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PubMed1996

A novel missense mutation in the amino-terminal domain of the human androgen receptor gene in a family with partial androgen insensitivity syndrome causes reduced efficiency of protein translation.

The role of the androgen receptor (AR) in male sexual differentiation is revealed in part by the analysis of naturally occurring mutations in families with androgen insensitivity syndrome (AIS). We have investigated a family with partial AIS affecting three generations and have identified a G to A substitution in the AR gene at the fourth position 3' from the A of the ATG initiation codon changing the second amino acid residue from glutamic acid to lysine (EK2). Transient expression of the mutant EK2-pCMVhAR expression vector in COS cells revealed decreased translation with a 20-50% reduction in mutant protein relative to wild type AR by immunoblot analysis. The rate of dissociation of [3H]methyltrienolone from the EK2 mutant (half-time [t1/2] = 1.7 +/- 0.08 SE h) was increased compared with wild type AR (t1/2 = 2.4 +/- 0.11 h). Cotransfection studies using an androgen responsive luciferase reporter vector demonstrated a 50% reduction in transcriptional activation by EK2. These functional alterations are consistent with the partial AIS phenotype in affected males, corroborate the AR amino-terminal domain effect on kinetics of androgen binding, and provide physiological evidence for earlier translation experiments identifying the nucleotide sequence for optimal translation initiation.

Adolescent↗

PubMed1995

Mapping domains in proteins: dissection and expression of Escherichia coli adenylyl cyclase.

We have used the pRE expression vector containing the Escherichia coli adenylyl cyclase gene (cya) with the unique NdeI restriction site CATATG at the initiation codon in conjunction with a family of self-complimentary oligonucleotides to create amino- and carboxyl-terminal domains in adenylyl cyclase. The three sets of oligonucleotides contain a TAA translation stop codon in all reading frames flanked by the NdeI restriction endonuclease sequence and one or two nucleotides (5' NNCATATGTTAATTAATTAACATATGNN 3'). Ligation of one of these annealed oligonucleotides into a restriction site or creation of 5' TAA/CATATG 3' translation stop/NdeI restriction site along a gene in the pRE expression vector facilitates the premature termination of protein synthesis thus yielding amino-terminal domains. Removal of a fragment of the gene corresponding to the amino-terminal portion by NdeI restriction and ligation brings the 3' end of the gene in frame with the initiator ATG. With this strategy, expression of the carboxyl-terminal domain of a protein is possible which is otherwise not as simple as the expression of the amino-terminal domain. The feasibility of expression of any domain of a protein is demonstrated using the cya gene to create several amino- and carboxyl-terminal domains of adenylyl cyclase.

Adenylyl Cyclases↗

PubMed1989

Mechanism of inhibition of eukaryotic translational initiation by the trinucleotide ApUpG.

ApUpG, the oligoribonucleotide homologous to the initiation codon, as well as the tetranucleotides ApUpGpA and ApUpGpG block initiation of protein synthesis in the rabbit reticulocyte lysate. These oligonucleotides are recognized as translational initiation sites by the ribosomes, leading to a very large accumulation of complete, but inactive, 80 S initiation complexes, containing methionylated initiator tRNA and ApUpG in a 1:1 stoichiometry. ApUpG appears to inhibit by competing with endogenous globin mRNA for 80 S ribosomal couples, since the inhibition of protein synthesis by ApUpG can be largely relieved by increasing the globin mRNA. The 80 S.Met-tRNAiMet.ApUpG complexes are not formed in the absence of hemin, demonstrating that their formation requires the active recycling of eukaryotic initiation factor 2. In addition the trinucleotide correctly directs the Met-tRNAiMet into the ribosomal donor site, since the methionyl residue is puromycin-reactive.

Animals↗

PubMedDate not supplied

Initiation of translation at internal AUG codons in mammalian cells.

Initiation of translation of eukaryotic mRNAs typically occurs at the first AUG triplet from the 5' end of the message, although several notable exceptions have been described. Using vectors which efficiently express the gene encoding the surface antigen of hepatitis B virus in monkey cells, we have studied the consequences of inserting ATG triplets in all three reading frames upstream of the usual translational initiation codon of this gene. In agreement with the scanning model for eukaryotic translation initiation, these additional codons can severely depress the initiation of translation at the 'authentic' start codon, although the extent of inhibition depends on sequences flanking the upstream AUG. Such inhibition can, however, be at least partially suppressed by the presence of a translation termination codon in-frame with the upstream AUG. These results raise the possibility that mammalian ribosomes can reinitiate translation at an AUG codon after previously initiating, and terminating, at an upstream site.

Animals↗

PubMed1993

Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.

To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.

Amino Acid Sequence↗

PubMed1989

Influence of the 5'-end region of aldehyde dehydrogenase mRNA on translational efficiency. Potential secondary structure inhibition of translation in vitro.

mRNA coding for rat liver mitochondrial aldehyde dehydrogenase was translated in rabbit reticulocytes 10 times more efficiently than was the corresponding bovine mRNA. Previous data showed that the mRNAs primarily differed in the noncoding 5'-end (K.-L. Guan and H. Weiner, manuscript submitted for publication to Arch. Biochem. Biophys.). Fusion mRNAs were constructed so that the 5'-untranslated region from one was attached to the coding sequence of the other. Furthermore, the mRNAs were mutated at position -3, a position implicated in translational regulation, so that both contained the same residues. Results indicated that the dramatic difference in translational rates between rat and bovine mRNAs was just partly due to the sequence around the initiation codon. Secondary structure prediction of mRNA indicated that stable secondary structure could exist in the 5'-end of bovine mRNA, whereas no such stable structure should exist in rat mRNA. Existence of the predicted secondary structure of bovine mRNA was substantiated by the observation that termination in DNA sequencing in the 5'-end of bovine cDNA occurred. It is concluded that stable secondary structure in the 5'-end region of bovine mitochondrial aldehyde dehydrogenase performs a major role in inhibiting translation. This inhibitory effect is related to the magnitude of free energy required to melt the secondary structure in order to reach the initiation AUG codon.

Aldehyde Dehydrogenase↗

PubMed1998

Identification of an alternative open reading frame ("hidden gene"?) stringently required for infectivity of poliovirus cDNA clones.

Translation of the uncapped poliovirus RNA starts at the AUG triplet spanning positions 741-743, and proceeds uninterrupted for almost the entire length of the genome. Such a cap-independent mechanism of internal initiation of translation determines that a long, extra-cistronic region extends between the 5'-end and the main open reading frame (ORF). We have identified 10 short ORFs initiated by the alternative translation initiation codons ACG, AUA, and GUG in the 5'-terminal extra-cistronic region (5'-ECR) of poliovirus RNA. Mutations introduced in all but one of these mini-cistrons had no effect on the infectivity of full-length cDNA clones, except when they modified a "hidden frame" spanning between nucleotides 157-192 (starting triplet: ACG). The mini cistron 157-192 is conserved in position, length and sequence in the genome of all types and strains of poliovirus. Adaptation to rat (Lansing) or mouse (variant of Sabin 2) is accompanied by a consistent pattern of changes in the primary sequence of this "hidden frame". The substitutions that abrogated the infectivity of cDNA clones were not expected to modify the predicted secondary structure of the 5'-ECR, and they did not alter the ability of the IRES to direct internal initiation of translation in bi-cistronic mRNAs. The infectivity of the mutated poliovirus cDNAs could be complemented in trans by co-transfecting the target COS-1 cells with an expression vector containing just the 5'-ECR of poliovirus type 2 (Lansing strain). The infectivity of poliovirus cDNA could be restored by co-transfecting short RNA transcripts of the wt 5'-ECR (Lansing), suggesting that the complementation in trans indeed requires the expression of the helper cDNA clone.

Animals↗

PubMed2006

Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context.

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.

5' Untranslated Regions↗

PubMed1996

Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution.

Translation of aphthovirus RNA is initiated at an internal ribosome entry site (IRES) element, preceding the first functional AUG initiation codon. The effect of mutations at the base of domain 3 of the aphthovirus IRES on translation activity has been analyzed by site-directed mutagenesis and expression of bicistronic RNAs in transfected cells. The results have shown that the enhanced IRES activity associated with a single pyrimidine transition fixed in a persistent aphthovirus variant (E. Martínez-Salas, J. C. Sáiz, M. Dávila, G. J. Belsham, and E. Domingo, J. Virol. 67:3748-3755, 1993) is base specific. Mutations predicted to destabilize the base of domain 3 were detrimental to IRES function, but subsequent restoration of the RNA structure gave rise to fully competent IRES. In contrast, single or multiple mutations that did not affect predicted helical structures modified the relative efficiency of translation by at most 10-fold, suggesting that primary sequence also plays a role in IRES activity. A correlation between the energy of stabilization of the IRES structure and the efficiency of translation has been noted. None of the 15 mutations studied reached a level of initiation of translation comparable to that of the IRES from the persistent variant. The results indicate a critical participation of the base of domain 3 in the activity of the aphthovirus IRES, with a strong effect of secondary or higher-order structures and minor effects of primary structure.

Animals↗

PubMed2000

Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum.

Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3-2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a "core" region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles.

Amino Acid Sequence↗

PubMed1996

Two novel point mutations causing receptor-negative familial hypercholesterolemia in a South African Indian homozygote.

Two novel point mutations have been identified in the low density lipoprotein receptor (LDLR) gene of a South African Indian patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, whose paternally-inherited allele has a single base substitution of A to T at position + 1. This conversion of the initiation codon ATG (methionine) to TTG (leucine) would abolish initiation of translation at the normal site, and consequently the synthesis of any normal LDLR molecules. The second mutation identified is a C to A base change at nucleotide position 1176 in exon 8, which creates a stop codon at cysteine-371. Except for previously-described polymorphisms in specific regions of the LDLR gene, the mutations identified in exons 1 and 8 were the only variants observed by screening enzymatically amplified genomic DNA comprising the entire coding and promoter region of the LDLR gene by combined heteroduplex-single-strand conformation polymorphism analysis and by direct sequencing. Cultured cells from the proband expressed no functional LDLR activity and contained no receptor protein that could be detected by antibody binding. These findings are consistent with the nature of the two base changes identified and provide evidence that the mutations cause FH in the proband and his affected family members. The mutations, designated M-21L and C371X, were absent in 17 apparently unrelated Indian hypercholesterolemics and 200 normal chromosomes screened.

Adolescent↗

PubMed2004

Loss of estrogen receptor-alpha expression is associated with hypermethylation near its ATG start codon in gastric cancer cell lines.

The proportion of gastric cancers positive for estrogen receptor (ER)-alpha expression is reported to be between 0-67%, depending upon the study. The role of ER-alpha in gastric carcinogenesis is unclear. The ER-alpha gene is located at chromosome 6q25.1, and the long arm of chromosome 6 has been known as a site with frequent loss of heterozygosity (LOH) in gastric cancer. ER expression is linked to suppression of cell proliferation in vitro. Epigenetic inactivation might explain the loss of ER-alpha gene expression in gastric cancer. Given there is no information available regarding the methylation status of the ER-alpha gene promoter region in gastric cancer, we investigated such methylation in 13 gastric cancer cell lines. Western blot analysis, reverse transcription-polymerase chain reaction (PCR), methylation-specific PCR (MS-PCR) and bisulfite sequencing analyses were used. ER-alpha protein was not detected in any cell line, although ER-alpha mRNA was detected in 1 of 13 gastric cancer cell lines. MS-PCR and bisulfite sequencing showed all 13 gastric cancer cell lines had methylated CpG regions in their ER-alpha gene promoters. In conclusion, inactivation of ER-alpha gene expression in gastric cancer cell lines appears associated with CpG island methylation near the TGA initiation codon of the ER-alpha gene.

Base Sequence↗

PubMed2001

New vascular endothelial growth factor isoform generated by internal ribosome entry site-driven CUG translation initiation.

We recently demonstrated that the very long 5'-untranslated region (5'-UTR) of the vascular endothelial growth factor (VEGF) mRNA contains two independent internal ribosome entry sites (IRES A and B). In the human sequence, four potential CUG translation initiation codons are located in between these IRES and are in frame with the classical AUG start codon. By in vitro translation and COS-7 cell transfections, we demonstrate that a high mol wt VEGF isoform [called large VEGF (L-VEGF)] is generated by an alternative translation initiation process, which occurs at the first of these CUG codons. Using a bicistronic strategy, we show that the upstream IRES B controls the translation initiation of L-VEGF. This isoform is 206 amino acids longer than the classical AUG-initiated form. With a specific antibody raised against this NH2 extension, we show that the L-VEGF is present in different mouse tissues or in transfected COS-7 cells. We also demonstrate that L-VEGF is cleaved into two fragments: a 23-kDa NH2-specific fragment and a fragment with an apparent size similar to that of the classical AUG-initiated form. This cleavage requires the integrity of a hydrophobic sequence located in the central part of the L-VEGF molecule. This sequence actually plays the role of signal peptide in the classical AUG-initiated form. The AUG-initiated form and the COOH cleavage product of the L-VEGF are both secreted. In contrast, the large isoform and its NH2 fragment present an intracellular localization. These data unravel a further level of complexity in the regulation of VEGF expression.

5' Untranslated Regions↗

PubMed2003

A bicistronic expression system for bacterial production of authentic human interleukin-18.

Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production.

Amino Acid Sequence↗

PubMed1994

Structure of the human APO-1 gene.

APO-1/Fas (CD95) is a type 1 transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor family characterized by cysteine-rich extracellular domains. Cross-linking of APO-1 mediates apoptosis in a variety of cells. In the present study we report the isolation and characterization of the human APO-1 gene spanning approximately 25 kb of human chromosome 10. The gene consists of nine exons (25 bp to > 1.44 kb) separated by eight introns (152 bp to approximately 12 kb). The boundaries of exon 2 to 5 encoding the extracellular region do not match the boundaries of the three APO-1 protein subdomains. Exon structure and functional protein domains correspond for exon 6 encoding the transmembrane region and for exon 9 encoding the "death domain". By a polymerase chain reaction-based approach we localized major transcriptional start sites in human spleen cells 77 and 73 nucleotides upstream of the translation initiation codon of the human APO-1 gene. Minor initiation sites were found at positions -128, -111, -91, and -74. The 5' flanking sequence of the human APO-1 gene is GC rich, contains a high number of CpG dinucleotides and lacks a consensus TATA box. Consensus binding sites for the transcription factors Sp1, AP-1, AP-2, GAF, NF-kappa B, and NF-AT were found. The elucidation of the human APO-1 gene structure will facilitate the study of its involvement in various diseases such as in autoimmunity.

Amino Acid Sequence↗

PubMed1987

Extracellular release of colicin A is non-specific.

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.

Base Sequence↗

PubMed1993

Molecular characterization of the 5'-flanking region of human genomic ETA gene.

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5'-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence of the lspA gene, the structural gene for lipoprotein signal peptidase of Escherichia coli.

The nucleotide sequence of the lspA gene coding for lipoprotein signal peptidase of Escherichia coli was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signal peptidase were consistent with those of the signal peptidase purified from cells harboring the lspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The lspA gene was preceded by the ileS gene coding for isoleucyl-tRNA synthetase and the tandem termination codons of the ileS gene overlapped with the initiation codon of the lspA gene.

Amino Acid Sequence↗