The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.     

Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyse rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobases (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. The abundance of a 7.5-kb Sm-C/IGF-I mRNA in poly(A+) RNAs from adult rat liver was 10-50-fold higher than in other adult rat tissues which provides further evidence that in the adult rat the liver is a major site of Sm-C/IGF-I synthesis and source of circulating Sm-C/IGF-I. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. Some samples of adult rat intestine contained the 4.7- and 3.9-kb IGF-II mRNAs and some samples of adult liver and lung contained the 4.7-kb mRNA. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.     

Complex structure and regulation of expression of the rat gene for inward rectifier potassium channel Kir7.1

Genomic organization of the rat inward rectifier K(+) channel Kir7.1 was determined in an attempt to clarify how multiple species of its mRNA are generated in a tissue-specific manner and how its expression is regulated. The rat Kir7.1 gene spans >40 kilobases (kb) and consists of eight exons; the first four exons encode the 5'-untranslated region that is unusually long ( approximately 3 kb). The coding region is located in exons 5 and 6. In the testis, exon 4 is processed as four exons (4a-4d), whereas it is recognized as a single exon in the small intestine. The three major species of rat Kir7.1 mRNA (1.4, 2.2, and 3.2 kb) were found to arise from alternative usage of the two promoters and polyadenylation signals and by alternative splicing of the 5'-noncoding exons. The splicing pattern of the 5'-noncoding exons is quite complex and highly tissue-specific, suggesting that complex mechanisms may operate to regulate the Kir7.1 expression. Deletion and mutational analysis of the promoter activity indicated that the rat Kir7.1 gene is regulated by cAMP through a CCAAT element. The cAMP induction was also demonstrated using the rat follicular cell line FRTL-5 endogenously expressing Kir7.1.     

Identification of RNA sequences and structures involved in site-specific cleavage of IGF-II mRNAs.

Insulin-like growth factor-II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region (UTR), rendering an unstable 5' cleavage product containing the coding region and a very stable 3' cleavage product of 1.8 kb consisting of the 3'-UTR sequence and the poly(A) tail. Previously, it was established that two widely separated elements in the 3'-UTR (elements I and II), that can form a duplex structure, are necessary and sufficient for cleavage. To further investigate the sequence and secondary structure requirements for cleavage, we have introduced a number of mutations around the cleavage site and assayed their effects on cleavage. Several recognition determinants involved in the endonucleolytic cleavage of IGF-II mRNAs were identified. Mutational analysis around the cleavage site revealed that cleavage is sequence specific and that the cleavage site must be in a single-stranded conformation to allow efficient cleavage. In addition, we have identified an accessory protein that specifically interacts with a stem-loop structure located 133 to 73 nt upstream of the cleavage site.