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PubMed2001

ABCD1 translation-initiator mutation demonstrates genotype-phenotype correlation for AMN.

BACKGROUND: Inherited mutations of the X-linked adrenoleukodystrophy (X-ALD) gene (ABCD1) cause two neuropathologically distinct disorders: cerebral adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN). The biochemical hallmark of these disorders is a reduction of very long chain fatty acid (VLCFA) beta-oxidation with accumulation of VLCFA esters in neural white matter. More than 300 mutations of the ABCD1 gene have been described. Genotype-phenotype correlation in X-ALD has not been demonstrated; indeed, the two disorders coexist in individual pedigrees and in homozygotic twin pairs. METHODS: The authors have identified one large kindred with a highly concordant AMN phenotype resembling an X-linked dominant hereditary spastic paraparesis. All obligate female carriers are clinically affected. The ABCD1 gene was examined by direct sequencing of genomic DNA and full-length cDNA. Mutant gene transcription was analyzed by reverse transcriptase PCR. ALD protein (ALDP) expression was tested by Western blotting and indirect immunofluorescence. VLCFA beta-oxidation was examined by in vitro assay. RESULTS: The authors have identified a novel deletion of the ABCD1 gene ATG translation initiation codon. The authors have demonstrated that an N-terminal truncated ALDP, missing the first 65 amino acids, is expressed by internal initiation of translation and is correctly trafficked to peroxisomes. They have documented complete penetrance of this mutant in all female carriers. They have also shown that VLCFA beta-oxidation is reduced to 20% of normal in association with this mutant ALDP. CONCLUSION: It appears that initiation of translation at an internal AUG codon generates a truncated ALDP that uniformly leads to an AMN phenotype in this family. Possible models for action of this truncated ALDP and full disease penetrance in heterozygotes are reviewed.

ATP Binding Cassette Transporter, Subfamily D, Mem↗

PubMed1991

Molecular characterization of the nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3.

The nisin resistance determinant of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3 was localized onto a 1.3-kb EcoRI-NdeI fragment by subcloning and interrupting the NdeI site by cloning random NdeI fragments into it; the nisin resistance determinant was then sequenced. The nucleotide sequence revealed a large open reading frame containing 318 codons. Putative transcription and translation signal sequences were located directly upstream from the initiation codon. Immediately downstream of the termination codon was a palindromic region resembling a rho-independent termination sequence. This 957-nucleotide open reading frame and its associated transcription and translation signal sequences were cloned into plasmid-free L. lactis subsp. lactis LM0230 and conferred an MIC of 160 IU of nisin per ml. This level of nisin resistance is equivalent to that of the initial nisin-resistant subclone, pFM011, used for further subcloning in this study. The inferred amino acid sequence would result in a protein with a molecular mass of 35,035 Da. This value was in agreement with the molecular mass of a protein detected after in vitro transcription and translation of DNA encoding the nisin resistance gene, nsr. This protein contained a hydrophobic region at the N terminus that was predicted to be membrane associated but did not contain a typical signal sequence cleavage site. No significant homology was detected when the DNA sequence of the nsr gene and the amino acid sequence of its putative product were compared with other available sequences. When subjected to Southern hybridization, a 1.2-kb DraI fragment encoding the nsr gene did not hybridize with the genomic DNA of the nisin-producing strain L. lactis subsp. lactis 11454.

Amino Acid Sequence↗

PubMed2000

Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins.

Human T-cell lymphotropic virus type I (HTLV-I) double-spliced mRNA exhibits two GUG and two CUG codons upstream to, and in frame with, the sequences encoding Rex and Tax regulatory proteins, respectively. To verify whether these GUG and CUG codons could be used as additional initiation codons of translation, two chimeric constructs were built for directing the synthesis of either Rex-CAT or Tax-CAT fusion proteins. In both cases, the CAT reporter sequence was inserted after the Tax AUG codon and in frame with either the Rex or Tax AUG codon. Under transient expression of these constructs, other proteins of higher molecular mass were synthesized in addition to the expected Rex-CAT and Tax-CAT proteins. The potential non-AUG initiation codons were exchanged for either an AUG codon or a non-initiation codon. This allowed us to demonstrate that the two GUG codons in frame with the Rex coding sequence, and only the second CUG in frame with the Tax coding sequence, were used as additional initiation codons. In HTLV-I infected cells, two Rex and one Tax additional proteins were detected that exhibited molecular mass compatible with the use of the two GUG and the second CUG as additional initiation codons of translation. Comparison of the HTLV-I proviral DNA sequence with that of other HTLV-related retroviruses revealed a striking conservation of the three non-AUG initiation codons, strongly suggesting their use for the synthesis of additional Rex and Tax proteins.

Base Sequence↗

PubMed1991

Clostridium botulinum types A, B, C1, and E produce proteins with or without hemagglutinating activity: do they share common amino acid sequences and genes?

Clostridium botulinum produce the antigenically distinct 150 kD neurotoxin serotypes (e.g., A, B, C1, and E) and simultaneously proteins, A Hn+, B Hn+, C Hn+, and E Hn-, that have high, low, and no hemagglutinating activity. A Hn+ and B Hn+ are serologically cross-reactive. A Hn+, B Hn+, and C Hn+ found as large aggregates (900-220 kD) can be dissociated on SDS-PAGE into multiple subunits, the smallest for A Hn+, B Hn+ is 17 kD and 27 kD for C Hn+. The 116 kD E Hn- does not aggregate. We determined the sequences of 10-33 amino terminal residues of the 17, 21.5, 35, and 57 kD subunits of A Hn+ and B Hn+. Each of these subunits have unique sequences, indicating that the larger units studies are not homomers or heteromers of smaller units. The subunits of A Hn+ and B Hn+ of comparable size have striking sequence identity (e.g., 21.5 kD subunits from the two are identical and 57 kD subunits have 80% identity). In vitro proteolysis of 116 kD E Hn- with different proteases did not impart hemagglutinating activity to the fragments. The 116 kD E Hn- and one of its proteolytic fragments (87 kD) were partially sequenced. Sixty-two base pairs downstream from the termination codon of the cloned 33 kD subunit of C Hn+, there is an initiation codon followed by an open reading frame for at least 34 amino acid residues (Tsuzuki et al., 1990). The derived amino acid sequence of this open reading frame, we found, has 73-84% sequence identity with those of the 17 kD subunits of A Hn+ and B Hn+ and significant identity with the N-terminal of E Hn-. These highly conserved sequences show existence of genetic linkage among the Hn+ and Hn- proteins.

Amino Acid Sequence↗

PubMed1983

Bacillus cereus 569/H beta-lactamase I: cloning in Escherichia coli and signal sequence determination.

The gene, penPC, for beta-lactamase I of Bacillus cereus 569/H has been cloned and its expression studied in Escherichia coli. The protein product from the in vitro translation of penPC was shown by gel electrophoresis to have an Mr of 36 000 which is larger than the in vivo products found in B. cereus and E. coli. The DNA sequence of the signal region was determined. It revealed that the smallest known mature form present in B. cereus culture fluids is preceded by 45-48 amino acids in pre-beta-lactamase I, considering that there are 3 initiation codons in the same reading frame, one or more of which might be initiating translation. Unlike the Bacillus licheniformis 749/C beta-lactamase, which has a membrane-bound thioether lipoprotein form, the single Cys residue in the B. cereus beta-lactamase I signal sequence is unmodified and a single processed form of the enzyme is present in E. coli cells carrying penPC.

Amino Acid Sequence↗

PubMed2005

Higher frequency of premature stop codon mutations at vpu gene of human immunodeficiency virus type 1 CRF01_AE compared with those of other subtypes.

Our previous study demonstrated the anti-apoptosis function of the human immunodeficiency virus type 1 (HIV-1) vpu gene product in normal CD4+ T lymphocytes. In this study, using sequences obtained from the HIV sequence database, we compared vpu sequences from 184 preparations of various subtypes of HIV-1 from diverse geographical regions. Our analysis revealed that CRF01_AE isolates had premature stop codon mutations at the vpu gene at a much higher rate (36%) than other subtypes (0-9%). The premature stop codon mutations in vpu existed mostly at two amino acid residues: the methionine initiation codon and the boundary between the transmembrane (TM) and cytoplasmic domains. The mutations at the latter site were more often detected in CRF01_AE. The higher mutation rates at vpu in CRF01_AE were confirmed by sequence comparison of polymerase chain reaction products newly obtained directly from the DNA extracted from peripheral blood mononuclear cells (PBMCs), but not from the RNA from the plasma, in CRF01_AE- and subtype B-infected individuals. This finding may indicate the possibility that the more abundant population of HIV-1 CRF01_AE is able to induce apoptosis in CD4+ T lymphocytes than the populations of other subtypes.

Codon↗

PubMed1986

Genetic definition of the translational operator of the threonine-tRNA ligase gene in Escherichia coli.

The Escherichia coli gene thrS that codes for threonine-tRNA ligase (tRNAThr ligase, formerly threonine-tRNA synthetase, EC 6.1.1.3) has previously been shown to be negatively autoregulated at the level of translation. Here we describe the use of several thrS-lac gene fusions to isolate cis-acting regulatory mutations that increase the translation but not the transcription of the thrS gene. These mutations lead to a total loss of control of repression and derepression of thrS. DNA sequence analysis locates the mutations between 10 and 40 base pairs upstream of the translation initiation codon of thrS and more than 100 base pairs downstream of the transcription initiation site. The mRNA region where these mutations are located shares primary and secondary structure homologies with specific parts of several isoacceptor tRNAThr species. These findings suggest that the ligase regulates its translation by binding to its mRNA at a place that shares some homology with its natural substrate.

Amino Acyl-tRNA Synthetases↗

PubMed1987

Stage-specific selection of alternative transcriptional initiation sites from the 5C actin gene of Drosophila melanogaster.

The transcription unit of the 5C actin gene exhibits a complex organization that is unique among the six actin genes of Drosophila melanogaster. Three different mRNA size classes showing distinct patterns of accumulation throughout development are detected on Northern blots. We have determined the structure of the various 5C actin transcripts by exon mapping using strand-specific RNA probes, primer extension analysis, and DNA sequences analysis of both cDNA and genomic clones. All the transcripts share a single protein-coding nucleotide sequence but are heterogeneous in the 5' and 3' untranslated regions. The 5' untranslated region of each transcript consists of either one of two small exons (exon 1 and exon 2) which are alternatively spliced to a single acceptor site 8 bp upstream from the translation initiation codon in exon 3. Results from primer extension analysis suggest that transcription can initiate from either exon 1 or exon 2, and also from a third site within exon 2. We detect an increase in the relative abundance of exon 1-containing transcripts at larval and pupal stages, as well as a change in the proportion of transcripts that initiate at either of the two exon 2 sites. Five polyadenylation sites have been found within three termination/processing regions that define the three size classes of polyadenylated transcripts. The results of our experiments indicate the existence in vivo of all possible combinations of 5' exon with 3' polyadenylation site. However, particular combinations of 5' initiation site and 3' polyadenylation site are preferred at certain developmental stages.

Actins↗

PubMed1994

Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase.

The thrC gene of Brevibacterium lactofermentum was cloned by complementation of Escherichia coli thrC auxotrophs. The gene was located by deletion mapping and complementation analysis in a 2.9-kb Sau3AI-HindIII fragment of the genome. This fragment also complemented a B. lactofermentum UL1035 threonine auxotroph that was deficient in threonine synthase. A 1,892-bp DNA fragment of this region was sequenced; this fragment contained a 1,446-bp open reading frame that encoded a 481-amino-acid protein having a deduced M(r) of 52,807. The gene was expressed in E. coli, by using the phage T7 system, as a 53-kDa protein. The promoter region subcloned in promoter-probe plasmids was functional in E. coli. A Northern analysis revealed that the gene was expressed as a monocistronic 1,400-nucleotide transcript. The transcription start point of the thrC gene was located by S1 mapping 6 bp upstream from the translation initiation codon, which indicated that this promoter was one of the leaderless transcription-initiating sequences. The threonine synthase overexpressed in B. lactofermentum UL1035 was purified almost to homogeneity. The active form corresponded to a monomeric 52.8-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme required pyridoxal phosphate as its only cofactor to convert homoserine phosphate into threonine.

Amino Acid Sequence↗

PubMed1993

Sequence analysis of scrA and scrB from Streptococcus sobrinus 6715.

The complete nucleotide sequences of Streptococcus sobrinus 6715 scrA and scrB, which encode sucrose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, have been determined. These two genes were transcribed divergently, and the initiation codons of the two open reading frames were 192 bp apart. The transcriptional initiation sites were determined by primer extension analysis, and the putative promoter regions of these two genes overlapped partially. The gene encoding enzyme IIScr, scrA, contained 1,896 nucleotides, and the molecular mass of the predicted protein was 66,529 Da. The hydropathy plot of the predicted amino acid sequence indicated that enzyme IIScr was a relatively hydrophobic protein. The gene encoding sucrose-6-phosphate hydrolase, scrB, contained 1,437 nucleotides. The molecular mass of the predicted protein was 54,501 Da, and the encoded enzyme was hydrophilic. The predicted amino acid sequences of the two open reading frames exhibited approximately 45 and 70% identity with those encoded by scrA and scrB, respectively, from Streptococcus mutans GS5. Homology also was observed between the N-terminal region of the S. sobrinus 6715 enzyme IIScr and other enzyme IIs specific for the glucopyranoside molecule, all of which generate glucopyranoside-6-phosphate during translocation and phosphorylation of the respective substrates. The sequence of the C-terminal domain of the S. sobrinus 6715 enzyme IIScr shared significant homology with enzyme IIIGlc from Escherichia coli and Salmonella typhimurium and with the C-terminal domain of enzyme IIBgl from E. coli, indicating that the two functional domains, enzyme IIScr and enzyme IIIScr, were covalently linked as a single polypeptide in S. sobrinus 6715. The deduced amino acid sequence of the gene product of S. sobrinus scrB shared strong homology with sucrase from Bacillus subtilis, Klebsiella pneumoniae, and Vibrio alginolyticus, suggesting conservation based on the physiological roles of these proteins.

Amino Acid Sequence↗

PubMed1984

Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues.

Partial sequence analysis of tryptic peptides has identified the E1B-495R (E1b-57K) (early transcription region 1B of 495 amino acid residues, with an approximate molecular weight of 57,000) protein of adenovirus 2 as encoded by the 495 amino acid open reading frame located in the adenovirus 2 DNA sequence between nucleotides 2016 and 3500. Additional proteins of 16,000 Mr and 18,000 Mr that are related to the E1B-495R protein were identified by cell-free translation of hybridization-selected mRNA. Analysis of [35S]methionine-containing amino terminal tryptic peptides by thin-layer chromatography showed that the E1B-495R, E1B-18K, and E1B-16K proteins all begin at the same initiation codon. The E1B-495R protein from 293 cells also has the same initial tryptic peptide, acetyl-methionyl-glutamyl-arginine. Sequence analysis of E1B-18K tryptic peptides indicated that this protein also has the same carboxy terminus as the E1B-495R protein and that it is derived from an mRNA that is spliced to remove sequences between nucleotides 2250 and 3269, resulting in a protein product of 155 amino acid residues. Analysis of E1B-16K tryptic peptides has not yet revealed the carboxy terminal structure of this protein. Both the E1B-495R and the E1B-155R (E1B-18K) proteins, as well as the E1B-16K protein, were precipitated from cell-free translations and from extracts of infected cells by antiserum against an amino terminal nonapeptide common to these proteins.

Adenovirus Early Proteins↗

PubMed1992

Chromosomal localization and organization of the murine genes encoding the beta subunits (AIC2A and AIC2B) of the interleukin 3, granulocyte/macrophage colony-stimulating factor, and interleukin 5 receptors.

Chromosomal genes for two mouse homologous beta subunits (AIC2A and AIC2B) of the interleukin-3, granulocyte/macrophage colony-stimulating factor, and interleukin-5 receptors were characterized. Both AIC2A and AIC2B genes were present on a 250-kilobase MluI restriction fragment and were mapped on murine chromosome 15 (these loci were provisionally designated as Il3rb-1 (AIC2A) and Il3rb-2 (AIC2B)), closely linked to the c-sis locus. Both genes consist of 14 exons and span about 28 kb each. The major transcription initiation sites of both genes were mapped at 194 bp from the initiation codon. These genes are 95% identical up to 700 bp from the transcription initiation sites. Potential recognition sequences for hemopoietic transcription factors including GATA-1 and PU.1 in addition to a TATA-like sequence are present in the 5'-flanking region. A stretch of 20 bp including the initiation site is homologous to the corresponding region of the erythropoietin receptor and the interleukin-7 receptor genes and to the initiator sequence of the adeno-associated virus P5 promoter, suggesting a possible role in transcription initiation. Comparison of the exon/intron boundaries of AIC2A and AIC2B genes with those of other members of the cytokine receptor superfamily reveals a conserved evolutionary structure. Isolation of various forms of AIC2 cDNAs reveals differential splicing of the transcripts.

Animals↗

PubMed1984

In vivo and in vitro transcription of the Escherichia coli glutaminyl-tRNA synthetase gene.

We have characterized the in vivo and in vitro transcription products of the Escherichia coli glnS gene which codes for the enzyme glutaminyl-tRNA synthetase. The in vivo glnS transcript is about 1.9 kilobases long. Sequence analysis of the 5'- and 3'-ends of glnS mRNA showed that transcription initiates approximately 30 bases upstream from the translation initiation codon AUG and terminates approximately 230 bases downstream from the termination codon UAA. Characterization of the in vitro transcripts of glnS revealed similar transcription initiation and termination sites as were found in the glnS mRNA produced in vivo. These results indicate that the Pribnow box structure upstream and the dyad symmetry terminator structure downstream of the glnS structural region are regulatory signals used for glnS expression. In vitro transcription of glnS is not autogenously regulated by glutaminyl-tRNA synthetase and glutamine; it is also not affected by the presence of tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Cloning, sequencing, and expression of the genes encoding subunits of Paracoccus denitrificans electron transfer flavoprotein.

The genes encoding the two subunits of Paracoccus denitrificans electron transfer flavoprotein (ETF) were identified by screening a genomic library constructed in pBluescript II SK+ with probes generated by amplification of genomic sequences by the polymerase chain reaction. Primers for the polymerase chain reaction were designed based on peptide sequences from purified Paracoccus ETF subunits. The genes are arranged in tandem in the genomic DNA with the deoxyadenylic acid residue in the TGA termination codon of the small subunit providing the deoxyadenylic acid residue for the ATG initiating codon of the large subunit. The deduced amino acid sequences of the ETF subunits exhibits extensive sequence identity with the human ETF subunits. The Paracoccus ETF is expressed from the pBluescript vector in Escherichia coli, yielding 30 mg of purified, catalytically active protein per liter of culture.

Amino Acid Sequence↗

PubMed1993

Expression of a globin gene in Caenorhabditis elegans.

Oligonucleotides related to parts of a globin-like sequence in the genome of Caenorhabditis elegans were used to probe a cDNA library from the same species. A complete globin-like sequence was found in the cDNA, showing that a globin gene appears to be expressed. The hypothetical protein was compatible with the conventional globin fold but may be truncated in the B helix, as in Chironomus globin III. An intron in the codon for residue E3 in the E helix was removed in expression. An initiation codon preceded the globin but the sequence upstream (extending for 30 nucleotides to the vector ligation site) had characteristics both of the code for a protein hydrophobic leader and of a trans-spliced RNA leader. The evidence indicates that C. elegans globin has a single domain, unlike some nematodes that express two tandem globin domains in a continuous translation product, and from its sequence may be predicted to have a high affinity for oxygen.

Amino Acid Sequence↗

PubMed1994

Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions.

In an attempt to understand how Escherichia coli ribosomes recognize the initiator codon on mRNAs lacking the Shine-Dalgarno (SD) sequence, we have studied 30S initiation complex formation in extension inhibition (toeprinting) experiments using (-SD)mRNAs which are known to be reliably translated in E. coli: the plant viral messenger A1MV RNA 4 and two chimaeric mRNAs coding for beta-glucuronidase (GUS) and bearing the 5'-untranslated sequence of TMV RNA (omega) or the omega-derived sequence (CAA)n as 5'-leaders. Ribosomal protein S1 and IF3 have been found to be indispensable for translational initiation. Protein S1 appears to be a key recognition element. S1 binds to sequences within the leaders of (-SD)mRNAs thus providing their affinity to E. coli ribosomes.

Alfalfa mosaic virus↗

PubMed2000

Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa.

We report a contiguous region of more than half (> 7,500 nt) of the mitochondrial genomes for Platynereis dumerii (Annelida: Polychaeta), Helobdella robusta (Annelida: Hirudinida), and Galathealinum brachiosum (Pogonophora: Perviata). The relative arrangements of all 22 genes identified for Helobdella and Galathealinum are identical to one another and to their arrangements in the mtDNA of the previously studied oligochaete annelid Lumbricus. In contrast, Platynereis differs from these taxa in the positions of several tRNA genes and in having two additional tRNA genes (trnC and trnM) and a large noncoding sequence in this region. Comparisons of relative gene arrangements and of the nucleotide and inferred amino acid sequences among these and other published taxa provide strong support for an annelid-mollusk clade that excludes arthropods, and for the inclusion of pogonophorans within Annelida, rather than giving them separate phylum status. Gene arrangement comparisons include the first use of a recently described method on previously unpublished data. Although a variety of alternative initiation codons are typically used by mitochondrial protein-encoding genes, ATG appears to be the initiator for all but one reported here. The large noncoding region (1,091 nt) identified in Platynereis has no significant sequence similarity to the noncoding region of Lumbricus, although each contains runs of TA dinucleotides and of homopolymers, which could potentially serve as signaling elements. There is strong bias for synonymous codon usage in Helobdella and especially in Galathealinum. In this latter taxon, 5 codons are completely unused, 13 are used three or fewer times, and G appears at third codon positions in only 26 of the 2,236 codons. Nucleotide composition bias appears to influence amino acid composition of the proteins.

Amino Acid Sequence↗

PubMed1997

The genomic organization of the murine Pax 8 gene and characterization of its basal promoter.

Lambda phage clones containing the murine Pax 8 gene were isolated from a C57BL/6 kidney genomic mouse library using mouse cDNA fragments as probes. A clone encompassing about 16 kb of the 5' untranslated region of the murine Pax 8 gene was isolated from a mouse embryonic stem cell (D3) library. The murine Pax 8 gene has a size of approximately 26 kb and contains the coding sequence for mRNA in 12 exons. The major and several minor transcription initiation sites were identified. Position +1 is located 488 nucleotides upstream of the ATG initiation codon and 24 bases downstream of a TATA-like sequence, ATAAAA. The translation initiation and termination sites are located in exons 2 and 12, respectively. Further analysis of 570 bases of the 5' flanking sequence revealed AP2, SP1, PEA3, zeste, NF-kappaB, and CCAAT consensus binding sites. Ribonuclease protection assays with a probe spanning the first two exons of mouse Pax 8 cDNA on total RNA samples isolated from different tissues of newborn mice show that the murine Pax 8 gene is predominantly expressed in kidney tissue. Low levels of Pax 8 gene expression were also found in the liver, spleen, lung, brain, and heart. The same transcription initiation sites are utilized in different tissues of newborn mice and embryo at Day 10.5 postconception. A FISH assay shows that the murine Pax 8 gene is located on chromosome 2, map position B.

Animals↗