Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme.

Area of Science:

• Molecular Biology
• Enzymology
• Genetics

Background:

• The EcoRII modification enzyme plays a role in DNA methylation.
• Understanding its gene sequence and expression is crucial for biotechnological applications.

Purpose of the Study:

• To clone and sequence the gene encoding the EcoRII modification enzyme.
• To investigate the expression and functional domains of the EcoRII methylase.

Main Methods:

• Gene cloning and nucleotide sequencing (1933 bp).
• Protein analysis (477 amino acids).
• Transcriptional start site mapping (S1 mapping).
• Site-directed mutagenesis (N-terminal deletion).
• Sequence homology comparison with other methylases.
• Gene expression studies using pUC and lambda PL promoter vectors.

Main Results:

• The complete nucleotide sequence of the EcoRII methylase gene was determined.
• Two transcriptional start sites were identified.
• A deletion mutant retaining partial enzyme activity was characterized.
• Homologous domains were identified among cytosine methylases.
• Gene cloning in pUC vectors enhanced expression 6-18 fold.
• A lambda PL promoter-based vector achieved 40-fold overproduction of EcoRII methylase.

Conclusions:

• The study successfully cloned, sequenced, and characterized the EcoRII methylase gene.
• Optimized expression strategies were developed, leading to significant protein overproduction.
• Comparative sequence analysis revealed conserved domains, aiding in understanding methylase function.