In vitro expression of two proteins from overlapping reading frames in a eukaryotic DNA sequence

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Overlapping reading frames (ORFs) are known to exist in various genomes.
• The expression of proteins from ORFs in rainbow trout (Oncorhynchus mykiss) has not been extensively studied.

Purpose of the Study:

• To investigate the in vitro expression of proteins from overlapping reading frames in rainbow trout genomic DNA.
• To characterize the synthesized proteins and their potential functions.

Main Methods:

• In vitro transcription of cloned DNA sequences using Salmonella phage 6 polymerase.
• In vitro translation using a rabbit reticulocyte lysate system.
• Hybridization and primer extension studies using a synthetic oligonucleotide probe.

Main Results:

• Two distinct and functional messenger RNAs (mRNAs) were synthesized: protamine mRNA and an overlapping mRNA termed 'Y'.
• The cell-free system produced corresponding protein products for both mRNAs.
• Pro-rich protein Y showed similarities to human salivary Pro-rich protein, avian sarcoma virus protein P19, and the myc oncogene product.
• Y-related mRNAs were detected in trout brain RNA using sensitive primer extension techniques.

Conclusions:

• Rainbow trout genomic DNA can express two distinct proteins from overlapping reading frames.
• The identified Pro-rich protein Y may have significant biological roles, potentially related to oncogenesis or viral proteins.
• Further research is warranted to elucidate the precise functions of protein Y and its expression regulation in vivo.