Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• Polyadenylation is a crucial post-transcriptional modification in eukaryotes.
• Mammalian poly(A) polymerase (PAP) is the key enzyme responsible for adding the poly(A) tail to mRNA.
• Understanding mammalian PAP structure and function is essential for gene expression regulation.

Purpose of the Study:

• To isolate and characterize cDNA clones encoding mammalian poly(A) polymerase.
• To investigate the functional properties of the cloned enzyme and its associated factors.
• To compare the mammalian PAP sequence with its yeast counterpart.

Main Methods:

• Polymerase chain reaction (PCR) using amino acid sequences from purified enzyme.
• cDNA cloning and expression in Escherichia coli.
• Biochemical assays for poly(A) polymerase activity.
• Northern blot analysis to detect mRNA transcripts.

Main Results:

• Isolated bovine cDNA clones encoding 82 kDa and 43 kDa polypeptides.
• Expressed 82 kDa protein in E. coli showed poly(A) polymerase activity dependent on CPF and AAUAAA signal.
• Identified mRNAs of 4.2, 2.4, and 1.3 kb, likely corresponding to PAP transcripts.
• The N-terminal half of bovine PAP shares 47% sequence identity with yeast PAP.

Conclusions:

• Successfully cloned and expressed functional mammalian poly(A) polymerase.
• Identified a specificity factor (CPF) and polyadenylation signal (AAUAAA) crucial for activity.
• Revealed evolutionary conservation between mammalian and yeast poly(A) polymerases.