Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

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The human insulin gene contains two intervening sequences, one is within the region transcribed into the 5'-untranslated segment of the mRNA and the other interrupts the C-peptide encoding region. A comparison of the human with the rat insulin genes indicates potential regulatory regions in the DNA segment preceding the gene and suggests that the ancestral form of the insulin gene had two intervening sequences.

In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5-to-3 exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5 and 3 ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.Messenger RNA degradation contributes significantly to the regulation of gene expression. In eukaryotes, elucidation of a number of mRNA degradation pathways is under way (for reviews, see refs 1-4). Presently, these pathways are better understood in yeast than in mammalian cells. Both in yeast and in mammalian cells, degradation of most polyadenylylated mRNAs appears to be initiated by poly(A) shortening: transcriptional pulse-chase experiments have shown that shortening of the poly(A) tail precedes mRNA degradation (5, 6). Some regulatory sequence determinants affect mRNA stability by modulating the rate of deadenylylation, whereas others modulate a later degradation step (5-7). This later step has been elucidated in yeast: deadenylylation at the 3Ј end triggers mRNA decapping at the 5Ј end, which is then followed by 5Ј-to-3Ј exonucleolysis (refs. 8 and 9 and references therein). In some specific circumstances, other degradation pathways are observed: exonucleolysis from the 3Ј end can occur when the 5Ј-to-3Ј exonuclease is inactive, and a decapping pathway independent of poly(A) tail shortening is involved in the degradation of mRNAs that show premature translation termination (9-11).In mammalian cells, the degradation pathway that follows deadenylylation is not well understood. Uncapped mRNAs are less stable than their capped counterparts in cell extracts, and enzymatic activities that catalyze mRNA decapping and 5Ј-to-3Ј exonucleolysis have been identified (refs. 1 and 3 and references therein). Furthermore, there is a conservation between yeast and mammalian cells of a functional interaction between the 5Ј and 3Ј ends of mRNAs: in both systems, these two ends contribute to translational control (e.g., see refs. 12 and 13). It is thus tempting to speculate that deadenylylation triggers a decapping-dependent degradation pathway in mammalian cells as well. However, a direct demonstration is lacking.We have developed a revers...

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Complementary DNAs for two distinct anglerfish somatostatin peptides (termed I and II) have been cloned in bacterial plasmids and sequenced. The nucleotide sequence for somatostatin I encodes a large percursor peptide (molecular weight 13,300) in which the somatostatin hormones is at the carboxyl terminus. The predicted 14-amino acid sequence for anglerfish somatostatin I is the same as mammalian somatostatin. Somatostatin II is also synthesized as part of a larger precursor (molecular weight 14,100) with the presumptive somatostatin hormone also at the carboxyl terminus. The 14-amino acid sequence of somatostatin II differs from somatostatin I at two internal residues (Tyr in place of Phe 7 and Gly in place of Thr 10). The two different somatostatins may have distinct biological activities. Homologies in the amino acid sequences of the two peptides outside the somatostatin moiety suggest other regions of the molecules have biological functions.

The structures of two remote glucocorticoid responsive units (GRUs) that cooperatively interact to promote cell-type specific glucocorticoid induction of rat tyrosine aminotransferase gene expression have been analyzed. DNAase I footprinting and gel mobility shift analyses reveal a complex array of contiguous and overlapping sites for cell type-specific DNA binding proteins. Apart from the glucocorticoid receptor, two liver-specific nuclear factors possess multiple binding sites in each of these GRUs: C/EBP and a newly identified liver-specific factor: HNF5. C/EBP possesses four binding sites in each GRU; a DNA-binding protein with similar binding specificity has been identified in fibroblasts; this protein could be related to AP-3. HNF5 possesses two binding sites in one GRU and four in the other. There are also HNF5 binding sites in numerous regulatory regions of other liver-specific genes. The interaction of HNF5 with DNA gives a characteristic DNAase I footprint with hypersensitive sites in the middle of the recognition sequence. Some of the C/EBP and HNF5 binding sites overlap in a conserved arrangement.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.