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PubMed2006

KDC2, a functional homomeric potassium channel expressed during carrot embryogenesis.

In Daucus carota, the model system for embryogenesis, it has been demonstrated that potassium and K(+) selective channels are involved in embryo development. Here, we report the isolation and cloning of a new carrot Shaker-like potassium channel, potassium D. carota channel 2 (KDC2), whose expression pattern during somatic embryogenesis proceeds along with the establishment of the polar axes and the settlement of the hypocotyl region. In plants, KDC2 transcript is localized at the shoot level, in the epidermis and guard cells, similarly to its Arabidopsis homolog KAT1. Electrophysiological assays indicated KDC2 as the first carrot subunit able to form homomeric functional channels in Xenopus oocytes, with properties similar to those of Arabidopsis KAT1.

Amino Acid Sequence↗

PubMed1999

Isolation, characterization and cDNA cloning of nicotianamine synthase from barley. A key enzyme for iron homeostasis in plants.

Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilize iron, plants have evolved at least two different strategies. The nonproteinogenous amino acid nicotianamine which is synthesized from three molecules of S-adenosyl-L-methionine, is an essential component of both pathways. This compound is missing in the tomato mutant chloronerva, which exhibits severe defects in the regulation of iron metabolism. We report the purification and partial characterization of the nicotianamine synthase from barley roots as well as the cloning of two corresponding gene sequences. The function of the gene sequence has been verified by overexpression in Escherichia coli. Further confirmation comes from reduction of the nicotianamine content and the exhibition of a chloronerva-like phenotype due to the expression of heterologous antisense constructs in transgenic tobacco plants. The native enzyme with an apparent Mr of approximately 105 000 probably represents a trimer of S-adenosyl-L-methionine-binding subunits. A comparison with the recently cloned chloronerva gene of tomato reveals striking sequence homology, providing support for the suggestion that the destruction of the nicotianamine synthase encoding gene is the molecular basis of the tomato mutation.

Alkyl and Aryl Transferases↗

PubMed2000

Human uroplakin Ib in ocular surface epithelium.

PURPOSE: To investigate the expression and localization of the human gene encoding uroplakin Ib in ocular surface epithelium. METHODS: The full-length cDNA of human uroplakin Ib was isolated from a cDNA library of human corneal epithelium, and the expression of uroplakin Ib in various tissues was examined by reverse transcription-polymerase chain reaction (RT-PCR). In cornea and conjunctiva, the expressions of uroplakin Ia, II, and III were also examined by RT-PCR. Finally, the localization of uroplakin Ib in the ocular surface was analyzed by immunofluorescence confocal microscopy, by using an antiserum against a synthetic peptide. RESULTS: Two mRNA isoforms, arising through two polyadenylation sites, were isolated. RT-PCR detected uroplakin Ib in cornea, conjunctiva, bladder, placenta, and kidney. Among other uroplakins, uroplakin II was also faintly detected in cornea and conjunctiva. Immunofluorescence confocal microscopy documented uroplakin Ib protein in the cell membranes of superficial and wing cells in the corneal epithelium. It was not found, however, in the most apical corneal epithelial cells. In limbus and conjunctiva, uroplakin Ib was also localized in the cell membranes of all epithelial layers, apart from the most apical cells. CONCLUSIONS: Uroplakin Ib is highly expressed in ocular surface epithelia. As in bladder epithelium, uroplakin Ib may protect the ocular surface from bacterial infection.

Amino Acid Sequence↗

PubMed2004

Prevalence of Enterocytozoon bieneusi in post-weaned dairy calves in the eastern United States.

Fecal specimens were obtained from 3- to 8-month-old post-weaned dairy calves on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, 59 of 452 calves (13%) from 11 farms in six states were found positive for Enterocytozoon bieneusi by PCR and DNA sequence analysis. Based on gene sequence data this genotype of E. bieneusi found in post-weaned calves was 100% identical to that found in pre-weaned calves in North America and differed by only two positions in 1,069 base pairs from specimens analyzed from humans. However, compared with previous reports, the prevalence of E. bieneusi was significantly higher in post-weaned than in pre-weaned calves from many of the same farms.

Animals↗

PubMedDate not supplied

Neural network method to analyze data compression in DNA and RNA sequences.

Neural network computations on RNA sequences are used to demonstrate that data compression is possible in these sequences. The result implies that a certain discrimination should be achievable between structured vs random regions. The technique is illustrated by computing the compressibility of short RNA sequences such as tRNA. The method should be valuable in measuring the information content of DNA, including noncoding DNA, which has been shown to display certain properties resembling natural language attributes.

Base Sequence↗

PubMed1997

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.

Amino Acid Sequence↗

PubMed1982

Two-dimensional graphic analysis of DNA sequence homologies.

We describe a computer program designed to facilitate the pattern matching analysis of homologies between DNA sequences. It takes advantage of a two-dimensional plot in order to simplify the evaluation of significant structures inherited in the sequences. The program can be divided into three parts, i) algorithm for search of homologies, ii) two-dimensional graphic display of the result, iii) further graphic treatment to enhance significant structures. The power of the graphic display is presented by the following application of the program. We conducted a search for direct repeats in the mouse immunoglobulin kappa-chain genes. Both the five J DNA sequences and other shorter repeats were found. We also found a longer stretch of homology that could indicate the presence of duplicated DNA in the J4, J5 region.

Base Sequence↗

PubMed1994

Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli.

Resistance to multiple antibiotics and certain oxidative stress compounds was conferred by three independently selected mutations (marR1, soxQ1, and cfxB1) that mapped to 34 min on the Escherichia coli chromosome. Mutations at this locus can activate the marRAB operon, in which marR encodes a putative repressor of mar transcription and marA encodes a putative transcriptional activator of defense genes against antibiotics and oxidants. Overexpression of the wild-type MarR protein reversed the phenotypes (antibiotic resistance and increased antioxidant enzyme synthesis) of all three mutants. DNA sequence analysis showed that, like marR1, the other two mutations were alterations of marR: a 285-bp deletion in cfxB1 and a GC-->AT transition at codon 70 (Ala-->Thr) in soxQ1. All three mutations cause increased amounts of mar-specific RNA, which supports the hypothesis that MarR has a repressor function in the expression of the marRAB operon. The level of mar RNA was further induced by tetracycline in both the marR1 and soxQ1 strains but not in the cfxB1 deletion mutant. In the cfxB1 strain, the level of expression of a truncated RNA, with or without tetracycline exposure, was the same as the fully induced level in the other two mutants. Overproduction of MarR in the cfxB1 strain repressed the transcription of the truncated RNA and restored transcriptional inducibility by tetracycline. Thus, induction of the marRAB operon results from the relief of the repression exerted by MarR. The marRAB operon evidently activates both antibiotic resistance and oxidative stress genes.

Adaptation, Physiological↗

PubMed2003

Tcoffee@igs: A web server for computing, evaluating and combining multiple sequence alignments.

This paper presents Tcoffee@igs, a new server provided to the community by Hewlet Packard computers and the Centre National de la Recherche Scientifique. This server is a web-based tool dedicated to the computation, the evaluation and the combination of multiple sequence alignments. It uses the latest version of the T-Coffee package. Given a set of unaligned sequences, the server returns an evaluated multiple sequence alignment and the associated phylogenetic tree. This server also makes it possible to evaluate the local reliability of an existing alignment and to combine several alternative multiple alignments into a single new one. Tcoffee@igs can be used for aligning protein, RNA or DNA sequences. Datasets of up to 100 sequences (2000 residues long) can be processed. The server and its documentation are available from: http://igs-server.cnrs-mrs.fr/Tcoffee/.

Consensus Sequence↗

PubMed2003

Cloning and characterization of the 3'-untranslated region of the human excitatory amino acid transporter 2 transcript.

The 3'-untranslated region (UTR) of the human excitatory amino acid transporter 2 (EAAT2) transcript was cloned and characterized. The full-length EAAT2 cDNA of 11 692 bp was found to contain 283 bp of 5' UTR, a 1725-bp open reading frame and an unusually long 3'-UTR of 9684 bp. The 3'-UTR of EAAT2 cDNA was well conserved among mammals, and human, macaque, rat and mouse cDNA had nearly identical 3' ends. The human EAAT2 transcripts were detected in brain, spinal cord, liver, adrenal gland, placenta and pancreas by northern hybridization, and many ESTs homologous to the human EAAT2 cDNA were found in numerous tissues. To investigate the role of human EAAT2 3'-UTR in gene expression, we constructed luciferase expression vectors containing 3'-UTR fragments spanning the entire length of the region. The individual fragments varied in their effects on reporter gene expression in human astrocytes by a factor of eight to ten suggesting a complex role of the 3'-UTR in post-transcriptional regulation of EAAT2 gene expression.

3' Untranslated Regions↗

PubMed1999

Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase.

Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids.

Alkyl and Aryl Transferases↗

PubMed2001

DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity.

Acyl CoA:diacylgycerol acyltransferase (EC; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity, were isolated from the lipid bodies of the oleaginous fungus Mortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals, which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer to the M. ramanniana genes as MrDGAT2A and MrDGAT2B.

Acyltransferases↗

PubMed2003

A candidate olfactory receptor subtype highly conserved across different insect orders.

Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. Here we describe the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86-88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors revealed a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects.

Amino Acid Sequence↗

PubMed2005

Isolation and characterization of a polymorphic stigma-specific class III peroxidase gene from Senecio squalidus L. (Asteraceae).

A novel stigma-specific class III peroxidase gene, SSP (Stigma-Specific Peroxidase), has been isolated from the self-incompatible daisy Senecio squalidus L. (Asteraceae). Expression of SSP in flower buds is developmentally regulated, with maximal levels of expression coinciding with anthesis, when stigmas are most receptive to pollen and when self-incompatibility is fully developed. In situ hybridization revealed SSP expression to be localized exclusively to the specialized secretory epidermal cells (papillae) of the stigma, which receive and discriminate pollen. SSP is therefore the first tissue-specific and cell-specific peroxidase gene identified in a plant. SSP belongs to a distinct clade of class III plant peroxidases that possess two introns, instead of the more normal situation of three conserved introns. The deduced amino acid sequence of SSP revealed a 27 amino acid signal peptide, suggesting that the SSP protein is secreted to the cell wall of the stigmatic papillae. In-gel peroxidase activity assays showed that SSP has relatively low peroxidase activity compared to other, as yet uncharacterized, peroxidases present in stigmatic extracts. Six SSP alleles have been cloned from different lines of S. squalidus carrying a range of self-incompatibility (S)-alleles but there was no consistent association between the presence of a particular SSP allele and S-genotype indicating that SSP is not the female determinant of SSI in S. squalidus. Nevertheless, the precise expression of SSP in stigmatic papillae suggests that it may have a more general function in pollen-stigma interactions, or alternatively in protection of stigmas from pathogen attack. Extensive database screens have identified homologues of SSP in other plant species, but available expression data for these genes indicates that none are flower-specific, suggesting that SSP represents a new functional type of class III peroxidase specific to the stigma. We discuss the possible function(s) of S. squalidus SSP in pollen-stigma interactions and in protection of stigmas from pathogen attack.

Alleles↗

PubMed2005

An approach towards experimental cDNA sequence determination of predicted genes: an example from Arabidopsis U3-55k homologues.

Experimental cDNA sequence determinations lag behind in silico gene structure predictions in some recently sequenced genomes. This may be due in part to low transcript abundance and/or the severely spatio-temporarily restricted expression pattern of some genes. Here we characterize the predicted repressed gene of Arabidopsis thaliana (At4g21130) that encodes a homologue of the Arabidopsis U3-55K-like protein (At4g05410) and of the U3-55K (RNU3IP2, Rrp9p) proteins from other eukaryotes. In man and yeast, U3-55K is involved in the processing of the pre-ribosomal RNA. Here we show that treatment with inhibitors of histone deacetylases (trichostatin A, sodium butyrate) or DNA methyltransferases (5-aza-2'-deoxycytidine) induces a low but distinct level of mRNA from the repressed Arabidopsis At4g21130 locus, which can be detected by RT-PCR amplification. Direct sequencing of PCR products reveals the open reading frame that differs, in part, from the hypothetical one and encodes a seven-WD-repeat protein highly conserved when compared to U3-55K proteins from various eukaryotic species. This suggests the conservation of its function. The described approach may help to determine the nucleotide sequences of transcripts from predicted genes with a low level of expression.

Amino Acid Sequence↗

PubMed2000

RT-PCR, nucleotide, amino acid and phylogenetic analyses of enterovirus type 71 strains from Asia.

A specific and sensitive method based on RT-PCR was developed to detect enterovirus 71 (EV71) from patients with hand, foot and mouth disease, myocarditis, aseptic meningitis and acute flaccid paralysis. RT-PCR primers from conserved parts of the VP1 capsid gene were designed on the basis of good correlation with sequences of EV71 strains. These primers successfully amplified 44 strains of EV71 including 34 strains isolated from Singapore in 1997 and 1998, eight strains from Malaysia isolated in 1997 and 1998, one Japanese strain and the neurovirulent strain EV71/7423/MS/87. RT-PCR of 30 strains of other enteroviruses including coxsackievirus A and B, and echoviruses failed to give any positive amplicons. Hence, RT-PCR with these primers showed 100% correlation with serotyping. Direct sequencing of the RT-PCR products of 20 EV71 strains revealed a distinct cluster with two major subgroups, thus enabling genetic typing of the viruses. The genetic heterogeneity of these strains culminated in amino acid substitutions within the VP1, VP2 and VP3 regions. The sequencing of a 2.9 kb fragment comprising the capsid region and the major part of 5' UTR of two Singapore strains revealed that they belonged to a group distinct from the prototype EV71/BrCr strain and the EV71/7423/MS/87 strain. The dendrogram generated from 341 bp fragments within the VP1 region revealed that the strains of Singapore, Malaysia and Taiwan belong to two entirely different EV71 genogroups, distinct from the three genogroups identified in another recent study.

Amino Acid Sequence↗

PubMed2002

KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen.

The KAP-2 protein that binds to the H-box (CCTACC) element in the bean CHS15 chalcone synthase promoter was purified, and internal peptide sequence used to design primers leading to the cloning of KAP-2 from bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula). KAP-2 shares sequence similarity to the large subunit of mammalian Ku autoantigen, a protein proposed to be involved in control of DNA recombination and transcription. KAP-2 sequences were present in genomic DNA from a range of legumes, and a related protein is found in Arabidopsis thaliana. Recombinant KAP-2 expressed in insect cells showed the same binding specificity for the CHS15 H-box as the protein purified from bean cell extracts. In vitro transcription assays confirmed that KAP-2 stimulates transcription from a promoter harboring the H-box cis element.

Acyltransferases↗

PubMed2001

Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner.

Glutamine:fructose-6-phosphate aminotransferase (GFAT; EC 2.6.1.16) expression is tightly regulated in the context of amino sugar synthesis in many organisms from yeast to humans by transcriptional and post-translational processes. We have cloned the cDNA of the GFAT1 of Drosophila melanogaster (Dmel/Gfat1). One of the two putative protein kinase A (PKA) phosphorylation sites proposed for the regulation of human GFAT1 [Zhou, Huynh, Hoffmann, Crook, Daniels, Gulve and McClain (1998) Diabetes 47, 1836-1840] is conserved in Dmel/GFAT1. In the other one the reactive serine has been converted to a cysteine, making further access by PKA unlikely. The Dmel/Gfat1 gene is localized at position 81F on the right arm of chromosome 3. By whole-mount in situ hybridization specific expression of Dmel/GFAT1 was detected in embryonic chitin-synthesizing tissues and in the corpus cells of salivary glands from late third larval instar. Expressing Dmel/GFAT1 in yeast we showed that Dmel/GFAT1 activity is controlled by UDP-N-acetylglucosamine and PKA in the yeast total protein extract system. We propose a model for the independent regulation of the Dmel/GFAT1 enzyme by feedback inhibition and PKA.

Amino Acid Sequence↗