Expression of functional recombinant human factor IX in milk of mice.

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk beta-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.

Molecular structure and alternative splicing of the human carboxypeptidase M gene.

Using RACE technology the 5' and 3' ends of human carboxypeptidase M (CPM) mRNA were determined and found to be divergent from the published sequence. With these results the complete structure of the human CPM gene was established based on the human genome sequence in the GenBank database. The gene was shown to contain 9 exons comprising at least 75 kb of genomic sequence. A novel first exon of 30 bp was identified and an upstream promoter sequence containing several transcription factor binding sites was found by computer analysis. Furthermore, the ATG starting codon was detected defining an open reading frame of 1329 bp that codes for a protein of 443 residues. Additionally, the polyadenylation site was discovered, determining a 3' noncoding region of 2000 nucleotides. The exon-intron boundaries diverged substantially compared to those of the other basic carboxypeptidases, CPD, CPE, CPN, and AEBP1. Cloning and sequencing of RT-PCR products from different tissues revealed alternative splicing of exons 3 and 5, which results in the generation of four different mRNA isoforms. RNA extracted from tumor tissues contained more CPM mRNA than control tissue, suggesting an upregulation of CPM expression in tumors and raising the question of the role of this enzyme in cancer.

Transcriptional regulation of the rat bradykinin B2 receptor gene: identification of a silencer element.

Kinins are involved in a variety of physiological and pathophysiological processes related to cardiovascular homeostasis, inflammation, blood flow, and nociception. Under physiological conditions, the bradykinin B2 (BKB2) receptor is constitutively expressed and mediates most of kinins' actions. However, the mechanisms regulating BKB2 receptor gene expression are still poorly understood. In this study, 4.6 kilobases of the 5'-flanking region from the rat BKB2 receptor gene were sequenced, and computer analysis revealed several sites for transcriptional factors. Nine promoter mutants were cloned in luciferase reporter gene vectors and transfected in NG108-15 cells and rat aorta vascular smooth muscle cells (VSMCs), showing several positive and negative regulatory elements. A classical silencer with 56 base pairs (bp) caused a decrease in reporter gene activity in NG108-15 cells and VSMCs and was able to inhibit the thymidine kinase promoter. Using electrophoretic mobility shift assay and surface plasmon resonance assay, protein-DNA interactions in the silencer region were determined and specific sets of protein-silencer complexes were detected in both cell types. More intense complexes were observed in the central 21 bp of the silencer and mutation in a putative SRE-1 site strongly impaired the protein-DNA binding. Down-regulation of the BKB2 receptor population in NG108-15 cells promoted by N(6), 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate was paralleled by an increase in the amount of nuclear proteins bound to the silencer sequence showing an inverse relationship between protein-silencer complexes and the transcription of the BKB2 receptor gene. In summary, these data highlight the cell-specific regulation of the BKB2 receptor and the importance of a silencer element present in the regulatory region of the gene.