The Mapping of Transcription Factor Binding Sites in the Turkey Prolactin Gene

Abstract

The cessation of egg-laying during the incubation period of the turkey hen is a source of major economic loss to the turkey industry. In August of 2000 there were approximately 2.7 million turkey breeder hens in the United States. Since the value of one fertile turkey egg is $0.62, the loss of only one egg per hen per year would cost the industry $1.7 million. A number of management procedures have been implemented to control egg production and prevent incubation. However, these methods are labor intensive.

The anterior pituitary hormone prolactin (PRL) is involved in the onset of incubation in the turkey hen. Levels of circulating PRL and PRL mRNA are 10X greater in photostimulated hens than in photorefractory hens, 20X greater in laying hens, and 100X greater in incubating hens. It would be useful to determine the molecular mechanisms controlling regulation of the turkey (t) PRL gene. This information could be used to modulate the release of PRL and thereby prevent the induction of the incubation period in turkey hens.

Approximately 2 kilobases (kb) of the tPRL 5'-flanking region were examined by the electrophoretic mobility shift assay (EMSA) using nuclear extracts from turkey pituitaries and liver. Within this 2 kb fragment, only three regions of the tPRL gene were identified that participate in tissue- and sequence-specific DNA-protein interactions with nuclear extracts from turkey pituitaries. These are the regions from nucleotides (nt) -41 to -73, -105 to -137, and -175 to -199, named tprl-1, tprl-2 and tprl-3, respectively. Three shifted bands were observed using tprl-1 and tprl-2 while two shifted bands were seen using tprl-3.

Competition EMSAs done on these three regions showed that in the presence of unlabeled, excess, specific competitor DNA, the proteins bound to competitor DNA and no shifted bands were observed. If the competitor was a nonspecific DNA sequence, then there was no effect on the shifted bands. When using labeled tprl-2 and unlabeled tprl-1 as competitor DNA, no shifted bands were observed. However, when using labeled tprl-1 and unlabeled tprl-2 as competitor DNA, only one of three shifted bands was eliminated. These data indicate that tprl-1 and tprl-2 bind both common and specific pituitary nuclear proteins and have different affinities for pituitary nuclear proteins. A supershift EMSA involving the addition of rabbit-anti-rat Pit-1 indicated that tPit-1 is a common pituitary nuclear protein that is bound to tprl-1 and tprl-2. However, this interaction may not occur in the turkey in vivo. The mapping of transcription factor binding sites in the tPRL 5'-flanking region is the first step toward the identification and isolation of factors that bind to and regulate transcription of the PRL gene.