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Primary structure of the putative human oncogene, pim-1.

A full-length (6.1-kb) human genomic pim-1 gene, together with its immediate 5'-upstream promoter sequence (Ppim) was isolated and sequenced. The human pim-1 gene shares an overall nucleotide (nt) sequence identity of 53% with the previously reported murine pim-1 gene. It consists of six exons and five introns and contains a protein-coding region that is identical in nt sequence to a full-length human pim-1 cDNA. The gene codes for a predicted Pim-1 protein of 313 amino acids (aa) with an Mr of 35,690 and a pI of 5.7. The deduced aa sequence of the human Pim-1 has 94% identity with the murine Pim-1 whereas the nt sequences of the two genes are 88% identical. All of the conserved aa residues of the mouse pim-1 gene, which are homologous to known protein kinases are conserved in the predicted human protein. The human Ppim region is very G + C-rich (69%) and shares greater than 80% identity with the murine Ppim. The Ppim has no TATA- or CAAT-box sequences but does contain a number of nt sequences similar to the putative binding sites of several presumptive transcription factors.

Amino Acid Sequence↗

Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.

Amino Acid Sequence↗

Experimental investigation and nuclear model calculations on proton-induced reactions on highly enriched 114Cd at low energies.

The excitation functions of both Cd(p,n)(114m)In114 and Cd(p,2n)(113m)In114 reactions were evaluated experimentally by the stacked-foil technique on highly enriched (114)Cd isotope. Two stacks were irradiated by proton beam with energies 14.7 and 18MeV on the MGC-20 cyclotron at Nuclear Research Center, AEA, Egypt. The present results support the previous measurements. The theoretical interpretations of the measured excitation functions were done by EMPIRE-II (v2.18 Mondovi) code. Compatibility with predictions of hybrid Monte Carlo simulation (HMS) pre-equilibrium mechanism was obtained.

Journal Article↗

Comparative analysis of iron regulated genes in mycobacteria.

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.

Amino Acid Sequence↗

Insight into the genome of Aspergillus fumigatus: analysis of a 922 kb region encompassing the nitrate assimilation gene cluster.

Aspergillus fumigatus is the most ubiquitous opportunistic filamentous fungal pathogen of human. As an initial step toward sequencing the entire genome of A. fumigatus, which is estimated to be approximately 30 Mb in size, we have sequenced a 922 kb region, contained within 16 overlapping bacterial artificial chromosome (BAC) clones. Fifty-four percent of the DNA is predicted to be coding with 341 putative protein coding genes. Functional classification of the proteins showed the presence of a higher proportion of enzymes and membrane transporters when compared to those of Saccharomyces cerevisiae. In addition to the nitrate assimilation gene cluster, the quinate utilisation gene cluster is also present on this 922 kb genomic sequence. We observed large scale synteny between A. fumigatus and Aspergillus nidulans by comparing this sequence to the A. nidulans genetic map of linkage group VIII.

Aspergillus fumigatus↗

Construction and validation of a Sinorhizobium meliloti whole genome DNA microarray: genome-wide profiling of osmoadaptive gene expression.

Based on the complete Sinorhizobium meliloti genome sequence we established DNA microarrays as a comprehensive tool for systematic genome-wide gene expression analysis in S. meliloti 1021. For these PCR fragment-based microarrays, called Sm6kPCR, a collection of probes for the 6207 predicted protein-coding genes consisting of 6046 gene-specific PCR fragments and 161 70 mer oligonucleotides was arrayed in high density on glass slides. To obtain these PCR fragments primer pairs were designed to amplify internal gene-specific DNA fragments of 80-350 bp. Additionally, these primers were characterized by a 5' extension that allowed for reamplification using standard primers after the first amplification employing the specific primers. In order to ascertain the quality of the Sm6kPCR microarrays and to validate gene expression studies in S. meliloti parallel hybridizations based on RNA samples obtained from cells cultured under identical conditions were performed. In addition, gene expression in S. meliloti in response to an osmotic upshift imposed by the addition of 0.38 M NaCl was monitored. 137 genes were identified showing significant changes in gene expression resulting from the osmotic upshift. From these genes 52 were induced and 85 genes were repressed. Among the genes displaying different RNA levels some functional groups could be identified that are particularly remarkable. Repression was observed for 8 genes related to motility and chemotaxis, 7 genes encoding amino acid biosynthesis enzymes and 15 genes involved in iron uptake whereas 14 genes involved in transport of small molecules and 4 genes related to polysaccharide biosynthesis were induced.

Chromosome Mapping↗

Occurrence of blaOXA-72 in a clinical isolate of carbapenem-resistant Acinetobacter pittii ST206 in Japan.

The development of carbapenem resistance in Acinetobacter spp., which are recognized as significant opportunistic pathogens, is of critical importance as it poses challenges to therapy and the control of healthcare-associated infections in clinical settings. This study investigated the genetic characteristics of a carbapenem-resistant A. pittii clinical isolate from a university hospital using whole-genome sequencing. The A. pittii strain SU8507, which was detected in the abdominal drainage fluid of a patient, exhibited resistance to imipenem (MIC: 128 μg/mL) and meropenem (MIC: 64 μg/mL) and produced positive results by the CIMTris method. A. pittii SU8507, belonging to ST206, harbored the blaOXA-72 and new variants of intrinsic blaOXA-213-like gene blaOXA-1222, which lacks an upstream insertion sequence element, and blaADC-1-like gene blaADC-343. The blaOXA-72 gene, flanked by XerC/XerD-like recombination sites, was located on a plasmid pSU8507, sized at 10,913 bp, carrying 13 predicted protein-coding genes. Complete pSU8507 containing mobA, repB, and the yoeB-yefM toxin-antitoxin genes, showed 98.9% nucleotide sequence identity and 75% coverage with plasmid pA2702 of the A. baylyi strain A2702, but a low BLAST MAX score. SU8507 harbored virulence genes involved in biofilm formation, types II and VI secretion systems, type IV pilus system, and serum resistance. This study describes the first isolation of an OXA-72-producing, carbapenem-resistant A. pittii clinical isolate in Japan. Given that the isolation rate of carbapenem-resistant Acinetobacter spp. Remains low in Japan, it is crucial to expand the scope of rapid, accurate carbapenemase detection to include not only A. baumannii, but also non-baumannii Acinetobacter spp.

Humans↗

Alternative transcription of the mouse Gh gene identifies an immune-associated transcript with species-specific structural divergence.

Growth hormone (GH) in mice is primarily expressed in the anterior pituitary, although Gh expression has been reported in extrapituitary tissues, including immune organs. However, the structure of immune-associated Gh transcripts remains poorly characterized. To determine whether splenic Gh transcripts differ from pituitary Gh mRNA, 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses were performed. While 3' RACE showed a shared polyadenylation site, 5' RACE identified a novel exon located approximately 2 kb upstream of the conventional exon 1, generating a transcript (spl-Gh mRNA) with a distinct first exon but shared downstream exons with pituitary Gh mRNA (pit-Gh mRNA). RT-PCR analysis revealed that spl-Gh mRNA is predominantly expressed in immune tissues such as spleen and bone marrow, and its distribution did not correlate with Pit-1 mRNA expression. Quantitative RT-PCR further demonstrated that spl-Gh mRNA was expressed at levels comparable to those of pit-Gh mRNA in the mouse spleen, indicating that spl-Gh is one of the major Gh transcript forms in this tissue. Sequence analysis indicated that spl-Gh mRNA is predicted to retain coding potential for a GH protein. Comparative genomic analyses further demonstrated that genomic features associated with the spl-Gh transcriptional unit are conserved only in a subset of closely related Mus species. In contrast, although a spl-Gh-related transcript was detected in rat spleen, no properly spliced mouse-like transcript was identified under the present experimental conditions. The detected transcript exhibited intron retention and an in-frame stop codon, suggesting that it is unlikely to produce a functional GH protein. These findings identify a distinct immune-associated Gh transcript generated through alternative transcription of the mouse Gh gene and suggest that immune-associated Gh transcriptional mechanisms have undergone species-specific divergence among rodents. Together, these findings reveal previously unrecognized complexity in Gh gene regulation and highlight species-specific differences in immune-associated Gh transcripts.

Animals↗

Molecular cloning and nucleotide sequence of human pancreatic prechymotrypsinogen cDNA.

The cDNA clone encoding human prechymotrypsinogen was isolated from a human pancreas cDNA library and its nucleotide sequence was determined. The sequence consists of a 16 bp 5' non-coding region, a 789 bp amino acid coding region and a 60 bp 3' non-coding region. The predicted product consists of 263 amino acids, including 18 amino acids for a signal peptide and 15 amino acids possible for an activation peptide. Southern blot analyses using the cloned cDNA as a probe revealed that human genomic DNA carries at least two genes that are related to chymotrypsinogen.

Amino Acid Sequence↗

Deaths related to lead poisoning in the United States, 1979-1998.

This study was conducted to describe trends in US lead poisoning-related deaths between 1979 and 1998. The predictive value of relevant ICD-9 codes was also evaluated. Multiple cause-of-death files were searched for records containing relevant ICD-9 codes, and underlying causes and demographic characteristics were assessed. For 1979-1988, death certificates were reviewed; lead source information was abstracted and accuracy of coding was determined. An estimated 200 lead poisoning-related deaths occurred from 1979 to 1998. Most were among males (74%), Blacks (67%), adults of age >/=45 years (76%), and Southerners (70%). The death rate was significantly lower in more recent years. An alcohol-related code was a contributing cause for 28% of adults. Only three of nine ICD-9 codes for lead poisoning were highly predictive of lead poisoning-related deaths. In conclusion, lead poisoning-related death rates have dropped dramatically since earlier decades and are continuing to decline. However, the findings imply that moonshine ingestion remains a source of high-dose lead exposure in adults.

Adolescent↗

The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea).

The complete mitochondrial genome sequences were determined for two species of human hookworms, Ancylostoma duodenale (13,721 bp) and Necator americanus (13,604 bp). The circular hookworm genomes are amongst the smallest reported to date for any metazoan organism. Their relatively small size relates mainly to a reduced length in the AT-rich region. Both hookworm genomes encode 12 protein, two ribosomal RNA and 22 transfer RNA genes, but lack the ATP synthetase subunit 8 gene, which is consistent with three other species of Secernentea studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T, but low in G and C. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. For both hookworm species, genes were arranged in the same order as for Caenorhabditis elegans, except for the presence of a non-coding region between genes nad3 and nad5. In A. duodenale, this non-coding region is predicted to form a stem-and-loop structure which is not present in N. americanus. The mitochondrial genome structure for both hookworms differs from Ascaris suum only in the location of the AT-rich region, whereas there are substantial differences when compared with Onchocerca volvulus, including four gene or gene-block translocations and the positions of some transfer RNA genes and the AT-rich region. Based on genome organisation and amino acid sequence identity, A. duodenale and N. americanus were more closely related to C. elegans than to A. suum or O. volvulus (all secernentean nematodes), consistent with a previous phylogenetic study using ribosomal DNA sequence data. Determination of the complete mitochondrial genome sequences for two human hookworms (the first members of the order Strongylida ever sequenced) provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance.

Amino Acid Sequence↗

Biological implications of complementary hydropathy of amino acids.

The principle of complementary hydropathy predicts that peptides coded for by opposing DNA strands will bind one another because highly hydrophilic amino acids will be complemented by hydrophobic ones and vice versa. This paper provides the chemical plausibility for such interactions. It is suggested that exons coding for interacting peptides were juxtaposed and co-evolved together. Present day genes are no longer thus arranged because of duplications and exon shuffling.

Amino Acids↗

Molecular cloning of cDNA encoding the alpha unit of CNGC gene from human fetal heart.

Cyclic nucleotide-gated ion channels (CNGCs) play crucial roles in visual and olfactory signal transduction. As a first step to explore the presence of a CNGC gene in human heart, we cloned a human heart CNGC gene. The sequence consists of 111 bp 5' non-coding region and a 2064 bp open reading frame which is followed by a 459 bp 3' non-coding region. The predicted protein consists of 688 amino acids with a short highly charged segment rich in lysine and glutamate. Sequence comparison indicates that the human heart cDNA is almost identical to the retinal rod photo receptor CNGC cDNA. However, the human cardiac cDNA is lacking a 205 bp Alu fragment in the 5'-uncoding region, has a glutamic acid residue at amino acid position 129, and has a replacement of glutamic acid with a lysine residue at amino acid position 99. Data obtained with northern blot analysis confirm the presence of RNA for the CNGC alpha chain. This channel might play a role in cyclic nucleotide-mediated cellular processes, such as the inotropic effect in the heart.

Amino Acid Sequence↗

Coat protein of the Ectocarpus siliculosus virus.

Ectocarpus siliculosus virus, EsV, multiplies in sporangia and gametangia of the marine brown alga Ectocarpus siliculosus. We describe an improved method for the isolation of morphologically intact and infectious virus from diseased plants. We show that treatment of virus particles with high concentrations of CsCl results in a substantial loss of structural proteins. One of the proteins which resists CsCl treatment is glycoprotein-1, the largest of the three viral glycoproteins. We have isolated an EsV genomic fragment with an open reading frame encoding glycoprotein-1. The predicted amino acid sequence is rich in hydrophilic amino acids, but contains hydrophobic regions close to the amino and carboxy termini. A discrepancy between the molecular weight predicted from the coding region and the molecular weight determined by gel electrophoresis suggests that proteolytic processing is required for the maturation of the protein.

Amino Acid Sequence↗

Isolation of a Toxoplasma gondii cyclin by yeast two-hybrid interactive screen.

GAL4-based yeast two-hybrid cDNA libraries from Toxoplasma gondii RH strain were constructed and screened for interactors of a putative T. gondii cdc2-related kinase, TgCRK2. A screen of 3.2 million transformants yielded a single yeast clone that harbored a protein fusion capable of specifically interacting with TgCRK2. Sequencing revealed the cDNA insert (TgCYC1) had homology to the cyclin class of proteins. The TgCYC1 cDNA fragment was used to probe a conventional T. gondii cDNA library and a 2.65 kb cDNA coding for a predicted protein of 582 amino acids was obtained. Based on comparison with a 5'-RACE product from tachyzoite mRNA, the 2.65 kb cDNA for TgCYC1 appeared to be complete. TgCYC1 had the highest similarity to Plasmodium falciparum CYC1 and displayed sequence characteristics that place it in the cyclin H class of eukaryotic cyclins. In synchronous tachyzoite populations the level of TgCYC1 mRNA was unchanged indicating it is not cell cycle regulated at the mRNA level. TgCYC1 rescues the G(1)/S cyclin cell cycle defect in S. cerevisiae strain DL1 demonstrating that this apicomplexan cyclin can function in an established heterologous model system.

Amino Acid Sequence↗

Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain.

A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.

Adenocarcinoma↗

Isolation of a novel human cDNA (rhoHP1) homologous to rho genes.

A novel full-length cDNA showing homology with rho genes was isolated from a human placenta cDNA library. Sequencing of a total of 1086 nucleotides of this clone revealed an open reading frame of 630 nucleotides (210 amino acids). In view of its degree of homology to members of the Rho family of molecules (50-54% identical amino acids, 60-63% identical nucleotides within the coding region), the predicted product was designated RhoHP1(Rho-related protein HP1). Northern analysis indicated that a message about 1.2-kb long is expressed in human heart, placenta, liver, skeletal muscle, and pancreas and, with weaker intensity, in several other tissues.

Amino Acid Sequence↗

Isolation of spermidine synthase gene (spsA) of Dictyostelium discoideum.

The gene encoding spermidine synthase (spsA) was isolated from Dictyostelium discoideum using the technique of insertional mutagenesis. Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 h of development. Sequencing of the genomic DNA and a full-length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids. The sequence is highly conserved, with amino acid identities compared to spermidine synthases of humans, 59.5%, to mouse, 61.3%, and to yeast, 58.1%. A null mutant of the spsA gene is unable to grow in the absence of exogenous spermidine. Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that have abnormally short stalks.

Amino Acid Sequence↗