CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.     

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.     

Cloning and characterization of zinc finger protein 161 in Rattus norvegicus.

Zinc finger protein 161 (ZFP161) belongs to the large Kruppel type ZFP family, contains five consecutive zinc fingers, and may be involved in development. Here, we present the novel cloning and characterization of the ZFP161 cDNA from Rattus norvegicus. The rat ZFP161 has an open reading frame of 1347 bp and encodes a ZFP of 449 amino acids with a predicted mass of 51 kDa. Sequence alignment shows that there is a high homology among the deduced amino acid sequences of the rat, mouse and human ZFP161 proteins. We determined that both the mouse and rat coding sequences are contained within a single exon, with the start codon (ATG) contained in a separate exon. RT-PCR revealed that the ZFP161 mRNA is expressed at high levels in rat spleen, brain, lung and kidney, and at much lower levels in muscle and heart.     

TTG serves as an initiation codon for the ribosomal protein MvaS7 from the archaeon Methanococcus vannielii.

The ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is a protein of 188 amino acids, i.e., it is 42 amino acids longer than previously suggested. The triplet TTG serves as a start codon. The methanogenic translation initiation region that includes the rare TTG start codon is recognized in Escherichia coli.     

Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I)

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.     

Characterization of rat β-glucan receptor dectin-1

Dectin-1 is a small C-type lectin receptor for fungal cell wall β-glucan. Its homologues in some species, including mouse and human, have been characterized, and their importance in antifungal immunity has also been clarified. However, its homologue in the rat has not yet been identified. In this study, DNA/amino acid sequences of rat dectin-1 were analyzed by rapid amplification of cDNA ends. The sequence of rat dectin-1 was found to be highly homologous to that of the mouse. It possesses essential motifs for the recognition of β-glucan and signal transduction. However, the position of the start codon in the detected sequence was not conserved, and its cytoplasmic tail was shorter than that observed in other species. Similar to mouse dectin-1, two major isoforms of rat dectin-1 that were generated by alternative splicing were identified: a full-length isoform and a shorter isoform deficient in the stalk domain. It was also demonstrated that rat dectin-1 is capable of binding fungal β-glucan and activating nuclear factor-kappa B via Syk and the CARD9/Bcl10-mediated pathway. These results suggest that rat dectin-1 also plays essential roles in immune responses against fungi.     

Characterization of rat beta-glucan receptor dectin-1

Dectin-1 is a small C-type lectin receptor for fungal cell wall beta-glucan. Its homologues in some species, including mouse and human, have been characterized, and their importance in antifungal immunity has also been clarified. However, its homologue in the rat has not yet been identified. In this study, DNA/amino acid sequences of rat dectin-1 were analyzed by rapid amplification of cDNA ends. The sequence of rat dectin-1 was found to be highly homologous to that of the mouse. It possesses essential motifs for the recognition of beta-glucan and signal transduction. However, the position of the start codon in the detected sequence was not conserved, and its cytoplasmic tail was shorter than that observed in other species. Similar to mouse dectin-1, two major isoforms of rat dectin-1 that were generated by alternative splicing were identified: a full-length isoform and a shorter isoform deficient in the stalk domain. It was also demonstrated that rat dectin-1 is capable of binding fungal beta-glucan and activating nuclear factor-kappa B via Syk and the CARD9/Bcl10-mediated pathway. These results suggest that rat dectin-1 also plays essential roles in immune responses against fungi.     

Molecular characterization of the murine argininosuccinate synthetase locus.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.     

Mutations at a Zn(II) finger motif in the yeast eIF-2 beta gene alter ribosomal start-site selection during the scanning process

We have genetically reverted HIS4 initiator codon mutants in yeast and identified three unlinked genes, sui1, sui2, and SUI3 (suppressors of initiator codon mutants), which when mutated confer the ability to initiate at HIS4 despite the absence of an AUG start codon. Molecular and biochemical characterization shows that SUI3 encodes the beta-subunit of the eukaryotic translation initiation factor eIF-2. SUI3 suppressor genes contain single base changes at a Zn(II) finger motif. This motif is present in a cDNA sequence encoding the human eIF-2 beta gene product. Mutations in SUI3 suppressor alleles change amino acids that are conserved in the yeast and human motifs. Protein sequence analysis shows that a mutant beta-subunit allows initiation at a UUG codon in the absence of an AUG start codon at HIS4. Taken together, these data implicate a nucleic acid-binding domain of eIF-2 as an important component of the "scanning" ribosome that participates in recognition of a start codon.     

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs.

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.