Structural analysis of an annexin gene regulated by prolactin in the pigeon cropsac

Youssef Samir Hitti, Marquette University


In an effort to understand the mechanisms by which prolactin (PRL) regulates specific gene expression, I undertook the structural analysis of an annexin I gene (cp35) which is induced by PRL in the Columbid cropsac. Annexins are Ca$\sp{2+}$-dependent actin- and membrane-binding proteins which include major substrates for receptor-associated tyrosine kinases and protein kinase C. Naive and PRL-treated cropsacs express cp35 cross-hybridizing mRNAs differing in their 5$\sp\prime$ termini as revealed by northern analysis of cropsac RNA with a cp35 cDNA and 5$\sp\prime$end-specific oligonucleotide probes. The question whether these mRNAs arise by the differential processing of the transcript of one gene, or are products of two genes was considered. Southern analysis of genomic DNA reveals a cp35 sequence complexity consistent with a very large gene or multiple related genes. Six independent clones were isolated from a pigeon liver genomic library, and were characterized by restriction mapping and Southern analysis. One 13 Kb clone, W41, compromises 85% of the coding DNA and 3 Kb of 5$\sp\prime$ flanking DNA, but not the terminal 3$\sp\prime$ coding and UT regions. The latter were contained in overlapping clones. Restriction fragments of W41 DNA were subcloned in the bacteriophage M13. Sequencing and S1 protection assays of a 5 Kb HindIII subclone show that exon 1 (47 bp) encodes most of the 5$\sp\prime$ UTR, flanked by a canonical TATA promoter 31 bp upstream of a putative cap site and by 1.4 Kb of upstream DNA. Introns of 1.6 and 1.1 Kb, respectively, separate exons 1 and 2 (79 bp), and exons 2 and 3 (94 bp). Exons 4 (95 bp), 5 (114 bp), 6 (91 bp), and part of 7 ($>$58 bp) were also mapped by sequencing other subclones. Additional exons were identified downstream by S1 protection assays, yielding a total of 12-13 exons, and a gene size of about 14 Kb. The data strongly suggest the existence of more than one annexin I gene in the pigeon, in contrast to the human gene. Sequence and exon organization analysis of the cp35 gene suggests that it arose recently in a duplication/inversion mechanism leading to the diversification of the annexin I gene in Columbid birds. This event resulted in the recruitment of new coding sequences in the 5$\sp\prime$ region of the gene and the loss of potential phosphorylation sites in that region. Regulatory elements which brought the gene under the control of PRL might also have been simultaneously recruited.

Recommended Citation

Hitti, Youssef Samir, "Structural analysis of an annexin gene regulated by prolactin in the pigeon cropsac" (1990). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI9101415.