Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

A double mutant allele,csr1-4, of Arabidopsis thaliana encodes an acetolactate synthase with altered kinetics

A comparison is made of the kinetic characteristics of acetolactate synthase (EC 4.1.3.18) in extracts from Columbia wild type and four near-isogenic, herbicide-resistant mutants of Arobidopsis thaliana (L.) Heynh. The mutants used were the chlorsulfuron-resistant GH50 (csr1-1), the imazapyr-resistant GH90 (csr1-2), the triazolopyrimidine-resistant Tzp5 (csr1-3) and the multiherbicide-resistant, double mutant GM4.8 (csr1-4), derived from csr1-1 and csr1-2 by intragenic recombination (G. Mourad et al. 1994, Mol. Gen. Genet. 243, 178–184). and V _max values for the substrate pyruvate were unaffected by any of the mutations giving rise to herbicide resistance. Feedback inhibition by L-valine (L-Val), L-leucine (L-Leu) and L-isoleucine (L-Ile) of acetolactate synthase extracted from wild type and mutants fitted a mixed competitive pattern most closely. K_i values for L-Val, L-Leu and L-Ile inhibition were not significantly different from wild type in extracts from csr1-1, csr1-2, and csr1-3. K _i values were significantly higher than wild type by two- and five-fold, respectively, for csr1-4 with L-Val and L-Leu but not L-Ile. GM4.8 (csr1-4) plants were also highly resistant in their growth to added L-Val and L-Leu. The data suggest that (i) single mutational changes occurred that affected the binding of herbicides to the acetolactate synthase molecule without influencing the binding of substrates and feedback inhibitors (e.g. csr1-1, csr1-2 and csr1-3) and (ii) bringing two of these single mutations (csr1-1 and csr1-2) together in a double mutant (csr1-4) gave rise to an enzyme with altered characteristics as well as plants with changed growth in response to added L-Val and L-Leu. The implications of these conclusions for genetic transformation using these herbicide-resistant genes are discussed.Abbreviations ALS acetolactate synthase - L-Val L-valine - L-Leu L-leucine - L-Ile L-isoleucine This work was supported in part by a grant-in-aid of research from the Natural Sciences and Engineering Research Council of Canada to J.K. and by a research grant award from Purdue University to G.M.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.     

De novo activation of the transposable element Tam2 of Antirrhinum majus

Summary The nivea locus of Antirrhinum majus encodes the enzyme chalcone synthase required for the synthesis of red anthocyanin pigment. The stable allele niv-44 contains an insertion in the nivea gene (Tam2) which has all the structural features of a transposable element. We have shown that this insertion can excise from the nivea locus when niv-44 is combined with another allele (niv-99) in a heterozygote. Activation of Tam2 excision is caused by a factor tightly linked to the niv-99 allele and may be due to complementation between Tam2 and a related element, Tam1. Factors which repress the excision of Tam2 and Tam1 are also described. Repression is not inherited in a simple mendelian way. Many stable mutations may be due to the insertion of transposable elements. Our data suggest that their stability may be due to the absence in the genome of activating factors and to the presence of repressors.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

TFS1: A suppressor of cdc25 mutations in Saccharomyces cerevisiae

Summary The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Molecular analysis of the loss of somatic instability in the bz2::mu1 allele of maize

Summary Multiple genetic and epigenetic changes were detected within one plant generation at the bz2:: mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10^–4 per gamete. One germinal excision event was found that contained an 8 by deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75–77 by deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.     

Two new mutations of the human BCHE gene (IVS3-14T>C and L574fsX576)

The genetic variation of human butyrylcholinesterase is associated with the majority of prolonged cases of apnea in patients submitted to the muscle relaxant succinylcholine. The present study reports two new mutations of the BCHE gene in 346 Euro-Brazilians: IVS3-14T>C found in five heterozygotes (allele frequency: 0.72 ± 0.32%) and L574fsX576 found in one heterozygote (allele frequency: 0.14 ± 0.14%). These two variants were not found in 85 Guarani Amerindians. It is not expected that the IVS3-14T>C mutation may interfere in the splicing process and that the mutation found in exon 4 (L574fsX576) may disturb BChE tetramerization and activity.     

ore2, a mutation affecting proline biosynthesis in the yeast Saccharomyces cerevisiae, leads to a cdc phenotype

Summary We report here the isolation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae which exhibit cdc phenotypes. The recessive mutations defined four complementation groups, named ore1, ore2, ore3 and ore4. At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle. The wild-type allele of the gene altered in ore2 mutants was cloned. The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues. The deduced amino acid sequence showed 25% identity with that of the Escherichia coli 1-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline. The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline. In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the PRO3 gene which encodes the S. cerevisiae ¹-pyrroline-5-carboxylate reductase.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.     

Cell selection and inheritance of imidazolinone resistance in sugarbeet (Beta vulgaris)

 Sugarbeets are sensitive to imidazolinone herbicide residues applied to rotational crops. Two imidazolinone-resistance (IMI-R) sugarbeet traits were developed by somatic cell selection to overcome rotation restrictions for sugarbeets where imidazolinones have been applied. Sir-13 is an IMI-R/SU-S (sulfonylurea-sensitive) variant selected from an imidazolinone-sensitive (IMI-S) sugarbeet clone, REL-1. A second variant, 93R30B, resistant to imidazolinone as well as to sulfonylurea herbicides (IMI-R/SU-R), was selected from a plant homozygous for a previously described sulfonylurea-specific resistance trait, Sur (IMI-S/SU-R). The IMI-R alleles (Sir-13 and 93R30B) were found to be corresponding allelic variants at the same ALS locus and both were tightly associated with the Sur allele. Each resistant allele is dominant to the sensitive wild-type allele; however, incomplete dominance is shown among resistance alleles. Diploid sugarbeet contains a single ALS gene copy, limiting the ability to stack these resistance traits in the same plant by traditional breeding. Received: 1 May 1997 / Accepted: 30 June 1997     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

A cycloheximide-resistant mutant of Tetrahymena pyriformis

A mutant of Tetrahymena pyriformis, syngen 1, resistant to cycloheximide was obtained after mutagenesis (with N-methyl-N′-nitro-N-nitrosoguanidine) followed by a cross (to obtain macro-nuclear expression of the mutant phenotype). A genetic analysis has shown that cycloheximide resistance in the mutant strain is due to a dominant nuclear allele, designated chx-1. Heterozygotes (chx-1/chx⁺) are initially resistant but segregate stable, sensitive cell lines during vegetative growth, demonstrating that allelic exclusion occurs with this determinant, as with many others in syngen 1. This feature, coupled with the selective advantage conferred by the chx-1 allele in the presence of cycloheximide, makes this mutation a useful genetic tool. A strain homozygous for the chx-1 allele exhibits an exponential growth rate identical to that of the wild type in proteose peptone-yeast extract medium in the absence of cycloheximide. In 10 μg/ml of the drug, the resistant cells grow at a somewhat lower rate, after an initial lag and adaptation to the presence of the drug. This concentration causes complete inhibition of growth and eventual lysis of wild-type cells. The cellular basis for cycloheximide resistance and adaptation in the mutant is presently under investigation.     

Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults

In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64×10⁻⁶. The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 (P<0.05) which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity.     

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli

We have characterized 202 lacI⁻ mutations, and 158 dominant lacI^−d mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The −(G:C) frameshifts were the dominant mutational class in the lacI⁻ collections of both NR6112 and EE125, and in the lacI^−d collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI^−d collection. This study completes a comprehensive analysis of 1194 lacI⁻ and 348 lacI^−d mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.