Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.

Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.

SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.