Large-scale mutational analysis of EMS-induced mutation in the lacI gene of Escherichia coli

Pieńkowska, Małgorzata; Glickman, Barry W.; Ferreira, A. Santos; Anderson, Marshall W.; Zieleńska, Maria

doi:10.1016/0027-5107(93)90214-z

Cited by 18 publications

(7 citation statements)

References 28 publications

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“…The sequences from three independently derived clones were identical to the sequence of the SSII gene from wild-type peas reported by Dry et al (1992; EMBL accession number X88790), except at position 2260, where the G/C pair in the wild-type sequence had been changed to an A/T. This change is typical of those caused by alkylating reagents (Pienkowska et al, 1993) and is consistent with results for mutations at the r locus generated in the same mutation program (MacLeod, 1994). The change converts tryptophan 651 into a stop codon and would thus result in premature termination of translation.…”

Section: The Rug5-a Mutation Lies In the Ssii Genesupporting

confidence: 61%

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos

1998

Current Opinion in Plant Biology

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Mutations at the rug5 ( rug osus 5 ) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B 2 and B 3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B 2 and B 3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed. INTRODUCTIONThe starch synthases that catalyze the synthesis of the branched amylopectin component of the starch granule are poorly understood. It is well established that a specific class of granule-bound starch synthases (known as granulebound starch synthase I or GBSSI) is responsible for the synthesis of the unbranched amylose component of the granule. Two or more distinct isoforms other than GBSSI are present in storage organs of the species examined to date, and these together with starch branching enzymes (SBEs) are responsible for the synthesis of amylopectin. However, it is not clear whether different isoforms play qualitatively distinct roles in amylopectin synthesis . Study of the roles of such isoforms is hampered by a lack of mutations that affect them specifically and exclusively. Analysis of transgenic potato plants in which activities of specific isoforms have been reduced (Edwards et al., 1995;Abel et al., 1996;Marshall et al., 1996) has thus far not revealed whether these isoforms have qualitatively distinct roles.In this study, we elucidate the role of the major isoform of starch synthase present in the soluble fraction of the developing pea embryo. Starch synthase II (SSII) is a protein of 77 kD that accounts for 60 to 70% of the soluble starch synthase activity of the pea embryo. It is also present within the matrix of the starch granule (Smith, 1990;Denyer and Smith, 1992; Dry et al., 1992; Denyer et al., 1993;Edwards et al., 1996). Analysis of mutant lines of peas from which GBSSI is absent has shown conclusively that SSII is not involved in amylose synthesis (Denyer et al., 1995a). It is reasonable to assume, therefore, that SSII is important in the synthesis of amylopectin.Amylopectin is a highly branched polymer consisting of linear chains of ␣ (1,4)-linked glucose residues joined together by ␣ (1,6)-linkages. Within the granule, the chains are thought to be arranged in clusters at intervals of 9 nm, within which chains associate t...

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Section: The Rug5-a Mutation Lies In the Ssii Genesupporting

confidence: 61%

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos

1998

Current Opinion in Plant Biology

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“…Aligned data were passed through a filter to reduce spurious SNP calls caused by sequencing and alignment errors (Online Methods). As a result, we found that mutants harbor 1,001 (Hit1917-pl1) and 1,339 (Hit0813-pl2) transition-type (G→A and C→T) SNPs with high-quality scores (Supplementary Table 4), presumably caused by ethyl methanesulfonate mutagenesis 7 .…”

Section: Mutmap Applied To Pale Green Leaf Mutantsmentioning

confidence: 90%

Genome sequencing reveals agronomically important loci in rice using MutMap

et al. 2012

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“…It is known from previous studies (see ref. 27 for a large-scale mutational analysis of ethyl methanesulfonate (EMS)-induced mutations) that there are 23 sites sensitive to GC+AT transitions in the N-terminal region of thelaclgene. GC-+AT transitions induced by CCNU were at 21 of these 23 sites; the exceptions were positions 129 and 179.…”

Section: Resultsmentioning

confidence: 99%

“…This result suggests that the efficiency of the repair of 06-chloroethylated guanine lesions by bacterial alkyltransferases may be poor at sites surrounded by A:T base-pairs. In this respect, it is noteworthy to comment that positions 75 and 120 are two well-known hot-spot sites for GC+AT transitions induced by simple monofunctional ethylating agents (position 75 for EMS and position 120 for N-ethyl-N-nitrosourea) in Uvr-bacteria only, and the frequencies of mutation at these two positions are significantly reduced in cells fully capable of excision repair [27,31,321.…”

Section: Discussionmentioning

confidence: 96%

Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in the Escherichia coli lacl gene of O⁶‐alkylguanine‐DNA alkyltransferase‐proficient and ‐deficient strains

Jurado

Ferrezuelo

Pueyo

1995

Molecular Carcinogenesis

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Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the lacl gene of Escherichia coli were recovered from bacteria proficient (Ogt+ Ada+) and deficient (Ogt- Ada-) in O6-alkylguanine-DNA alkyltransferase activity. A CCNU dose of 1 mM was selected for DNA sequence analysis. A total of 245 induced mutations were characterized. The mutations were almost exclusively (95%) GC-->AT transitions, indicating that CCNU-induced mutations arose in bacteria primarily from misreplication of O6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents. The distribution of CCNU-induced GC-->AT mutations was significantly altered by the presence of DNA alkyltransferase activity (P = 0.01). In the Ogt+ Ada+ mutational spectrum, guanines flanked on both sides by A:T base-pairs were on average 2.8 times more likely to mutate than those flanked by G:C base-pairs on at least one side. This bias disappeared in the Ogt- Ada- genetic background, thereby providing evidence that O6-chloroethylated guanines adjacent to G:C base-pairs are better targets for bacterial alkyltransferase than those not adjacent to G:C base-pairs. We recently reported a similar bias for ethyl methanesulfonate, strengthening the idea that CCNU is acting as a simple ethylating compound. In summary, this paper presents for the first time evidence that DNA repair by O6-alkylguanine-DNA alkyltransferases plays a major role in removing lesions responsible for GC-->AT transitions induced by CCNU, influencing their ultimate distribution with respect to sequence context.

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Large-scale mutational analysis of EMS-induced mutation in the lacI gene of Escherichia coli

Cited by 18 publications

References 28 publications

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos

Genome sequencing reveals agronomically important loci in rice using MutMap

Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in the Escherichia coli lacl gene of O⁶‐alkylguanine‐DNA alkyltransferase‐proficient and ‐deficient strains

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Large-scale mutational analysis of EMS-induced mutation in the lacI gene of Escherichia coli

Cited by 18 publications

References 28 publications

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos

Genome sequencing reveals agronomically important loci in rice using MutMap

Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in the Escherichia coli lacl gene of O6‐alkylguanine‐DNA alkyltransferase‐proficient and ‐deficient strains

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Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in the Escherichia coli lacl gene of O⁶‐alkylguanine‐DNA alkyltransferase‐proficient and ‐deficient strains