Mass spectrometric characterization of full-length rat selenoprotein P and three isoforms shortened at the C terminus. Evidence that three UGA codons in the mRNA open reading frame have alternative functions of specifying selenocysteine insertion or translation termination

Selenoprotein P is an abundant extracellular glycoprotein. Its mRNA contains 10 UGAs in an open reading frame terminated by a UAA. This predicts that full-length selenoprotein P will contain 10 selenocysteine residues. Full-length selenoprotein P and three smaller isoforms that have identical N termini have been demonstrated. Selenoprotein P was purified from rat plasma, and the four isoforms were separated by heparin chromatography and SDS-PAGE. Mass spectrometric peptide analysis of the full-length isoform verified 357 of its 366 predicted amino acid residues, including its C terminus and all 10 selenocysteines. The C termini of the smaller isoforms were characterized by mass spectrometry. The shortened isoforms terminated where the second, third, and seventh selenocysteine residues were predicted to be. This suggests that all isoforms arise from the same mRNA and that the UGAs that specify the second, third, and seventh selenocysteines in full-length selenoprotein P can alternatively serve to terminate translation, producing the shorter isoforms.     

The developmentally regulated type X collagen gene contains a long open reading frame without introns

Type X collagen is a recently discovered product of hypertrophic chondrocytes that is localized to presumptive mineralization zones of hyaline cartilage. Thus, in the epiphyseal growth plate of long bones it is present only in the zone of hypertrophic chondrocytes and absent in the resting and rapidly growing cartilage and in bone. Type X collagen represents, therefore, a transient and developmentally regulated collagen which is synthesized by a subpopulation of chondrocytes. We report here the isolation and characterization of cDNA and genomic clones specific for the chicken protein. The results demonstrate that the polypeptide chains of this collagen contain three distinct domains: a short non-collagenous, amino-terminal region, a collagenous domain of 460 amino acid residues, and a non-collagenous, carboxyl-terminal domain of 170 amino acid residues. The nucleotide sequence of the gene shows that these domains are encoded by a long open reading frame that is not interrupted by introns. Examination of the amino acid sequence derived from this nucleotide sequence reveals the presence of a hydrophobic segment localized 10 amino acid residues upstream from the translational stop codon. The length and sequence characteristics of this segment raise the intriguing possibility that Type X collagen polypeptides may contain a transmembrane segment.     

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.     

Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame

The suppressor of cytokine signalling 1 protein (SOCS-1) belongs to a novel family of cytokine inducible factors which function as inhibitors of the JAK/STAT pathway. While SOCS-1 previously has been described as a single-exon gene, here we present evidence for an additional 5' exon, separated by a 509 bp intron from exon 2. Exon 1 and part of exon 2 contain an open reading frame of 115 nt, ending one nucleotide upstream of the major reading frame. Using SOCS-1-promoter/luciferase constructs, we investigated which sequences are involved in the regulation of SOCS-1 expression. Serial promoter deletion clones indicate the localization and functionality of SP1, interferon-stimulated responsive elements (ISRE), and interferon-gamma-activated sites (GAS) promoter elements in the SOCS-1 5' flanking region. We present evidence that the upstream open reading frame (uORF) represses the translation of the downstream major open reading frame (mORF). Mutating the start codon of the uORF relieves this repression. Our data indicate that expression of the SOCS-1 protein is repressed on translational level by a mechanism, which bears similarities to that postulated for genes like retinoic acid receptor beta2 (RARbeta2), S-adenosylmethionine-decarboxylase (AdoMetDC), Bcl-2, and others.     

The upstream open reading frame of cyclin-dependent kinase inhibitor 1A mRNA negatively regulates translation of the downstream main open reading frame

The skeletal muscle cells are one of the main sites of glucose uptake through glucose transporter 4 (GLUT4) in response to insulin. In muscle cells, 5' adenosine monophosphate-activated protein kinase (AMPK) is known as another GLUT4 translocation promoter. Natural compounds that activate AMPK have a possibility to overcome insulin resistance in the diabetic state. Piceatannol is a natural analog and a metabolite of resveratrol, a known AMPK activator. In this study, we investigate the in vitro effect of piceatannol on glucose uptake, AMPK phosphorylation and GLUT4 translocation to plasma membrane in L6 myocytes, and its in vivo effect on blood glucose levels in type 2 diabetic model db/db mice. Piceatannol was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by piceatannol of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector. Piceatannol suppressed the rises in blood glucose levels at early stages and improved the impaired glucose tolerance at late stages in db/db mice. These in vitro and in vivo findings suggest that piceatannol may be preventive and remedial for type 2 diabetes and become an antidiabetic phytochemical.     

Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: identification of three in-phase TGA selenocysteine codons.

The mitochondrial selenoprotein is a major structural protein of the keratinous mitochondrial capsule in mammalian sperm, a structure that functions in shaping mitochondria into the helical sheath surrounding the flagellum. A cDNA clone (Kleene et al., 1990) was isolated previously encoding a protein whose predicted size and amino acid content of > 20% cysteine and proline closely resembled a selenoprotein in the bull mitochondrial capsule. The sequences of additional cDNAs and genomic DNA reported here reveal that the mouse mitochondrial capsule selenoprotein reading frame begins 54 codons further upstream than previously reported. Significantly, these 54 codons contain three in-phase UGA codons, which normally signify stop but encode selenocysteine in bacterial and mammalian selenoproteins. The coding region of the mitochondrial capsule selenoprotein gene is interrupted by a single intron. S1 mapping and primer extension demonstrate that the vast majority of MCS mRNAs are spliced using consensus 5' and 3' slice junctions in mammalian cells. However, two cDNAs have been identified that apparently represent rare mRNA variants produced by use of cryptic splice sites.     

The unique sequence of the herpes simplex virus 1 L component contains an additional translated open reading frame designated UL49.5.

We present evidence for the existence of an additional herpes simplex virus 1 gene designated UL49.5. The sequence, located between genes UL49 and UL50, predicts a hydrophobic protein with 91 amino acids. Attempts to delete UL49.5 were not successful. To demonstrate that UL49.5 is expressed, we made two recombinant viruses. First, we inserted in frame an oligonucleotide encoding a 15-amino-acid epitope known to react with a monoclonal antibody. This gene, consisting of the authentic promoter and chimeric coding domain, was inserted into the thymidine kinase gene of wild-type virus and in infected cells expressed a protein which reacted with the monoclonal antibody. The second recombinant virus contained a 5' UL49.5-thymidine kinase fusion gene. The protein expressed by this virus confirmed that the first methionine codon of UL49.5 served as the initiating codon. The predicted amino acid sequence of UL49.5 is consistent with the known properties of NC-7, a small capsid protein whose gene has not been previously mapped. A homolog of UL49.5 is present in the genome of varicella-zoster virus, located between homologs of UL49 and UL50.     

The self-splicing intron in the Neurospora apocytochrome b gene contains a long reading frame in frame with the upstream exon.

We have determined the DNA sequence of intron 1 and flanking exons in the mitochondrial apocytochrome b gene of the Neurospora laboratory strain 74A and the natural isolate North Africa. In contrast to a previous report, we find that this intron contains an open reading frame (ORF) of 951 bases in frame with the upstream exon. The putative intron-encoded protein resembles those of other intron ORFs with respect to length, calculated isoelectric point, and proportion of basic, acidic, polar, and non-polar amino acids; however, no amino acid sequences resembling the "decapeptides" characteristic of maturase-like ORFs were found. Coupled with the previous finding that this intron is capable of self-splicing in vitro in the absence of proteins, the observations discussed here raise the possibility that other introns with long, in-frame ORFs may also be capable of RNA-catalyzed splicing in vitro.     

The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.     

Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum

Selenoprotein P is the second plasma selenoprotein to be purified. It is a glycoprotein and has been shown to be distinct from plasma glutathione peroxidase. This study characterizes selenoprotein P further. Deglycosylation of the protein shifts its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from Mr 57,000 to Mr 43,000, indicating it has a substantial carbohydrate component. Measurement of selenium indicates a selenium content of 7.5 +/- 1.0 atoms/molecule based on a polypeptide weight of 43,000. Amino acid analysis accounts for all the selenium as selenocysteine. The protein is also rich in cysteine (17 residues) and histidine (23 residues). Fragmentation of selenoprotein P by trypsin and by cyanogen bromide produces peptides with varying selenium content. This indicates that selenium-rich regions of the protein exist. The concentration of selenoprotein P determined by radioimmunoassay in serum from control rats is 26.3 +/- 4.5 micrograms/ml and in serum from selenium-deficient rats it is 2.7 +/- 0.8 micrograms/ml. Depletion of selenoprotein P from control serum using an immunoaffinity column indicates that over 60% of serum selenium in the rat is contained in this protein. These results demonstrate that selenoprotein P is the major form of selenium in rat serum. It is the first selenoprotein described which has more than one selenium atom/polypeptide chain.     

The expression of the open reading frame of Arabidopsis CAX1, but not its cDNA, confers metal tolerance in yeast

The biochemical properties and regulation of several plant CAX (CAtion eXchanger)-type vacuolar Ca2+/H+ exchangers have been extensively analysed in yeast expression assays. In the present study, we compare and contrast the phenotypes of yeast cells expressing the CAX1 cDNA and open reading frame (ORF). We report that the CAX1 ORF, but not the cDNA containing the 3′-untranslated region (UTR), was able to confer Ca2+ tolerance when expressed in a Ca2+-sensitive yeast mutant. Additionally, only yeasts expressing the N-terminal truncated CAX1 ORF were able to grow on high Mn2+ media, suggesting that removal of the 3′-UTR altered activity. However, removal of the 3′-UTR from another CAX did not alter the yeast phenotypes. Expression studies demonstrated that expressing the CAX1 ORF in yeast elevates CAX1 RNA and protein levels. Our results suggest that the 3′-UTR modulates expression of CAX1 in yeast.     

Maintenance of the correct open reading frame by the ribosome

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon–anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.     

Prokaryotes that grow optimally in acid have purine-poor codons in long open reading frames

In nucleic acids the N-glycosyl bonds between purines and their ribose sugar moities are broken under acid conditions. If one strand of a duplex DNA segment were more vulnerable to mutation than the other, then the archaeon Picrophilus torridus, with an optimum growth pH near zero, could have adapted by decreasing the purine content of that strand. Yet, P. torridus has an optimum growth temperature near 60°C, and thermophiles prefer purine-rich codons. We found that, as in other thermophiles, high growth temperature correlates with the use of purine-rich codons. The extra purines are often in third, non-amino acid determining, codon positions. However, as in other acidophiles, as open reading frame lengths increase, there is increased use of purine-poor codons, particularly those without purines in second, amino acid-determining, codon positions. Thus, P. torridus can be seen as adapting (a) to temperature by increasing its purines in all open reading frames without greatly impacting protein amino acid compositions, and (b) to pH by decreasing purines in longer open reading frames, thereby potentially impacting protein amino acid compositions. It is proposed that longer open reading frames, being larger mutational targets, have become less vulnerable to depurination by virtue of pyrimidine for purine substitutions.