Isolation and characterization of the chicken cystatin-encoding gene: mapping transcription start point and polyadenylation sites

Abstract

A 6.9-kb fragment containing coding sequences for chicken egg white cystatin (CsnEW) was isolated from a chicken genomic library using the CsnEW cDNA as a probe. The gene is approximately 2.4 kb in length; it contains three exons, two introns and two polyadenylation signals. The exon-intron arrangement corresponds exactly with those of other members of the Csn superfamily. The sequence of the 5' flanking region contains two SP1-binding sites and a high G+C content suggestive of a housekeeping gene. All tissues studied express CsnEW mRNA; Northern analysis showed that CsnEW mRNA levels are most abundant in lung and least abundant in liver and spleen. Mapping of the 3' end of the CsnEW mRNA isolated from brain tissue resolved two CsnEW mRNA species. The larger transcript resulting from the use of the second polyadenylation signal was more abundant than the smaller transcript. Determination of the transcription start point (tsp) of CsnEW mRNA by primer extension and RNase protection assays showed that CsnEW mRNA from a number of chicken tissues was approximately 40-50 nucleotides shorter than that predicted from the CsnEW cDNA isolated from chicken oviduct.