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p53 mutations in lung cancer following radiation therapy for Hodgkin's disease.

High risks of lung cancer occur after successful treatment of Hodgkin's disease. In addition to tobacco smoking, other risk factors include radiotherapy, chemotherapy, and immunosuppression, although the relative contributions of each are unknown. We conducted p53 mutational spectrum analysis in second lung cancers after radiation therapy for Hodgkin's disease in the Netherlands and in Ontario, Canada. Lung cancer tissues from 11 patients were analyzed by p53 immunohistochemistry and DNA sequence analysis. All were male cigarette smokers, all received radiation therapy, and six also received chemotherapy. The lung cancers occurred 9.8 years (mean) after treatment. Radiation doses to lung lobes that developed the tumors averaged 5.7 Gy (range, 3.7-11.7 Gy). Sequence analysis showed four missense and two silent p53 point mutations in five patients. There were four G:C-->A:T transitions; three of four mutated deoxyguanines occurred on the coding strand, and one was a CpG site. There were two transversions: one G:C-->C:G and one A:T-->C:G. Despite moderate or heavy smoking histories in all patients, the mutational spectrum appears to differ from usual smoking-related lung cancers in which G:C-->T:A transversions predominate. The absence of G:C-->T:A mutations and the prominence of G:C-->A:T transitions, which are characteristic of radiation and oxidative damage, suggest that radiotherapy might have caused some of the p53 mutations. These data illustrate the potential of mutation analysis to determine causes of human cancer. If confirmed in a larger series, these results imply that some radiation-induced cancers can be distinguished from those caused by other factors.

Adult↗

Screening for mutations in BMP4 and FOXC1 genes in congenital anomalies of the kidney and urinary tract in humans.

Recent studies have demonstrated in mice that bone morphogenetic protein 4 (BMP4) and forkhead transcription factor 1 (FOXC1) are involved in the organogenesis of the kidney and urinary tract and that derangement of either gene, BMP4 or FOXC1, leads to development of congenital anomalies of the kidney and urinary tract (CAKUT). In order to determine whether human CAKUT is associated with abnormalities in BMP4 or FOXC1, we established a PCR-based methodology for the DNA sequence analysis of BMP4 and FOXC1 in humans. Our initial screening identified an insertion mutation in FOXC1 with a triplet GGC in three of the seven patients with CAKUT. In the present study, no mutation was detected in the coding sequence of BMP4.

Animals↗

Hemoglobin S/hemoglobin Osler: a case with 3 beta globin chains. DNA sequence (AAT) proves that Hb Osler is beta 145 Tyr-->Asn.

A 13-year-old African-American female with erythrocytosis and three different beta globins on electrophoresis beta A, beta S, and beta Osler, raised the possibility that one chromosome 11 might contain a duplicated beta globin gene, since there are normally only 2 beta globin genes. DNA sequence analysis showed GTG at codon 6 in exon 1, corresponding to Hb S and AAT at codon 145 in exon 3, indicating a substitution of Asn for Tyr. Thus, Hb Osler undergoes spontaneous post-translational deamidation, beta 145 Asn-->beta 145 Asp. Unmodified Hb Osler (Asn) co-migrates with Hb A on electrophoresis and co-elutes with Hb A on HPLC; therefore it has not been identified previously. All previous studies have incorrectly identified the mutation as being beta 145 (HC 2) Tyr-->Asp.

Adolescent↗

Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources.

The aerobic degradation of phenol, chlorobenzene and dichlorobenzene as a sole carbon source has been observed in bacterial Gram-positive strain G2PT isolated from a wastewater bioprocessor. Cells display branching mycelia fragmenting into rod and coccoid elements when grown on TSA. Aerial hyphae formation occurs when grown on phenol and chlorinated aromatics as the sole carbon source. Growth was observed at up to 0.75% phenol as a sole carbon source, indicating a strong tolerance for the compound. The 16S rRNA gene sequence shares the greatest similarity with members of the Rhodococcus genus, with the closest shared nucleotide identity of 98% with the aromatic toxin degrading bacteria Rhodococcus zopfii DSM 44108T. Neighbor-joining and parsimony analysis of Corynebacterineae 16S rRNA gene sequences consistently places strain G2PT in a clade shared with R. zopfii within the Rhodococcus rhodochrous subclade. Based on a unique polyphasic profile involving phenotypic, ribosomal DNA sequence analysis, DNA-DNA hybridization, mol% DNA G+C content and fatty acid composition, G2PT is proposed to represent a previously uncharacterized, novel species in the genus Rhodococcus. The name Rhodococcus phenolicus is proposed for the isolate with the type strain G2PT (= DSM 44812) (= NRRL B-24323) [corrected]

Bioreactors↗

Molecular analysis of a major soluble egg protein in the scleractinian coral Favites chinensis.

Soluble proteins were extracted from eggs of the scleractinian coral Favites chinensis, and analyzed using SDS-polyacrylamide gel electrophoresis. Two major proteins, named FcEP-1 and 2, were detected, and two partial amino acid sequences of FcEP-1 were determined. A cDNA encoding FcEP-1 was identified, using reverse transcription PCR with degenerate oligonucleotide primers designed based on the amino acid sequences, and rapid amplification of cDNA ends. Upon translation of the cDNA, FcEP-1 was predicted to consist of 648 amino acids, and the protein sequence exhibited similarity to vertebrate and invertebrate vitellogenins. FcEP-1 transcripts were already present approximately 6 months before spawning, when the size of oocytes was approximately 1/60 of the mature egg, and could be detected throughout the vitellogenic period. These observations suggest that detection of FcEP-1 transcripts may be useful to monitor the vitellogenic activity in F. chinensis.

Amino Acid Sequence↗

Plant low-molecular-weight phospholipase A2S (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa).

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A2 (PLA2) from developing elm seed endosperm. This represented the first purified and characterized PLA2 from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 1 19 amino acids (PLA2-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA2-II. preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA2. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca(2+)-binding loop and active-site motif, which are characteristic of animal secretory PLA2s. A soluble PLA2s activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA2s. The purified rice PLA2 consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA2-I and PLA2-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA2s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.

Amino Acid Sequence↗

The phylogenetic positions of the conifer genera Amentotaxus Phyllocladus, and Nageia inferred from 18S rRNA sequences.

To determine the evolutionary positions of the conifer genera Amentotaxus, Phyllocladus, and Nageia, we obtained 18S rRNA sequences from 11 new taxa representing the major living orders and families of gymnosperms. With the published Chlamydomonas as an outgroup, phylogenetic analyses of our new data and available sequences indicate that (1) the Gnetales form a monophyletic group, which is an outgroup to the conifers, (2) the conifers are monophyletic, (3) Taxaceae, Cephalotaxaceae, Cupressaceae, and Taxodiaceae form a monophyletic group, (4) Amentotaxus is closer to Torreya than to Cephalotaxus, suggesting that Amentotaxus is better to be classified as a member of Taxaceae, (5) Phyllocladus, Dacrycarpus, Podocarpus, and Nageia form a monophyletic group, and (6) Pinaceae is an out-group to the other families of conifers. Our finding that Phyllocladus is a sister group of the Podocarpaceae disagrees with the suggestion that the phylloclade of the genus is an ancient structure and that the genus is a terminal taxon within the Podocarpaceae. The genus Nageia is more closely related to Podocarpus than to Dacrycarpus and was derived from within the Podocarpaceae. In conclusion, our data indicate that in conifers, the uniovulate cone occurred independently in Taxacaeae and Cephalotaxaceae, and in Podocarpaceae after the three families separated from Pinaceae, and support the hypothesis that the uniovulate cone is derived from reduction of a multiovulate cone.

DNA, Complementary↗

A thrombin inhibitor from the ixodid tick, Amblyomma hebraeum.

A novel thrombin inhibitor named Amblin was identified from the haemolymph of the ixodid (hard) tick, Amblyomma hebraeum, and the coding cDNA was isolated from a tick cDNA library. This cDNA codes for a preprotein of 166 amino acids, including a predicted signal peptide composed of 15 amino acids N-terminal to the mature Amblin. The 151-amino-acid mature Amblin contains 14 cysteines and two Kunitz-like domains. It displays high sequence similarity with a tissue factor pathway inhibitor (TFPI), Ixolaris, from the ixodid tick, Ixodes scapularis, which has 10 cysteines, and a thrombin inhibitor, Boophilin, from the ixodid tick, Boophilus microplus, which has 12 cysteines. Recombinant Amblin specifically inhibited thrombin as efficiently as native Amblin did. This is the first report of a thrombin inhibitor from tick haemolymph.

Amino Acid Sequence↗

An [Fe] hydrogenase from the anaerobic hydrogenosome-containing fungus Neocallimastix frontalis L2.

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic eukaryotes scattered throughout the eukaryotic tree. All of the eukaryotic enzymes characterized so far are iron-only [Fe] hydrogenases. In contrast, it has previously been suggested that hydrogenosomes of the best-studied anaerobic fungus Neocallimastix frontalis L2 contain an unrelated iron-nickel-selenium [NiFeSe] hydrogenase. We have isolated a gene from strain L2 that encodes a putative protein containing all of the characteristic features of an iron-only [Fe] hydrogenase, including the cysteine residues required for the co-ordination of the unique 'hydrogen cluster'. As is the case for experimentally verified hydrogenosomal matrix enzymes from N. frontalis, the [Fe] hydrogenase encodes a plausible amino terminal extension that resembles mitochondrial targeting signals. Phylogenetic analyses of an expanded [Fe] hydrogenase dataset reveal a complicated picture that is difficult to interpret in the light of current ideas of species relationships. Nevertheless, our analyses cannot reject the hypothesis that the novel [Fe] hydrogenase gene of Neocallimastix is specifically related to other eukaryote [Fe] hydrogenases, and thus ultimately might be traced to the same ancestral source.

Amino Acid Sequence↗

New contributions towards the understanding of the phylogenetic relationships among economically important fruit flies (Diptera: Tephritidae).

Fruit flies (Diptera: Tephritidae) are a species-rich and economically important group. The phylogenetic relationships among the many taxa are still to be fully resolved and the monophyly of several groups is still to be confirmed. This paper reports a study of the phylogenetic relationships among 23 economically important tephritid species (representing several major lineages of the family) which examines the sequence of a region of mitochondrial DNA encompassing the cytb, tRNA(Ser) and ND1 genes. Substitutions characteristic of particular taxa were found that could help classify members of the family at any developmental stage. The trees obtained by the maximum parsimony, neighbour joining and maximum likelihood methods were generally compatible with present morphological classification patterns. However, the data reveal some characteristics of the phylogenetic relationships of this family that do not agree with present classifications. The results support the probable non-monophyletic nature of the subfamily Trypetinae and suggest that Bactrocera cucurbitae (Coquillet) is more closely related to the genus Dacus than to other species of Bactrocera.

Animals↗

Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli.

Genetic data have suggested that TolC, AcrA and AcrB constitute a major antibiotic efflux system in Escherichia coli. Through reversion analysis of an unstable and antibiotic-sensitive TolC mutant (TolCP246R,S350C), we isolated extragenic suppressors that mapped within the acrRAB loci. DNA sequence analysis revealed that 18 isolates contained 10 different missense mutations within the acrA gene, whereas a single isolate had a missense mutation within the acrR gene, which codes for the acrAB repressor. Besides reversing the hypersensitivity phenotype of TolCP246R,S350C, AcrA and AcrR alterations elevated the mutant TolC protein level, thus indicating that the mechanism of suppression involves the stabilization of an unstable mutant TolC protein. Eight of the 10 AcrA alterations were clustered in the 202-265 region of the mature protein, whereas the other two suppressors affected residues 30 and 146. Based on the recently solved crystal structure of MexA, an AcrA counterpart from Pseudomonas aeruginosa, the regions encompassing residues 30 and 202-265 constitute the alpha+beta-domain of AcrA (MexA), whereas that of 146 form the alpha-domain. The data suggest that residues of these two AcrA domains either directly or indirectly influence interactions with TolC. Curiously, the stability of three mutant AcrA proteins, bearing an L222Q, L222R or P265R substitution, became dependent on the presence of either wild-type or mutant TolC. This dependence of the mutant AcrA proteins on TolC further supported the notion of a direct physical interaction between these two proteins. Because a mutation in acrR or acrAB expression from a multicopy plasmid also suppressed the TolCP246R,S350C defects, it indicated that wild-type AcrA when produced in high levels presumably establishes similar interactions with the mutant TolC protein as do the suppressor forms of AcrA produced from the chromosomal copy. The AcrA-mediated suppression of mutant TolC phenotypes and the stabilization of mutant TolC protein were dependent on AcrB, reflecting the existence of a functional complex between TolC and AcrAB in vivo.

Anti-Bacterial Agents↗

Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli.

The pbp3 gene encoding PBP3 of Bacillus cereus was cloned and sequenced. For this purpose, PBP3 was first purified from B. cereus ts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereus ts-4 pbp3 gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3 structural gene was ligated into the pET17 x b expression vector and pET-pbp3 was constructed. A protein was produced by the cells of E. coli carrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.

Amino Acid Sequence↗

Characterization of an interleukin-15 like (IL-15L) gene from zebrafish (Danio rerio).

In fish, interleukin (IL)-2, IL-21 and IL-15 genes have recently been identified by in-silico cloning. Fish IL-15 gene is similar to counterparts from mammals and other vertebrates. A zebrafish genomic database-search initiated to find IL-2 and IL-21 genes from zebrafish (Danio rerio) led to the identification of an IL-15 like gene (IL-15L). This gene was cloned by prediction and the transcripts were subsequently cloned by PCR. The predicted translation yielded a 162 amino acid protein with a 42 amino acid-long signal peptide. This protein shared identities of 28.4% to 31.5% with other mammalian and vertebrate IL-2, IL-15 and IL-21 genes. The gene occupies 7.7 kb of the genomic DNA and the coding region spans into four exons and is interrupted by three introns, which is similar to the genomic structure of IL-2 gene. The chromosomal synteny and phylogenetic analyses support our view that this IL-15L gene is specific to teleosts. Furthermore, two alternatively spliced forms have been identified with differential exon usage translating for proteins of 108 and 120 amino acids in length. The analysis of the alternative splicing suggests it may play an important role in regulating the function of this novel gene. Analyses by RT-PCR and in situ hybridization show gene expression in lymphoid tissues like intestine, gills, spleen, pancreas and kidney suggestive of a role in immunity of fish.

Alternative Splicing↗

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.

Alternative Splicing↗

The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase.

The Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells). The L. innocua gene encoding the above protein, termed ferritin from Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5alphaF'. DNA sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein. Primer extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start codon, respectively. Each transcriptional startpoint is preceded by suitably located -10 and -35 elements, which match the sigma(A) (proximal) and sigma(B) (distal) consensus sequences.In L. innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more markedly, under low-iron growth conditions. The effect of iron is apparent in the exponential and stationary phases of growth. An up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several members of the Dps family. The unusual regulation by iron of the Listeria ferritin gene provides further support to the evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of ferritin in the physiology of this bacterium.

Amino Acid Sequence↗

A new soluble 10kDa monoheme cytochrome c-552 from the anammox bacterium Candidatus "Kuenenia stuttgartiensis".

The chemolithoautotrophic anammox bacterium Candidatus "Kuenenia stuttgartiensis" grows anaerobically using ammonium as electron donor for nitrite reduction. More than 10% of the proteins in cell extracts of "K. stuttgartiensis" consist of c-type heme proteins. A 10kDa soluble cytochrome c was purified from cell extracts using ultracentrifugation and anion exchange chromatography. The UV/Vis spectrum of the reduced cytochrome showed the gamma, beta and alpha absorption maxima at 419, 522 and 552nm, respectively. The N-terminal amino acid sequence and peptide fragments of the tryptic digest of the protein were used to identify the corresponding gene. Analysis of the gene product showed that the protein was preceded by a 30 amino acids long leader sequence and that it belonged to the low-spin class ID cytochrome c. The CXXCH motive was located at the N-terminal site of the protein. The gene organization of the cytochrome showed some resemblance to cytochrome c clusters of unknown function in the genome of Nitrosomonas europaea and Geobacter sulfurreducens PCA.

Amino Acid Sequence↗

A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity.

The virB operon of the Agrobacterium tume-faciens pTiA6NC plasmid likely plays a role in directing T-DNA transfer events at the bacterial membrane, as determined previously by mutagenesis and cellular fractionation studies and by DNA sequence analysis of the approximately 12-kilobase-pair operon. The DNA sequence analysis also revealed consensus mononucleotide binding domains in the deduced virB5 and virB11 gene products, suggesting that one or both of these proteins couple energy, by means of nucleotide triphosphate (NTP) hydrolysis, to T-DNA transport. In this report, the product of virB11, an essential virulence gene, was overproduced in Escherichia coli and purified by using immunoaffinity chromatography. The immunoaffinity purified protein, as well as NaDodSO4/polyacrylamide gel-eluted protein, bound and hydrolyzed ATP in the absence of DNA effectors. VirB11 protein also demonstrated in vitro autophosphorylation activity. VirB11 protein was localized primarily to the cytoplasmic membrane by immunoblot analysis of membrane fractions. The deduced VirB11 protein exhibits sequence similarity to comG ORF1, a protein required for uptake of DNA by competent Bacillus subtilis cells. These findings suggest that phosphorylation may serve to activate a component(s) of the A. tumefaciens T-DNA transport apparatus and may also represent a general activation mechanism of other bacterial DNA transport systems.

Adenosine Triphosphatases↗