Biomaterial Database

A method for selection of forward mutations in supF gene carried by shuttle-vector plasmids. 1993

R R Ariza, and T Roldán-Arjona, and C Hera, and C Pueyo

Departmento de Genética, Facultad de Ciencias, Universidad de Córdoba, España.

The supF gene of Escherichia coli has been widely used as a mutagenic target in several shuttle-vector plasmids. Mutations in this gene are usually screened by a colony colour assay based on the suppression of a lacZ amber mutation in an appropriate bacterial indicator strain. This screening method cannot measure the low mutation frequencies usually detected in prokaryotes, and therefore precludes the use of supF gene for studying mutational spectra in bacteria. In this paper we report the development of a simple method for the selection of supF forward mutations in shuttle-vector plasmids. The method has implied the construction of an araD- araC(Am) mutant strain (MBL50) of E.coli. The L-arabinose sensitivity caused by the accumulation of a toxic intermediate in araD- mutants is abolished in MBL50 because the araC(Am) mutation blocks the L-arabinose catabolic pathway. Strain MBL50 becomes sensitive to L-arabinose when transformed with a supF+ plasmid but remains resistant upon transformation with a supF- mutant. This new L-arabinose resistance selection method was able to detect supF- mutant fractions up to three orders of magnitude below those determined with the colony colour screening assay. The method was further validated by carrying out in vivo mutagenesis experiments with N-methyl-N-nitrosourea (MNU) and a shuttle-vector-bearing strain (UC2109) completely defective in O6-methylguanine (O6meG) alkyltransferase repair capacity. The DNA sequence alterations of 22 independent supF- mutants induced by MNU were determined. All mutations were G:C-->A:T transitions in agreement with the predicted significance of the mispairing potential of the O6meG lesion. A preference for the sequence 5'-GG-3' was detected, revealing a 5'-flanking base influence. The accumulation of all 22 MNU-induced mutations in three sites of the supF genes might be related to the lack of O6meG alkyltransferase repair capacity of strain UC2109. The L-arabinose resistance method described in this paper allows rapid scoring and sequencing of forward mutations in the supF gene on shuttle-vectors, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria. Since shuttle-vectors replicate both in bacteria and mammalian cells, this method makes it possible to compare prokaryotic and eukaryotic mutational spectra.

UI	MeSH Term	Description	Entries
D008770	Methylnitrosourea	A nitrosourea compound with alkylating, carcinogenic, and mutagenic properties.	Nitrosomethylurea,N-Methyl-N-nitrosourea,NSC-23909,N Methyl N nitrosourea,NSC 23909,NSC23909
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D010641	Phenotype	The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment.	Phenotypes
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005798	Genes, Bacterial	The functional hereditary units of BACTERIA.	Bacterial Gene,Bacterial Genes,Gene, Bacterial
D005822	Genetic Vectors	DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.	Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012343	RNA, Transfer	The small RNA molecules, 73-80 nucleotides long, that function during translation (TRANSLATION, GENETIC) to align AMINO ACIDS at the RIBOSOMES in a sequence determined by the mRNA (RNA, MESSENGER). There are about 30 different transfer RNAs. Each recognizes a specific CODON set on the mRNA through its own ANTICODON and as aminoacyl tRNAs (RNA, TRANSFER, AMINO ACYL), each carries a specific amino acid to the ribosome to add to the elongating peptide chains.	Suppressor Transfer RNA,Transfer RNA,tRNA,RNA, Transfer, Suppressor,Transfer RNA, Suppressor,RNA, Suppressor Transfer