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PubMed2000

The ORF RTL1 transcript of fowl adenovirus type-8 is spliced and truncated at late stages of the virus replication cycle.

Two transcription products were found for the open reading frame (ORF) RTL1 located near the right terminus of the fowl adenovirus type-8 genome. The larger transcript, which was transcribed mostly during the early stage of the virus infection, contains the complete sequence (933 nucleotides) of the predicted ORF from the genomic DNA sequence encoding a 311 amino acid (aa) polypeptide. In contrast, the shorter transcript, which was more predominant at the late stage of the infection, was missing 580 nucleotides (from nucleotide 117 to 696). A premature stop codon was introduced at 210 nucleotides downstream from the start codon and the shorter transcript would encode a 70 aa polypeptide. This observation indicates that the ORF RTL1 may produce two different proteins, which function differently at different stages of the virus infection. Another possibility is that the virus may use alternative splicing as a mechanism to control the expression of the ORF, since the spliced transcript was prematurely terminated at the late stage of the infection.

Alternative Splicing↗

PubMed2006

Independent evolution of bitter-taste sensitivity in humans and chimpanzees.

It was reported over 65 years ago that chimpanzees, like humans, vary in taste sensitivity to the bitter compound phenylthiocarbamide (PTC). This was suggested to be the result of a shared balanced polymorphism, defining the first, and now classic, example of the effects of balancing selection in great apes. In humans, variable PTC sensitivity is largely controlled by the segregation of two common alleles at the TAS2R38 locus, which encode receptor variants with different ligand affinities. Here we show that PTC taste sensitivity in chimpanzees is also controlled by two common alleles of TAS2R38; however, neither of these alleles is shared with humans. Instead, a mutation of the initiation codon results in the use of an alternative downstream start codon and production of a truncated receptor variant that fails to respond to PTC in vitro. Association testing of PTC sensitivity in a cohort of captive chimpanzees confirmed that chimpanzee TAS2R38 genotype accurately predicts taster status in vivo. Therefore, although Fisher et al.'s observations were accurate, their explanation was wrong. Humans and chimpanzees share variable taste sensitivity to bitter compounds mediated by PTC receptor variants, but the molecular basis of this variation has arisen twice, independently, in the two species.

Alleles↗

PubMed1998

Exon-intron structure, analysis of promoter region, and chromosomal localization of the human type 1 sigma receptor gene.

Sigma receptor is a protein that interacts with a variety of psychotomimetic drugs including cocaine and amphetamines and is believed to play an important role in the cellular functions of various tissues associated with the endocrine, immune, and nervous systems. Here we report on the structure and organization of the human gene coding for this receptor. The gene is approximately 7 kbp long and contains four exons, interrupted by three introns. Exon 3 is the shortest (93 bp), and exon 4 is the longest (1,132 bp). Among the introns, intron 3 is the longest (approximately 1,250 bp). Exon 2 codes for the single transmembrane domain present in the receptor. 5' rapid amplification of cDNA end reactions with mRNA from the JAR human trophoblast cell line have identified 56 bp upstream of the translation start codon as the initiation site for transcription. This transcription start site has been confirmed by RNase protection analysis. Structural analysis of the 5' flanking region has revealed that the gene is TATA-less. This region, however, contains a CCAATC box in the reverse complement and several GC boxes that are recognition sites for SP1. There are also consensus sequences for the liver-specific transcription factor nuclear factor-1/L, for a variety of cytokine responsive factors, and for the xenobiotic responsive factor called the arylhydrocarbon receptor. Southern blot analysis of the genomic DNA from Chinese hamster-human and mouse-human hybrid cell lines and fluorescent in situ hybridization with human metaphase chromosome spreads have shown that the gene is located on human chromosome 9, band p13, a region known to be associated with different psychiatric disorders.

Amino Acid Sequence↗

PubMed1982

Structures of the genes for the beta and epsilon subunits of spinach chloroplast ATPase indicate a dicistronic mRNA and an overlapping translation stop/start signal.

A 2,4-kilobase-pair region of spinach chloroplast DNA adjacent to the gene for the large subunit of ribulosebisphosphate carboxylase has been analyzed by RNA hybridization, in vitro transcription/translation, and DNA sequence determination. The analysis indicates that this region carries the genes for the beta and epsilon subunits of chloroplast ATPase and that the two genes are cotranscribed into a dicistronic mRNA with 4-base-pair overlap between the stop codon of the beta-subunit gene and the start codon of the epsilon-subunit gene. The ATPase and carboxylase genes are transcribed divergently with respect to each other. The deduced amino acid sequences of the beta and epsilon subunits from spinach show 67% and 26% homology, respectively, with the published sequences of the beta and epsilon subunits of Escherichia coli ATPase.

Journal Article↗

PubMed1991

Treponema phagedenis encodes and expresses homologs of the Treponema pallidum TmpA and TmpB proteins.

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase II and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive alpha-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.

Amino Acid Sequence↗

PubMed2002

Expression system for high levels of GAG lyase gene expression and study of the hepA upstream region in Flavobacterium heparinum.

A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

Bacterial Proteins↗

PubMed1997

The carboxy-terminal two-thirds of the cowpea chlorotic mottle bromovirus capsid protein is incapable of virion formation yet supports systemic movement.

Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.

Bromovirus↗

PubMed1994

Hepatic lipase gene is transcribed in rat adrenals into a truncated mRNA.

Rat adrenals contain a lipase activity that is indistinguishable from hepatic lipase (HL) present in liver. Expression of HL mRNA in adrenals was studied using the method of reverse transcription-polymerase chain reaction (RT-PCR). A 596-bp fragment of HL cDNA spanning exons 5 to 8 was amplified when using total RNA from rat adrenals and liver, but not from heart or kidney. The abundance of HL mRNA was quantified by competitive RT-PCR using a standard RNA that was generated in vitro by transcription from a deleted HL cDNA construct. Adrenals contained 0.4 attomoles of HL mRNA per microgram of total RNA, compared to 16 attomoles in liver. In hypertrophic adrenals isolated from corticotrophin-treated rats, the abundance also amounted to 0.4 attomoles of mRNA per microgram of total RNA. However, amplification of full-length cDNA from either control or hypertrophic adrenals was never observed. Detailed analysis by PCR using different combinations of primers indicated that exons 3 to 9 including the 3'-untranslated region were expressed in adrenal RNA, but not the first two coding exons. The upstream part of the adrenal lipase mRNA was cloned after rapid amplification of cDNA ends (RACE). The resulting clones showed a unique 126-bp sequence 5' of the exon 2-exon 3 junction. This sequence contained multiple termination codons in all three reading frames but lacked a potential start codon. RT-PCR using an HL-specific primer and an oligonucleotide directed against this 5'-sequence showed that it is not only expressed in RNA from adrenals but also in liver. Pulse-labeling of freshly isolated adrenocortical cells with [35S]methionine followed by immunoprecipitation with anti-HL antibodies failed to show synthesis of mature HL, but indicated the synthesis of immunoreactive proteins in the 40-45 kDa range that remained mainly intracellular. Hence, the HL gene is transcribed in adrenals but results in an mRNA species with a unique 5'-end. Translation from an internal start site may produce an intracellular HL isoform that differs markedly from the liver-type lipase previously identified in adrenals.

Adrenal Cortex↗

PubMed1988

Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase.

The nucleotide sequence of the Bacillus anthracis edema factor (EF) gene (cya), which encodes a calmodulin-dependent adenylate cyclase, has been determined. EF is part of the tripartite protein exotoxin of B. anthracis. An ATG start codon, immediately upstream from codons which specify the first 15 amino acids (aa) of EF, was preceded by an AAAGGAGGT sequence which is its probable ribosome-binding site. Starting at this ATG codon, there was a continuous 2400-bp open reading frame which encodes the 800-aa EF-precursor protein with a Mr of 92,464. The mature, secreted protein (767 aa; Mr 88,808) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. A consensus amino acid sequence (Gly-X-X-X-X-Gly-Lys-Ser,X = any aa), which was part of the presumed ATP binding site for EF, was also present. The codon usage of the EF gene reflected the high A + T (71%) base composition for its DNA. B. anthracis EF was not related to the Escherichia coli or yeast adenylate cyclases, but was related to the Bordetella pertussis calmodulin-dependent adenylate cyclase.

Amino Acid Sequence↗

PubMed1994

Translation initiation from non-AUG codons in COS1 cells is mRNA species dependent.

Sendai virus P/C mRNA, human erythrocyte membrane protein 4.1 mRNA and PDGF-B chain mRNA were used to test whether translation initiation from non-AUG codons in COS1 cells was mRNA species dependent. Site-directed mutants of the authentic translation start sites of these mRNAs to alternate start codons showed that while P/C mRNA is capable of initiating translation from non-AUG start sites the other two mRNAs are not. Our study shows that translation initiation from non-AUG codons is mRNA species dependent and suggests that higher order structure of an mRNA determines the non-AUG translation start site.

Animals↗

PubMed1991

The nucleotide sequence and deduced amino acid composition of the haemagglutinin and fusion proteins of the morbillivirus phocid distemper virus.

The amino acid composition of the two surface proteins of the recently isolated morbillivirus phocid distemper virus (PDV) were deduced from the nucleotide sequence. The fusion (F) protein of PDV exhibited characteristics similar to those of other morbillivirus F proteins. The overall amino acid similarity with its closest homologue, canine distemper virus (CDV), was 72%. From the context of the starting codons and the requirement for a hydrophobic signal peptide, it is likely that translation of the PDV F mRNA starts at the third AUG, corresponding to codon 95 in the long open reading frame of the PDV F gene. After removal of the signal peptide, F0 starts at amino acid 105. From this position the F protein of PDV and CDV exhibit 84% amino acid similarity. The PDV haemagglutinin (H) protein showed 74% amino acid similarity with CDV H protein and highly conserved features responsible for the tertiary structure. Despite these similarities, the two H proteins show marked antigenic differences when probed with monoclonal antibodies. Earlier studies have indicated that rinderpest virus (RPV) is the prototype virus of the morbillivirus genus, from which first CDV/PDV and later measles virus (MV) evolved. From the close relationship shown in this study, it is likely that the divergence of CDV and PDV occurred after MV evolved from RPV.

Amino Acid Sequence↗

PubMed2025

The plastid genome of the critically endangered Valeriana trinervis (= Centranthus trinervis) and insights from comparison with other Valeriana plastomes (Caprifoliaceae).

The first complete plastid genome of the critically endangered species Valeriana trinervis was sequenced, assembled and compared with other published Valeriana plastomes. In this study, we assembled the plastid genome of the critically endangered, endemic species Valeriana trinervis (= Centranthus trinervis) and compare it with all published plastomes of Valeriana. We found not only differences in the inverted repeats boundaries, in the type and abundance of repeats, but also similarities in codon usage and microsatellite numbers. We detected non-canonical start codons in several genes and identified variation in several regions that could be useful for phylogenetic and phylogeographic studies. The phylogenetic tree inference based on both full plastomes and coding sequence data indicated that V. trinervis is sister to all Eurasian Valeriana accessions confirming the phylogenetic position recently investigated. This is the first plastome available for a species of the Mediterranean clade of Valeriana previously known as Centranthus, and it adds further data to understand the evolution and diversification of this systematically debated genus.

Genome, Plastid↗

PubMed1986

The beta-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis.

The nucleotide sequence of a 1583-bp DNA fragment containing gene bg1A for endo-beta-1,3-1,4-glucanase (EC 3.2.1.73) of Bacillus amyloliquefaciens strain BE20/78, a high producer of secreted enzymes, has been determined. The gene bg1A comprises an open reading frame (ORF) of 717 bp (= 239 codons) starting with ATG at 469 up to the translation stop codon TAA at 1188. Upstream from the translation initiation codon ATG, the ribosome-binding sequence 5'-AAAAAAGGGGG-3' and two putative bglA promoters have been identified. A box of eleven AT out of twelve base pairs (bp) precedes the -35 region of promoter P1. Beyond the translation stop codon UAA, a sequence of 69 bp can be folded into a hook-like stem-loop structure which probably functions as a transcriptional terminator. The ORF region of the gene bglA reveals about 90% homology with another beta-glucanase gene, bglS of Bacillus subtilis C120 sequenced by Murphy et al. (1984). Three regions of frequent amino acid (aa) changes are indicated. However, the major difference between these is a set of deletions within the non-coding region separating the bglA gene from an unknown preceding ORF and by one deletion shortening the proposed signal peptide by three aa (Pro-Tyr-Leu-). The putative transcription terminator of gene bglA completely lacks homology with a B. subtilis bglS gene. The signification of deletions erasing the 'sacR-homology region' in B. amyloliquefaciens, which have been detected in proximity of the beta-glucanase gene of B. subtilis by Steinmetz and Aymerich (1986), is discussed.

Bacillus↗

PubMed1993

Cloning and sequencing of the Haemophilus influenzae aroA gene.

This study was designed to clone the aroA gene (encoding 5-enolpyruvylshikimate-3-phosphate synthase [EPSPS]) from Haemophilus influenzae using a mixed-primer polymerase chain reaction (PCR). The mixed primers were based on a back translation of previously reported blocks of amino acid homology between different cloned EPSPS proteins. The PCR-produced probe from H. influenzae DNA was used to identify a recombinant lambda phage from which the rest of H. influenzae aroA was subcloned and sequenced. The sequence of H. influenzae aroA is highly homologous to all other aroA genes sequenced to date. The upstream sequence of aroA, when translated, results in a protein with strong homology to the purN gene product, glycinamide ribonucleotide transformylase. The stop codon of the putative H. influenzae purN gene overlaps the start codon for H. influenzae aroA, suggesting that the two genes may form an operon and that they may be translationally coupled.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1998

Cloning and transcriptional analysis of the nifUHDK genes of Trichodesmium sp. IMS101 reveals stable nifD, nifDK and nifK transcripts.

Trichodesmium spp. are marine filamentous, non-heterocystous cyanobacteria capable of aerobic nitrogen fixation. In this study, the nitrogenase structural genes (nifHDK) and nifU gene of Trichodesmium sp. IMS101 were cloned and sequenced. The Trichodesmium sp. IMS101 nifH, nifD and nifK amino acid sequences showed only 79%, 66% and 68% identity, respectively, to those of Anabaena sp. strain PCC 7120. A potential transcription start site for nifH was found 212 bases upstream of the nifH start codon. Promoter-like nucleotide sequences upstream of the transcription start site were identified that were very similar to those identified for the nitrogenase genes of Anabaena spp. Sequence analysis revealed regions of DNA that may form stem-loop structures in the intercistronic regions downstream of nifH and nifD. RNA analysis by Northern hybridization revealed the presence of transcripts corresponding to nifH, nifHD and nifHDK. Surprisingly, Northern hybridization also revealed the presence of transcripts that corresponded to nifD, nifDK and nifK, which have not been previously reported as transcripts in contiguous nifHDK genes of cyanobacteria. Transcription of the nifHDK genes was not significantly repressed in the presence of nitrate at a final concentration of 20 mM or at oxygen concentrations of up to 40%, whereas ammonium and urea inhibited nifHDK transcription. The transcription of the nifHDK genes was not affected by darkness, which suggests that transcription of these genes in Trichodesmium is not directly regulated by light.

Base Sequence↗

PubMed1989

Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine.

The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.

Anticodon↗

PubMed1995

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

Aerobiosis↗

PubMed1995

Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level.

A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.

Amino Acid Sequence↗