Nucleotide sequence of the E coli gene coding for dihydrofolate reductase

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The fol gene encodes dihydrofolate reductase (DHFR), a crucial enzyme in folate metabolism.
• Understanding the genetic structure and expression of the E. coli fol gene is important for various biological processes.

Purpose of the Study:

• To map the fol gene on plasmid pLC1437a.
• To determine the nucleotide sequence of the fol gene and its flanking regions from E. coli strain RSO.
• To characterize the RNA transcripts of the fol gene.

Main Methods:

• Gene mapping using restriction endonuclease cleavage sites.
• DNA sequencing of the fol gene and flanking regions.
• Analysis of in vivo RNA transcripts using Northern blotting or similar techniques.
• Development of a new 3' end DNA labeling procedure.

Main Results:

• The fol gene was successfully mapped on plasmid pLC1437a.
• The complete nucleotide sequence of the fol gene and flanking regions from strain RSO was determined.
• Predicted amino acid sequence shows minor variations compared to E. coli B DHFR.
• Major in vivo RNA transcripts of approximately 550 and 590 nucleotides were identified, along with longer transcripts.

Conclusions:

• The study provides a detailed genetic and transcriptional analysis of the E. coli fol gene.
• The determined sequence offers insights into DHFR structure-function relationships.
• A novel and efficient method for 3' end labeling of blunt-ended DNA fragments was developed.