1. Sexual Preference in Rams: Investigating the Role of Prolactin Tari Tan Dr. Fred Stormshak Dept. of Animal Science (Dr. Charles Roselli, OHSU) HHMI 2006

2. Male-Oriented Rams 8% of domestic ram population http://www.nature.com/news/2001/010208/images /sheep_200.jpg Exclusively mount other rams

3. Observing the Behavior Preference Testing: RAM

4. Biological Basis for the Behavior? Medial preoptic area-anterior hypothalamus (MPOA-AH) Male-oriented rams more closely resemble ewes Ovine sexually dimorphic nucleus (oSDN)

5. The Presence of Prolactin K110-14 K98-6 MPOA prolactin over-expressed in male-oriented rams (compared to female-oriented rams)

6. The Prolactin Gene Over 300 biological activities LactationReproduction Development of mammary glandImmune Response Angiogenesis (development of blood vessels) Homeostasis http://sheepandgoat.com/images/ewenursinglamb.jpg

7. Transcription and the 5 ’ Promoter Serves as a control for initiating transcription of mRNA Has been sequenced for bovine, human, and rat prolactin genes, but not sheep Goal: sequence the ovine 5’ promoter region measure of “gene expression”

8. DNA Amplification Polymerase chain reaction (PCR) known sequence unknown known Need to use a modified technique… ?

9. Bubble PCR - Preparation 1. Obtain purified genomic DNA 2. Design “bubble”, hybridize top and bottom strands complementary 5’ 3’ 5’ not complementary 3. Digest genomic DNA with restriction enzyme (Bam HI) unknown known 5’ 3’ 5’ 1 2 4. Ligate bubble to genomic DNA fragments 1 2

10. Bubble PCR General Scheme: bubble promotercoding region 5’ 3’ 5’ 3’ 5’ 5’ 3’ (red and blue indicate complementarity) Step 1: Coding primer creates complementary strand 5’ 3’ 5’ Step 2: Once complementary strand has been synthesized, bubble primer primes 5’ 3’ 5’

11. The Ligation Ligation of the bubble construct to digested genomic DNA BEFORE AFTER digests ligations Differences: -greater bubble excess -longer bubble hybridization time digest

12. Successful Amplification Product ~1100 base pairs long

13. Preliminary Sequence 5’ 3’ Reads from bubble construct: bubble coding Next step: obtain sequence reading from coding 5’3’ bubble coding

14. Preliminary Sequence ATGCGTCGANCNNNCGATCGACCTGACTTAAGGANAAATTGCANTCNAGGTTAAGCG GCATANCGCGCTCAGCATNACTCTCGAANGGAGACGAGGNGNTCCNGGGAACTTNN NNCACATTTCCAAAAGNNCNGCCGAAATGACCCCTTTNNATTCNNCAAGGGAANNA GATTTTCNNTGNNCTGCNTNNACCTCCANGNAAATGGATCCNCTNCNNCCCCGGGNG GNNANNGCTCNNGNATNTTCTCAAAGTNGTCGGNGAGGAGAGTCTCGAGTTNAAAG ACGNGACNTCAAAGGANCCGCATNNNNTGGTCNNNGGAAAGGACTGNCCNCATGTG ATNTTCCCGGTGGGCTTTAAAAATTCCNNNCCNGCNNATGTNCGGGACTAAGACNNN GTNCGNNGNCGAGACCCNAAACTNACNNTTCNANTCCTGTGNTGACTGGNAANNNG GGCTACTTNTGGAGNCCTCCNCATGGNAATNNGTNTCTCGTCNCGNATNCGGGNNTG ANACTTAATTCTTNANNCCCCANNNGNAACNCNAATGNCAACTGGNCNTTCC…

15. Future Research Sequence both strands of PCR product Purify DNA from our male-oriented and female- oriented rams to compare promoter DNA sequences 2 Scenarios: 1. DNA sequences differ 2. DNA sequences are the same Analyze transcription factors involved in the regulation of the prolactin gene

16. Acknowledgements HHMI Fred Stormshak, Charles Roselli Kevin Ahern and Indira Rajagopal The Stormshak Lab: Caitlin, Reed, Jared, Cecily…and the sheep