mutations are also temperature sensitive, again an unexpected result. The two multisite mutations are both temperature sensitive and osmotic remedial. For mutants at this locus osmotic remediability and temperature sensitivity cannot be considered diagnostic criteria for missense mutations.