Clone And Expression Of Immediate-Early Gene Arc/Arg3.1 In Some Amphibian And Reptilian Species

Immediate-early genes (IEG) belongs to the inducible gene group that can be used as markers to investigate neural activities. Unlike most IEG that are transcription factors, products of IEG Arc/Arg3.1 gene are cellular structure elements and have been suggested to play an important role in synaptic plasticity during the learning and memory processes. At present study, i amplified and sequenced the Arc/Arg3.1 cDNA from a snake (Dinodon rufozonatum) and African clawed frog (Xenopus laevis) and then predicted the amino acid sequence. Arc/Arg3.1 genes were pulled out from the genomes of the carolina anole (Anolis carolinensis), tropical clawed frog (Xenopus tropicalis) and Asian elephant (Elephas maximus) based on their relative conversation comparing with all sequences available to date. With the nucleotide sequence we also predicted the amino acid sequences of the Arc/Arg3.1 from these species. I analyzed the important structural sites of carolina anole Arc/Arg3.1 DNA and predicted this protein's 2D structures, inferred the potential phosphorylated sites of these two reptilian , two amphibian, two avian and four mammalian species by Ca²⁺/calmodulin-dependent protein kinase II (CaMkII) and protein kinase C (PKC), closely-related to the learning and memory functions. I also compared the Arc/Arg3.1 predicted amino acid residues of these ten species to determine the degree of conservation among species. A phylogenetic tree has been made based on the amino acid sequence of Arc/Arg3.1 in 15 species to show the evolutionary relationships. With the amplified Arc/Arg3.1 sequence of snake, I obtained a highly specific DNA fragment about 300bp from it to make RNA probes. By giving the snake some environmental stimuli such as light or infrared, in situ hybridization with the RNA probes, I found the expression of Arc/Arg3.1 in the brain of snake. The main results and conclusions we obtained are as follows:1. I amplified and sequenced 1188bp fragment of the Arc/Arg3.1 cDNA from red banded snake (Dinodon rufozonatum), and predicted the translated product, i.e. 396 amino acid residues, and obtained 1023bp fragment of African clawed frog (Xenopus laevis) Arc/Arg3.1 cDNA, predicted to be 341 amino acid residues. In addition, completed DNAs to code the Arc/Arg3.1 were pulled out from the genomes of the carolina anole (Anolis carolinensis) and Asian elephant (Elephas maximus) by deleting introns based on their relative conversation comparison with all sequences available to date,respectively, I obtained 1227bp encoded 409 amino acids for Carolina anole and 1188bp encoded 396 amino acids for Asian elephant. Near completed sequence with 1023bp of Arc/Arg3.1 was also pulled out from the genomes of tropical clawed frog (Xenopus tropicalis), encoded 341 amino acids.2. From the carolina anole genomic library a 2862 bp DNA sequence was obtained, the DNA sequence show that the 3' non-coding region included a single AATTTA sequence, the fragment being implicated in destabilizing mRNAs, and a polyadenylation signal (AATAAA). Protein's 2D structures show the folded and hydrophobic domains. Phosphorylation sites for Arc/Arg3.1 genes of ten species suggested the avian species has the most sites and amphibians the least.3. There are two highly conserved regions in the Arc/Arg3.1 amino acid residue sequences in tetrapod species. By comparing the sequence, amphibians showed low sequence homology with other species, the phylogenetic tree also showed the consistent divergence, suggesting in the line of evolution amphibians had significant modifications in Arc/Arg3.1.4. By in situ hybridization, i found Arc/Arg3.1 highly expression in the brain of the snake after stimulation, almost focused in the diencephalon and mesencephalon.