The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region

We report the isolation and characterization of the human gene encoding islet amyloid polypeptide (IAPP). Previously characterized cDNA sequences correspond to three exons of which the first is noncoding. A functional promoter region was identified in the 5' flanking DNA; however, this was farther upstream than expected. Northern blot analysis of human insulinoma RNA revealed three IAPP mRNAs of sizes 1.2, 1.8 and 2.1 kb, in agreement with three polyadenylation signals present in the 3' end of the gene. In situ hybridization to metaphase chromosomes resulted in two distinct peaks on chromosome 12, at 12p12-p13 and 12q13-q14. Southern blot analysis of genomic DNA suggested a single IAPP locus but also indicated the presence of additional homologous sequences in human genomic DNA.     

Islet amyloid polypeptide: identification and chromosomal localization of the human gene

Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy-terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy-terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.     

Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed mouse cells.

The great majority of viral mRNAs in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV) have a common 3' end at the early polyadenylation site which is 23 nucleotides (nt) downstream of a canonical poly(A) consensus signal. Twenty percent of BPV mRNA from productively infected cells bypasses the early polyadenylation site and uses the late polyadenylation site approximately 3,000 nt downstream. To inactivate the BPV early polyadenylation site, the early poly(A) consensus signal was mutated from AAUAAA to UGUAAA. Surprisingly, this mutation did not result in significant read-through expression of downstream RNA. Rather, RNA mapping and cDNA cloning experiments demonstrate that virtually all of the mutant RNA is cleaved and polyadenylated at heterogeneous sites approximately 100 nt upstream of the wild-type early polyadenylation site. In addition, cells transformed by wild-type BPV harbor a small population of mRNAs with 3' ends located in this upstream region. These experiments demonstrate that inactivation of the major poly(A) signal induces preferential use of otherwise very minor upstream poly(A) sites. Mutational analysis suggests that polyadenylation at the minor sites is controlled, at least in part, by UAUAUA, an unusual variant of the poly(A) consensus signal approximately 25 nt upstream of the minor polyadenylation sites. These experiments indicate that inactivation of the major early polyadenylation signal is not sufficient to induce expression of the BPV late genes in transformed mouse cells.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

One of the copia genes is adjacent to satellite DNA in Drosophila melanogaster.

A method for purifying sequences adjacent to satellite DNA in the heterochromatin of D. melanogaster is described. A cloned DNA segment containing part of a copia gene adjacent to 1.688 g/cm3 satellite DNA has been isolated. The copia genes compose a repeated gene family which codes for abundant cytoplasmic poly(a)-containing RNA (Young and Hogness, 1977; Finnegan et al., 1978). We have identified two major poly (A)-containing RNA species [5.2 and 2.1 kilobases (kb)] produced by the copia gene family. The cloned segment contains copia sequences homologous to the 5' end of RNA within 0.65 kb of the 1.688 satellite DNA sequences. Seven different cloned copia genes from elsewhere in the genome have also been isolated, and a 5.2 kb region present in five of the clones was identified as copia by heteroduplex analysis. In addition, three ususual copies of copia were found: a "partial" copy of the gene (3.7 kb) which has one endpoint in common with the 5.2 kb unit; a copia gene flanked on one side by a 1.6 kb sequence and on the other by the same 1.6 kb sequence in the inverted orientation; and a copia gene flanked only on one side by the same sequence.     

Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs

Alternative polyadenylation leads to mRNAs with variable 3′ ends. Since a 3′-untranslated region (3′-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3′-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3′-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3′-UTR. CFIm is a heterodimeric 3′ end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3′-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.