›› 2006, Vol. 49 ›› Issue (5): 726-732.

• RESEARCH PAPERS • Previous Articles     Next Articles

Cloning and sequence analysis of a novel transcription-associated zinc ribbon protein gene from silkworm, Bombyx mori

FENG Shan, ZHANG Yao-Zhou   

  1. Institute of Biochemistry, Zhejiang SciTec University, Hangzhou 310018, China
  • Online:2006-11-07 Published:2006-10-20
  • Contact: ZHANG Yao-Zhou

Abstract:

The zinc ribbons are one group of diverse zinc finger proteins. Most zinc-ribbon domain is actually folded as three-stranded antiparallel β-sheets in their structure instead of finger-like helices. The zinc ribbon is a domain that functions as interaction modules binding to nucleic acids. The zinc ribbon proteins perform a broad range of functions such as replication and repair, transcription and translation as a transcriptional repressor.Through searching cDNA bank of silkworm pupae, a unique EST (GenBank accession no. DY230964) encoding the zinc ribbon protein was identified. In order to obtain full-length cDNA in Bombyx mori, we used this sequence as a probe and performed in silico cloning based on the B. mori EST database. A contig was thus assembled on the basis of several ESTs. It was also verified by RT-PCR. PCR product was purified and ligated intopGEM-T-easy vector. The recombinant clones were sequenced, which had the same sequence as the contig. We nominated this novel gene encoding zinc ribbon domain containing protein as BmZNRD1 (GenBank accession no. DQ432055). The full-length of the gene is 675 bp. It contains an ORF of 363 bp, 10 bp nucleotide sequence in 5′ UTR (untranslated region) and 302 bp nucleotide sequence in 3′ UTR (untranslated region). It encodes 120 amino acids and is highly homologous to those of eukaryotic organisms previously reported (about 60%), with their predicted mass 13.54 kD and isoelectric point 6.8. The deduced amino acid sequence is one of zinc ribbon proteins with two putative functional domains, the N-terminal domain Cx2Cx14Cx2C and the C-terminal domain Cx2Cx24Cx2C. The highly conserved amino acid sequence within the C-terminal domain is Cx2Cx6Yx3QxRSADEx2TxFx2Cx2C, which is well conserved through evolution,in organisms such as yeasts, Drosophila, nematodes, amphibians and mammals, and highly homologous to the yeast RNA polymerase A subunit 9 and transcription-associated proteins. An alignment of the cDNA sequence with the silkworm genome sequences revealed that there were 3 exons and 2 introns in the gene of  BmZNRD1, and all of them conformed the canonical GT-AG rules.

Key words: Bombyx mori, zinc ribbon protein gene, in silico cloning, gene cloning, sequence analysis