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PubMed1999

The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Amino Acid Sequence↗

PubMed1987

Nucleotide sequence of the genome region encoding the 26S mRNA of eastern equine encephalomyelitis virus and the deduced amino acid sequence of the viral structural proteins.

The 26S mRNA and most of the nsP4 encoding regions of the eastern equine encephalomyelitis (EEE) viral genome have been cloned. Excluding the poly(A) tail, the 26S mRNA region was determined to be 4139 nucleotides long and to share the same general organization as that of other alphaviruses. A highly conserved region of 19 nucleotides, the putative transcriptase recognition site for 26S mRNA synthesis, was present at the 26S/42S junction region of the 42S genomic RNA. Translation of the 26S mRNA began at the first AUG (positions 59 to 61) initiation codon and continued with an open reading frame that coded for a polyprotein of 1258 amino acids ending at a UAA ochre termination codon (positions 3776 to 3778). All four putative posttranslational cleavage sites used to generate the capsid, E3, E2, 6K and E1 proteins were conserved. Transmembrane domains present in the EEE virus structural polyprotein have been identified and their functions discussed. Pairwise comparison of the deduced amino acid sequences of the polyproteins of five alphaviruses (EEE, Venezuelan equine encephalitis, Sindbis, Semliki Forest and Ross River viruses) revealed EEE virus to be more closely related to VEE virus than to the other three viruses.

Alphavirus↗

PubMed1989

Analysis of the biotin-binding site on acetyl-CoA carboxylase from rat.

The biotin-binding site of acetyl-CoA carboxylase from rat was characterized as to its amino acid sequence and relative position in the enzyme molecule. Biotin binds to the lysyl residue in the tetrapeptide Val-Met-Lys-Met; this tetrapeptide is located in close proximity to the NH2 terminus. In all other biotin-containing enzymes, the conserved tetrapeptide Ala-Met-Lys-Met is the counterpart to that of rat acetyl-CoA carboxylase; and the lysyl residue is 35 residues from the COOH terminus. To examine the significance of these unusual features of the biotinylation site of animal acetyl-CoA carboxylase, cDNA fragments were expressed in a bacterial system and the effects of specific site-directed mutagenesis were examined. Replacement of Val by Ala in the conserved tetrapeptide abolished biotinylation of the expressed protein. However, introduction of a termination codon at residue 36, in such a way that the distance between the lysine on which biotin binds and the COOH-terminal amino acid was 35 residues and the penultimate amino acid was the hydrophobic residue leucine, increased the efficiency of biotinylation, provided a substantial portion of the NH2-terminal peptide was removed.

Acetyl-CoA Carboxylase↗

PubMed1985

Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3' flanking region.

A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, tissue expression of human xanthine dehydrogenase.

Xanthine dehydrogenase (XDH, EC 1.1.1.204) is a molybdenum iron-sulphur flavin hydroxylase which oxidizes a variety of purines, pterins and other heterogenic nitrogen compounds, serving as a rate-limiting enzyme in nucleic acid degradation. In this work, we have isolated and sequenced cDNA clones of human liver XDH. The obtained cDNA covers 4577 bases of human liver XDH mRNA with a 63 bp 5'-end untranslated region and a 515 bp 3'-end untranslated region. A termination codon TGA and a polyadenylation signal AATAAA were identified. An open reading frame encodes 1333 amino acid residues. The assignment of the N-terminal was confirmed by directly sequencing that region of purified human milk XDH. Northern blot analysis shows that the human XDH gene is widely expressed in human tissues.

Amino Acid Sequence↗

PubMed2006

Mutation specific therapy in CF.

CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. The mutations can be classified according to the mechanisms by which mutations disrupt CFTR function. This understanding of the different molecular mechanism of CFTR dysfunction provides the scientific basis for development of targeted drugs for mutation specific therapy of CF. Class I mutations are nonsense mutations that result in the presence of premature stop codon that leads to the production of unstable mRNA or the release from the ribosome of a short truncated protein that is not functional. The aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result the mutant CFTR is retained in the ER and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperons such as Sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or ATP binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome the affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the spicing machinery and generate both aberrantly and correctly spliced transcripts, the level of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increase of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that has to be reached in order to achieve normal function.

Colforsin↗

PubMed1996

Expression of a continuous open reading frame encoding subunits 1 and 2 of cytochrome c oxidase in the mitochondrial DNA of Acanthamoeba castellanii.

We have investigated the expression of a continuous open reading frame (ORF) present in the mitochondrial genome of Acanthamoeba castellanii and specifying the two largest subunits (COX1 and COX2) of the cytochrome c oxidase complex. Northern hybridization and primer extension analysis demonstrated that this ORF (cox1/2, 873 codons) is transcribed as part of a 4.7 kb RNA that also includes the upstream small subunit rRNA sequence. Between the cox1 and cox2 portions of the transcript, RNA sequence exactly matches gene sequence, excluding the possibility that a standard cox1 termination codon is created by post-transcriptional RNA processing or editing. Western analysis revealed an A. castellanii COX2 protein with a mobility matching that of mature COX2 from yeast (Saccharomyces cerevisiae) mitochondria. These observations indicate that although A. castellanii COX1 and COX2 are apparently translated from the same ORF, they do not exist in mature form as a COX1-COX2 "fusion" protein. Whereas translation of COX2 could potentially be initiated from an internal AUG codon in the cox1/2 ORF, COX1 must be generated either through an unusual translation termination mechanism acting between the cox1 and cox2 coding regions of the cox1/2 mRNA, or by co-translational or post-translational proteolytic processing of a translation product whose synthesis continues into the cox2 coding region. Because the cox2 nucleotide sequence predicts a COX2 protein considerably larger than that observed by Western analysis, A. castellanii COX2 may undergo additional post-translational processing to its final form.

Acanthamoeba↗

PubMed1988

Structural organization of the TRP1 gene of Phycomyces blakesleeanus: implications for evolutionary gene fusion in fungi.

The complete nucleotide (nt) sequence of the cloned TRP1 gene from Phycomyces blakesleeanus is reported. The gene encodes a trifunctional polypeptide that represents a sequential fusion of glutamine amidotransferase (TrpG), indoleglycerolphosphate synthetase (TrpC), and phosphoribosylanthranilate isomerase (TrpF) activities from the amino- to the carboxy terminus. This genetic organization is characteristic of filamentous fungi in general. The transcription start sites and the polyadenylation sites of the gene have been mapped. The two predominant TRP1 transcripts have a short leader of 13 and 18 nt, while minor species with significantly longer leaders are also detectable. Approximately 50 bp and 70 bp upstream from the major transcription start points, sequences that match the canonical eukaryotic TATA and CAAT boxes, respectively, are found. Two major polyadenylation signals are located approximately 50 and 70 nt downstream from the translational stop. Three closely clustered minor polyadenylation sites map to roughly 120-150 bp 3' to the termination codon. The TRP1 gene is the first and only Phycomyces gene that has been cloned and sequenced. The information regarding the promoter and terminator of a Phycomyces gene derived from these studies should benefit strategies aimed at gene manipulations in this organism.

Amino Acid Sequence↗

PubMed2001

Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa: effects on laminin-5 assembly, secretion, and deposition.

Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause junctional epidermolysis bullosa, a clinically and genetically heterogeneous blistering skin disease. Here, we describe a non-Herlitz junctional epidermolysis bullosa patient, compound heterozygote for two novel mutations affecting the LAMC2 gene. The mutation in the paternal allele is a de novo splice site mutation (522-1G-->A) that results in in-frame skipping of exon 4 and synthesis of a mutated gamma2 polypeptide (gamma2Delta4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation is a one base pair insertion (3511insA) in the 3' terminal exon of LAMC2 resulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a gamma2 polypeptide (gamma2t) disrupted in its alpha-helical C-terminal structure and truncated of the last 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of gamma2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investigate the biologic function of the laminin-5 molecules synthesized by the patient, mutant gamma2 cDNAs were transiently expressed in gamma2-null keratinocytes. Transfection of the gamma2Delta4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the gamma2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for the first time a functional alteration in a laminin-5 domain with a mild junctional epidermolysis bullosa phenotype.

Amino Acid Sequence↗

PubMed1997

Two novel mutations in the thyrotropin (TSH) receptor gene in a child with resistance to TSH.

The TSH receptor is a G protein-coupled receptor that mediates the effects of TSH in thyroid development, growth, and synthetic function. We report here that a child with features of TSH resistance, including markedly increased serum TSH concentrations and low normal thyroid hormone levels, is a compound heterozygote for two novel mutations in the TSH receptor gene. One allele has a G to A transition corresponding to an arginine to glutamine change at codon 109 (R109Q) in the extracellular domain of the receptor. The other allele has a G to A transition corresponding to a premature termination codon at tryptophan 546 (W546X) in the fourth transmembrane segment. Each parent is heterozygous for one mutation, and both parents have normal thyroid function. Cells transiently transfected with the R109Q mutant exhibited reduced membrane binding of [125I]TSH and impaired signal transduction in response to TSH. In contrast, the W546X mutant was nonfunctional, with negligible membrane radioligand binding. Our findings indicate that a single normal TSH receptor allele is sufficient for normal thyroid function, but that the compound abnormality in the proband leads to TSH resistance.

Amino Acid Sequence↗

PubMed1995

High-level expression of the human tumor necrosis factor-alpha.

Five expression plasmids for a total chemically synthesized gene of tumor necrosis factor-alpha (TNF-alpha) were constructed, which differ in two aspects: 1) distance (D) between the SD and the initiation codon ATG; 2) energy-delta G0f298 released to form the stable secondary structure in the translation initiation region. The plasmid with a (D) of 6bp has the lowest delta G0f298 (absolute value) and showed the highest expression level, up to 60% of the total bacterial proteins. Codons usage will impose much influence on expression level, so it is reasonable and understandable that the total chemically synthesized TNF-alpha gene with the codons preferable to E. coli use gave much higher expression level than the sc-TNF-alpha in which only partial N-terminal codons were modified.

Base Sequence↗

PubMed1994

Molecular cloning and characterization of the ctpA gene encoding a carboxyl-terminal processing protease. Analysis of a spontaneous photosystem II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803.

A nitrofurantoin enrichment technique was used to isolate a spontaneous photosynthesis-deficient mutant strain of the unicellular cyanobacterium Synechocystis sp. PCC 6803. This mutant, SK18, lacked any photosystem II (PSII) activity, but had normal photosystem I. The SK18 mutant strain could not be complemented with known genes encoding various structural proteins of PSII, but could be complemented with a recombinant plasmid pSL523 containing a 1.4-kilobase pair EcoRI fragment of the chromosomal DNA from wild-type Synechocystis 6803 cells. Determination of the nucleotide sequence of this DNA fragment revealed a previously unidentified open reading frame (ORF) encoding a 427-residue-long polypeptide. Hydrophobicity analysis of the amino acid sequence suggested that this protein is largely hydrophilic. A stretch of the first 31 amino-terminal residues of the polypeptide resembled a bacterial signal peptide and may be responsible for the translocation of this protein to the lumen space of the thylakoid membranes. The spontaneous mutation in the SK18 strain was identified to be a single nucleotide change introducing a premature termination codon in this ORF. The predicted sequence of the encoded protein showed significant similarity to that of the Prc protein, a carboxyl-terminal processing protease in Escherichia coli. We suggest that the cyanobacterial protein encoded by ORF427 is a similar processing protease and name the gene ctpA (carboxyl-terminal processing protease).

Algal Proteins↗

PubMed1991

Mutagenesis of the 43-kD postsynaptic protein defines domains involved in plasma membrane targeting and AChR clustering.

The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.

Animals↗

PubMed1990

Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli.

We analyzed protein expression from a cloned Mycoplasma hyorhinis genomic fragment that produces in Escherichia coli a set of related polypeptides of 110, 100, 65, and 55 kilodaltons from a coding region of just over 3.0 kilobases. Expression of these multiple products resulted from a mechanism operating at the translational level but not from truncation at UGA termination codons, which are known to encode tryptophan in several mycoplasma species. The structural relatedness of the proteins was demonstrated by two-dimensional tryptic peptic mapping, but their generation by posttranslational processing was ruled out by pulse-chase labeling analysis. Examination of proteins expressed from plasmid constructs and tryptic peptide analysis of these polypeptides and the original set of proteins revealed that they share carboxy-terminal regions, an observation inconsistent with truncation at UGA codons. Expression of proteins from this cloned fragment was not dependent on vector sequences and was observed when the coding region was placed under control of a T7 promoter, suggesting that all products were translated from a single message. Expression of related products in mycoplasmas was examined by immunoblot analysis of M. hyorhinis proteins with antiserum against overexpressed recombinant proteins. A single 115-kilodalton mycoplasma protein was detected, which is larger than any of the related proteins expressed in E. coli. Our analysis indicated that translation initiation sites are used in E. coli that are not active in mycoplasmas, thereby defining differences between the translational regulatory signals of mycoplasmas and eubacteria.

Bacterial Proteins↗

PubMed2003

Unusual multiple recoding events leading to alternative forms of hepatitis C virus core protein from genotype 1b.

In addition to its involvement in the formation of the capsid shell of the virus particles, the core protein of hepatitis C virus (HCV) is believed to play an important role in the pathogenesis and/or establishment of persistent infection. We describe here alternative forms of genotype 1b HCV core protein identified after purification of various products of core protein segment 1-169 expressed in Escherichia coli and their analysis by proteolysis, mass spectrometry, and amino acid sequencing. These proteins all result from a +1 frameshift at codon 42 (a different position than that previously reported in genotype 1a) and, for some of them, from a rephasing in the normal open reading frame at the termination codon 144 in the +1 open reading frame. To test the relevance of these recoding events in a eukaryotic translational context, the nucleotide sequences surrounding the two shift sites were cloned in the three reading frames into expression vectors, allowing the production of a C-terminally fused green fluorescent protein, and expressed both in a reticulocyte lysate transcription/translation assay and in culture cells. Both recoding events were confirmed in these expression systems, strengthening the hypothesis that they might occur in HCV-infected cells. Moreover, sera from HCV-positive patients of genotype 1a or 1b were shown to react differently against synthetic peptides encoded in the +1 open reading frame. Together, these results indicate the occurrence of distinct recoding events in genotypes 1a and 1b, pointing out genotype-dependent specific features for F protein.

Amino Acid Sequence↗

PubMed1996

Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits.

Multiple espilon subunits are major determinants of the diversity of the N-methyl-D-aspartate (NMDA) receptor channel. The four epsilon subunit mRNAs exhibit distinct expression patterns in the brain. In an attempt to elucidate the molecular basis of selective and characteristic expression of the NMDA receptor channel subunits, we have isolated the gene encoding the mouse NMDA receptor epsilon 3 subunit and have determined its structural organization. The epsilon 3 subunit gene spans 17.5 kb and consists of 14 exons. The major transcription start site is 439 bp upstream of the ATG initiation codon as determined by primer extension and S1 nucleas protection analyses. Two polyadenylation sites are 397 (or 398) and 402 bp downstream of the termination codon. The 5'-flanking region of the epsilon 3 subunit gene contains GC-rich segments including consensus sequences for binding of the transcription factors Spl and EGR-1. The murine chromosomal locations of the five NMDA receptor channel subunits, the epsilon 1 (Grin2a), epsilon 2 (Grin2b), epsilon 3 (Grin2c), epsilon 4 (Grin2d) and zeta 1 (Grinl) subunits, were determined using an interspecific backcross mapping panel derived from crosses of [(C57BL/6JxM. spretus) F1xC57BL/6J] mice. Each of these genes mapped to a single chromosome location. The mapping results assigned the five loci to five different mouse autosomes, indicating that they have become well dispersed among mouse chromosomes.

Amino Acid Sequence↗

PubMed1983

Characterization and nucleotide sequence of a chicken gene encoding an opal suppressor tRNA and its flanking DNA segments.

A naturally occurring opal suppressor serine tRNA has been purified from chicken liver and used as a probe to isolate the corresponding gene from a library of chicken DNA in bacteriophage lambda. This minor tRNA is encoded by a single-copy gene that is not part of a tRNA gene cluster. DNA sequence analysis of the gene and its flanking DNA segments shows that the gene is encoded in an 87-base-pair segment without intervening sequences and specifies a tRNA that reads the termination codon UGA. This gene has additional nucleotides in the 5' internal promoter region but has a normal 3' internal promoter sequence and the usual termination signal.

Animals↗

PubMed1989

Sequences downstream of translation start regulate quantitative expression of two petunia rbcS genes.

We investigated the basis for quantitative differences in leaf expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase. The most abundantly, SSU301, and the most weakly, SSU911, expressed petunia rbcS genes maintained their differential expression when transferred to tobacco, indicating that the determinants of quantitative expression are intrinsic to these rbcS genes. Analysis of chimeric genes in which the sequences of SSU301 and SSU911 had been exchanged at the translation start showed that sequences both 5' and 3' to the start codon contribute to differences in steady-state mRNA levels. The sequences 3' to the translation initiation codon were investigated by preparing chimeric genes in which sequences of the SSU301 and SSU911 were exchanged between each intron and at the translation termination codon. The results showed that sequences downstream of the coding region contribute to quantitative differences in expression of SSU301 and SSU911, and nuclear run-on transcription experiments indicated that the 3' sequences affect transcription rates of the rbcS genes.

Base Sequence↗