Molecular Cloning of a Cdna Encoding a Nerve Growth Factor Precursor from the Agkistrodon Halys Pallas

Abstract

NGF cDNAs from many sources have been cloned. Here we report a new practical method for cloning a nearly full-length NGF cDNA. Due to the high conservation in the 5’ and 3’ untranslated regions, oligonucleotides complementary to all sequences coding for them were synthesized. Total RNA was extracted from the Agkistrodon halys Pallas venom gland. Accordingly, the first strand of cDNA was synthesized and used as template. The RT-PCR amplified DNA manifested as a single band of predicted size after electrophoresis.

Nucleotide sequence analysis revealed an open reading frame of 726 bases encoding a 242 amino acid polypeptide, which includes Agkistrodon halys Pallas prepro-NGF, and whose sequence shows a high similarity to the prepro-NGF of cobra and krait Bungarus multicinctus. Comparison with known sequences shows that the 5’ and 3’ untranslated sequences show slightly higher conservation than the translated region. The prepro part of the precursor is less conserved. The Agkistrodon cDNA possesses the presumptive 18 residue amino terminal signal sequence. It also has two long highly conserved stretches, corresponding to functional domains, alternating with large divergent regions. Comparison of the mature NGF amino acid sequence revealed a high degree of sequence conservation of the structurally important residues. It suggested that the same overall three-dimension structure is adopted. The presence of aspartate introduces a potential N-glycosylation site, which doesn't exist in cobra NGF.

Additionally, because the major region of prepro NGF is encoded in one exon, human and mouse genomic DNA, used as a template for amplificating NGF cDNA, also proved to be competent. It revealed the feasibility of isolating and amplifying unknown genes using genomic DNA as a template for PCR.