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PubMed1998

Characterization of a new beta-spectrin gene which is predominantly expressed in brain.

We recently identified a gene which shows high similarity to the beta-spectrin gene but with a different chromosomal location from either of the two known beta-spectrin genes [T. Nagase, K.-I. Ishikawa, D. Nakajima, M. Ohira, N. Seki, N. Miyajima, A. Tanaka, H. Kotani, N. Nomura, O. Ohara, Prediction of the coding sequences of unidentified human genes: VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro, DNA Res. 4 (1997) 141-150]. In order to further characterize this new spectrin gene and its product, we isolated the rat counterpart of this gene and analyzed it in terms of its protein coding sequence, the tissue distribution of its mRNA and the product, and the regional distribution of the mRNA and the product in the brain. The results indicated that this gene was most abundantly transcribed in the brain and neurons were the predominant cell-type to express this gene. In particular, Purkinje cells were the richest in this gene product, and this new form of beta-spectrin was found more prominently in the dendrites than in the cell bodies. Since the expression pattern and the subcellular localization of this gene product were quiet distinct from those of the two beta-spectrin isoforms already characterized, this beta-spectrin gene would play an important role in neuronal membrane skeleton although it has been overlooked to date.

Amino Acid Sequence↗

PubMed2001

The gene structure of the Drosophila melanogaster homolog of the human proto-oncogene fos.

The Drosophila melanogaster homolog of the human proto-oncogene fos is Dfos. It is the only fos homolog in the Drosophila genome. Fos functions as a subunit of the heterodimeric transcription factor AP-1. There are two models of the Dfos gene. The first comes from a cDNA sequence of Dfos (Perkins et al., Genes Dev. 4 (1990) 822). The second is from the gene sequence published by the Drosophila genome project (Adams et al., Science 287 (2000) 2185), and there are notable contradictions between the two models. The promoter and the 5' end of the transcript sequence were not identified in either model. In this paper, we present the gene structure of Dfos and identify the promoter. This promoter has an initiator and a downstream promoter element sequence, but lacks a TATA box. Through comparison of the mRNA and genomic DNA sequences, three introns varying in length from 66 bp to 17.57 kb were found and verified by RT-PCR. The Dfos gene is 21.2 kb in length, giving a transcript of 3438 bp, coding for a predicted protein of 595 amino acids. The 3' untranslated region is confirmed to be 1092 bp in length.

3' Untranslated Regions↗

PubMed2003

Cloning of CnLYSO1, a novel extracellular lysophospholipase of the pathogenic fungus Cryptococcus neoformans.

We cloned a novel lysophospholipase (CnLYSO1) from Cryptococcus neoformans var. grubii by PCR amplification and a cDNA library screen. The open reading frame (ORF) of 1278 nucleotides coded for a predicted 426-amino-acid protein (CnLyso1p) with two highly conserved GXSXG lipase-specific catalytic motifs and a molecular weight of 48.3 kDa. CnLyso1p exhibited 14% and 21% identity to Arabidopsis thaliana and human lysophospholipases, respectively. Immunoprecipitation and Western blot analyses indicated that CnLyso1p was secreted as a high molecular weight protein of 97-140 kDa. CnLYSO1 expressed in a phospholipase B-null mutant of Saccharomyces cerevisiae demonstrated lysophospholipase and lysophospholipase transacylase activities at pH 4.0. Targeted disruption of CnLYSO1 did not affect growth, melanin or capsule production by C. neoformans. Secreted lysophospholipase and transacylase activities (pH 4.0) were 50% of wild type and CnLyso1p was undetectable on Western blots. Phospholipase B activity was reduced at pH 7.0 (P<0.006) and at pH 4.0 (P=NS). The amount of secreted Plb1p (the gene product of PLB1) was also reduced. Deletion of PLB1 abolished all three secreted activities at pH 4.0 and 7.0. These results are best explained by post-translational interaction, most likely the formation of a functional complex between the independently regulated gene products, CnLyso1p and CnPlb1p.

Amino Acid Sequence↗

PubMed1997

Cloning and sequencing of a new 15-hydroxyprostaglandin dehydrogenase related mRNA.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.

Amino Acid Sequence↗

PubMed1997

Leishmania braziliensis, molecular characterization of an elongation factor 1alpha gene.

The elongation factor EF-1alpha is one of the most studied components of the translation machinery owing to its abundance and possible role in other cellular functions. EF-1alpha mediates the correct coupling of the aminoacyl-tRNA on the A site of the ribosome in a GTP-dependent reaction. We have previously described an EF-1alpha DNA sequence in Leishmania amazonensis, pLEF11 (accession No. M92653), using PCR. In this paper we describe the DNA sequence and genomic organization of L. braziliensis EF-1alpha gene. Southern blot analysis revealed that EF-1alpha is organized as a 2 kb tandem repeat. The pLEF11 probe recognized a 1.8 kb mRNA from promastigotes in Northern blots. A clone containing the first copy and a half of the EF-1alpha tandem repeat was isolated by screening a L. braziliensis genomic library. Southern blot analysis showed that the isolated clone (lambda2.2) presented the same hybridization profile as that of a genomic blot. The partial sequencing of clone lambda2.2 spans 2959 nucleotides in length, which has two open reading frames separated by a putative non-coding region. The nucleotide and the predicted peptide sequence of the first coding region presented approximately 80% identity with other eukaryotic EF-1alpha genes. The sequence also displayed the four consensus motifs corresponding to the GTP-binding site (G1, G2, G3 and G4). Computer analysis of the sequence of both coding regions revealed three divergent nucleotides, which generated two changes at the amino acid level. One was found to be located in the G2 domain. The non-coding region of the EF-1alpha gene sequence showed potential regulatory elements such as polypyrimidine tracks, chi-homologous sequences and stem-loop forming sequences.

Amino Acid Sequence↗

PubMed2003

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

Actinobacillus pleuropneumoniae↗

PubMed2003

Determination of neutron flux distribution in an Am-Be irradiator using the MCNP.

A neutron irradiator has been assembled at IPEN facilities to perform qualitative-quantitative analysis of many materials using thermal and fast neutrons outside the nuclear reactor premises. To establish the prototype specifications, the neutron flux distribution and the absorbed dose rates were calculated using the MCNP computer code. These theoretical predictions then allow one to discuss the optimum irradiator design and its performance.

Americium↗

PubMed2006

Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa.

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.

Amino Acid Sequence↗

PubMed1992

Cloning and characterization of the casein kinase II alpha subunit gene from the lymphocyte-transforming intracellular protozoan parasite Theileria parva.

Theileria parva is an obligate intracellular protozoan parasite which is the causative agent of East Coast fever, an acute, leukemia-like disease of cattle. The intralymphocytic stage of the parasite induces blastogenesis and clonal expansion of quiescent bovid lymphocytes. Experiments in our laboratory have shown a marked increase of casein kinase II- (CK II-) like activity in T. parva-transformed lymphocytes. We have also detected CK II activity in purified T. parva schizonts. To explore the significance of this increase, we used a Drosophila melanogaster CK II alpha cDNA probe [Saxena et al. (1987) Mol. Cell Biol. 7, 3409-3417] to isolate a T. parva genomic clone encoding a CK II catalytic subunit. The clone contains a 1.3-kb open reading frame coding for a predicted protein of 420 amino acids (M(r) 50,200). Northern blot analysis revealed a single transcript of 1.65 kb. The deduced T. parva CK II catalytic subunit sequence shows, over 321 residues comprising the C-terminus of the molecule, extensive identity with CK II alpha and alpha' sequences from both vertebrate and invertebrate organisms. The T. parva CK II subunit amino acid sequence displays 68% identity with the Drosophila alpha subunit and 67% with the Caenorhabditis elegans alpha subunit but only 58% and 56% sequence identity with the Saccharomyces cerevisiae alpha and alpha' subunits, respectively. Comparison of the T. parva sequence with higher eukaryotic alpha and alpha' sequences reveals that it is most identical with the alpha subunit. A unique component of the T. parva CK II alpha subunit is a 99 amino acid sequence at the N-terminus, which contains a sequence motif with features characteristic of signal peptides.

Amino Acid Sequence↗

PubMed2004

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of zeins in mature maize kernels.

A high-throughput method has been developed to allow rapid analysis of maize seed storage proteins by matrix-assisted laser desorption time-of-flight mass spectrometry. The extraction solution containing an organic solvent, a reducing agent, and a volatile base has been optimized to enable extraction of all classes of zein proteins (alpha-, beta-, gamma-, and delta-). A near-saturating concentration of matrix, 2-(4-hydroxyphenylazo)benzoic acid, was necessary to obtain strong peaks for the most lipophilic zeins, the alpha-zeins. Zein proteins with small mass differences, difficult to separate by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were resolved through this analysis. Mass signals corresponding to the 10-kDa delta-, 15-kDa beta-, 16-kDa gamma-, 27-kDa gamma-, and several 19 and 22-kDa alpha-zeins were detected. The zein identities were further confirmed by the association of the number of cysteine residues in each zein MS peak, as determined by iodoacetamide derivatization, with the number predicted from its coding sequence. The relative zein abundance in the zein MS peaks was also correlated with the relative zein EST abundance among endosperm EST libraries. This method was utilized to examine the zein composition of a number of corn inbred lines and opaque mutants.

Hydrogen-Ion Concentration↗

PubMed1999

Evidence for factors regulating transfer cell-specific expression in maize endosperm.

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (< 100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.

Amino Acid Sequence↗

PubMed2000

Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully.

Amino Acid Motifs↗

PubMed2001

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis.

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.

Base Sequence↗

PubMed2000

Concreteness and relational effects on recall of adjective-noun pairs.

Extending previous research on the problem, we studied the effects of concreteness and relatedness of adjective-noun pairs on free recall, cued recall, and memory integration. Two experiments varied the attributes in paired associates lists or sentences. Consistent with predictions from dual coding theory and prior results with noun-noun pairs, both experiments showed that the effects of concreteness were strong and independent of relatedness in free recall and cued recall. The generally positive effects of relatedness were absent in the case of free recall of sentences. The two attributes also had independent (additive) effects on integrative memory as measured by conditionalized free recall of pairs. Integration as measured by the increment from free to cued recall occurred consistently only when pairs were high in both concreteness and relatedness. Explanations focused on dual coding and relational-distinctiveness processing theories as well as task variables that affect integration measures.

Adult↗

PubMed2002

Sequence and analysis of rice chromosome 4.

Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.

Arabidopsis↗

PubMed2006

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.

Azoarcus↗

PubMed2003

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Amino Acid Sequence↗

PubMed2026

Assessing the de novo paradigm in sporadic early-onset Alzheimer disease trios.

The genetic architecture of sporadic Early-Onset Alzheimer Disease (sEOAD, onset ≤65 years) remains largely unknown. To assess the de novo mutation (DNM) hypothesis, we performed a nationwide recruitment of 37 novel sEOAD patients-unaffected parents trios. After assessing known monogenic genes, we performed trio-based exome sequencing and jointly analyzed novel trios with 12 previously reported ones. Of these, we selected 16 trios for genome sequencing. We identified three patients with a pathogenic DNM in APP or PSEN1. Then, from the 46 remaining trios, we identified 38 non-synonymous coding DNM and 4 de novo copy number variants (CNVs) in exome data. Four DNM (2 novel, in SPHK2 and DDR1) and bi-allelic inherited variants in two genes affected Alzheimer disease-related genes. No significant burden of rare coding variants in exome/genome data from 5643 EOAD cases and 16097 controls was identified using nested windows centered on each DNM position, at the transcript level. From genome data, one non-coding DNM was predicted to affect splicing in an AD-associated gene, PINX1. Overall, 48% probands carried ≥1 inherited risk factor with odds ratio (OR) > 1.5 and GWAS-defined Genetic Risk Scores (GRS) distribution was more consistent with random distribution than enrichment in higher scores in probands. We confirm that DNMs in known monogenic genes explain sEOAD in a minority of cases, while candidate DNMs in other genes might account for a small proportion of additional cases. The majority of sEOAD patients may have a complex etiology including multiple inherited variants, however, GRS might not explain most of its genetic component.

Humans↗