Abstract
The insulin-, glucagon-, and somatostatin-producing cells in the pancreatic islets derive from a common precursor stem cell and differentiate sequentially during embryonic development, thereby providing an informative model for the study of the transcriptional mechanisms involved in the control of cell-specific gene expression. Relative to the early expression of the glucagon and insulin genes on embryonic days 10 and 12, respectively, the expression of the somatostatin gene is delayed (day 17). The relatively late expression of the somatostatin gene indicates the involvement of both negative and positive transcriptional control mechanisms. We show that the expression of the somatostatin gene in pancreatic islet cells is accomplished by the interplay of both positive and negative cis-regulatory DNA elements. We have characterized the functional properties of one of these positive control elements, the somatostatin gene upstream enhancer element (SMS-UE). The SMS-UE is a pancreatic islet D-cell-specific transcriptional regulator that acts synergistically with the cyclic AMP response element. Mutation-expression and cell-free transcription analyses show that the SMS-UE is a bipartite element with two interdependent functional domains. Our results indicate that the SMS-UE is part of a functional unit that includes other transcriptional control elements of the somatostatin gene proximal promoter, and that they act together to regulate the D-cell-specific transcription of the somatostatin gene in the islet cells of the pancreas.
Highlights
From the Laboratory of Molecular Endocrinology,Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston,Massachusetts 02114
Expression of the cells in the pancreatic islets derive from a common somatostatin gene occurs at day 17 of embryonic development precursor stem cell and differentiate sequentially dur- in cells that coexpress the insulin gene, which is subsequently ing embryonic development, thereby providing an in- repressed in maturesomatostatin-producing D-cells
Ratsomatostatin gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid resulted in the identification of a region spanning nucleotides -30 to -60 relative to thetranscription start site, that was apparently sufficient to confer cell-specific expression in cells derived from a thyroid medullary carcinoma [10]
Summary
Materials-Restriction, ligation, and other DNA-modifying enzymes were purchased from New England Biolabs (Beverly, MA) or Boehringer Mannheim. The plasmids were digested with therestriction enzyme AatII, whichrecognizes the sequence corresponding to the CRE octamer and generates 3’ end overhangs These were digested with T4 DNA polymerase to produce blunt ends. The reaction mixture contained 10 mM HEPES, pH 7.9, 60 mM KC1, 6 mMMgC12, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM creatine phosphate, and 0.5 mM each of ATP, GTP, CTP, and UTP Transcripts generated in this waywere collected by phenol-chloroform extraction followed by ethanol precipitation, and they were analyzed by primer extension [33] using a 19base oligonucleotide primer complementary to the coding strand of the chloramphenicol acetyltransferase gene in pOCAT (from nucleotides 2542 to 2560), and a 17-base oligonucleotide complementary to the adenovirus tripartite leader (from nucleotides 59 to 75). The sequences of the sense strand of the oligonucleotides used are: SMS-UE A domain, 5’GATCCTTCTTTGATTGATTTTGCGAGGA-3’; SMS-UEB domain, 5’-GATCCGCGAGGCTAATGGTGCGTA-3a’n;d heterologous control, 5’-GATCCAGGCACGAGCATCTGGTCA-3’
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
