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At least 1,261 records · Page 70Linked to original sources
PubMed1994

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor.

The protein kinase family can be subdivided into two main groups based on their ability to phosphorylate Ser/Thr or Tyr substrates. In order to understand the basis of this functional difference, we have carried out a comparative analysis of sequence conservation within and between the Ser/Thr and Tyr protein kinases. A multiple sequence alignment of 86 protein kinase sequences was generated. For each position in the alignment we have computed the conservation of residue type in the Ser/Thr, in the Tyr and in both of the kinase subfamilies. To understand the structural and/or functional basis for the conservation, we have mapped these conservation properties onto the backbone of the recently determined structure of the cAMP-dependent Ser/Thr kinase. The results show that the kinase structure can be roughly segregated, based upon conservation, into three zones. The inner zone contains residues highly conserved in all the kinase family and describes the hydrophobic core of the enzyme together with residues essential for substrate and ATP binding and catalysis. The outer zone contains residues highly variable in all kinases and represents the solvent-exposed surface of the protein. The third zone is comprised of residues conserved in either the Ser/Thr or Tyr kinases or in both, but which are not conserved between them. These are sandwiched between the hydrophobic core and the solvent-exposed surface. In addition to analyzing overall conservation in the kinase family, we have also looked at conservation of its substrate and ATP binding sites. The ATP site is highly conserved throughout the kinases, whereas the substrate binding site is more variable. The active site contains several positions which differ between the Ser/Thr and Tyr kinases and may be responsible for discriminating between hydroxyl bearing side chains. Using this information we propose a model for Tyr substrate binding to the catalytic domain of the epidermal growth factor receptor (EGFR).

Amino Acid Sequence↗
PubMed2001

Phylogeny of Bicyclus (Lepidoptera: Nymphalidae) inferred from COI, COII, and EF-1alpha gene sequences.

Despite the fact that Bicyclus anynana has become an important model species for wing-pattern developmental biology and studies of phenotypic plasticity, little is known of the evolutionary history of the genus Bicyclus and the position of B. anynana. Understanding the evolution of development as well as the evolution of plasticity can be attempted in this species-rich genus that displays a large range of wing patterns with variable degrees of phenotypic responses to the environment. A context to guide extrapolations from population genetic studies within B. anynana to those between closely related species has been long overdue. A phylogeny of 54 of the 80 known Bicyclus species is presented based on the combined 3000-bp sequences of two mitochondrial genes, cytochrome oxidase I and II, and the nuclear gene, elongation factor 1alpha. A series of tree topologies, constructed either from the individual genes or from the combined data, using heuristic searches under a variety of weighting schemes were compared under the best maximum-likelihood models fitted for each gene separately. The most likely tree topology to have generated the three data sets was found to be a tree resulting from a combined MP analysis with equal weights. Most phylogenetic signal for the analysis comes from silent substitutions at the third position, and despite the faster rate of evolution and higher levels of homoplasy of the mitochondrial genes relative to the nuclear gene, the latter does not show substantially stronger support for basal clades. Finally, moving branches from the chosen tree topology to other positions on the tree so as to comply better with a previous morphological study did not significantly affect tree length.

Animals↗
PubMed2000

Non-homologous recombination between Alu and LINE-1 repeats caused a 430-kb deletion in the dystrophin gene: a novel source of genomic instability.

Although large deletions in the dystrophin gene have been identified in more than two-thirds of Duchenne and Becker muscular dystrophy patients, the molecular mechanisms that lead to the generation of these deletions are largely unknown. Here, Alu and LINE-1 (L1) repetitive elements were shown to be present at one or other of the two ends, respectively, of a 430-kb deletion in the dystrophin gene. The breakpoint of the deletion, which stretches from exons 2 to 7, was defined more precisely by polymerase chain reaction (PCR) walking on introns 1 and 7. Finally, the region containing the breakpoint was amplified as a fragment of more than 10kb. Sequencing of the deletion endpoint revealed the presence of an Alu sequence in intron 1, 25 kb downstream from the 3' end of exon 1 that was joined directly to an L1 sequence in intron 7, 4.5kb downstream from the 3' end of exon 7. The deletion was calculated to be 430kb. To our knowledge, this is a novel recombination event joining non-homologous Alu and L1 repeats, and is the largest known intrachromosomal deletion that is thought to involve repetitive genetic elements. Sequence characteristics around the breakpoint are discussed.

Alu Elements↗
PubMed2004

Structure-activity relationship of synthetic truncated analogues of vasoactive intestinal peptide (VIP): an enhancement in the activity by a substitution with arginine.

In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.

Amino Acid Sequence↗
PubMed1981

18S defective interfering RNA of Semliki Forest virus contains a triplicated linear repeat.

The nucleotide sequence of a nearly full-length cloned cDNA copy of an 18S defective interfering (DI) RNA of Semliki Forest virus has been determined. This corresponded to a major virus-specific cytoplasmic RNA species at the 11th undiluted passage of the virus in BHK cells. The 1652-nucleotide-long sequence consists of a unique 5'-terminal sequence followed by three tandem 484-nucleotide repeat units derived from the 5' two-thirds of the viral genome and a unique sequence of 106 nucleotides preceding the poly(A) of the 3' terminus. One of the tandem 484-nucleotide repeat units contains an extra segment of 60 nucleotides. Hybridization experiments showed that the cloned cDNA was colinear with an 18S DI RNA and that it contained an approximately 320-nucleotide-long segment colinear with the viral genomic RNA. Analysis of 18S DI RNA oligonucleotide fingerprints revealed that the molecule studied and the heterogeneous DI RNA population contain similar repeated sequences. The mechanism by which the DI RNAs are generated is not known, but it seems likely that multiple internal deletions and duplications are involved.

Base Sequence↗
PubMed1975

N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O, and C.

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.

Amino Acid Sequence↗
PubMed2003

Cytotoxic T cell responses against immunoglobulin in malignant and normal B cells: implications for tumor immunity and autoimmunity.

There is compelling evidence in murine model systems and from human clinical trials that immunotherapy approaches can induce immune responses against tumors. The idiotypic determinants (Id) of the rearranged immunoglobulin proteins of malignant B cells were the first tumor-specific antigens to be recognized and provide a target of an immune mediated response against malignant B cells. Id was thought of as not only tumor-specific but also patient-specific since every patient's malignant B cell clone expresses a unique sequence that is determined by the third complementary determinant regions (CDRIII) of the Id. Although CD4(+) T cell responses against Id have been demonstrated, the role of CD8(+) T lymphocytes in induction of responses against Id has remained elusive. The requirements for peptides to bind to HLA molecules and elicit T cell responses have been studied extensively and are best characterized for HLA class I molecules that elicit CD8+ cytotoxic T lymphocyte (CTL) responses. We applied a bioinformatics approach to the immunoglobulin sequences of malignant and normal B cells. We identified and characterized epitopes recognized by CTLs within these sequences and could generate CTL responses against Id determinants of both normal B cells and malignant B cell. Class I binding immunoglobulin derived sequences had weak binding affinity but were capable in some cases of inducing CTL mediated immune response. More surprisingly, these sequences were not derived from unique sequences within the CDRIII region but were more often from framework region sequences that were often shared among patients and were also found in the immunoglobulin sequences of normal B cells. It was possible to increase binding affinity by single amino acid substitutions at the residues responsible for binding to HLA class I molecules. These "heteroclitic" peptides resulted in generation of T cells with increased ability to kill targets expressing not only the altered peptides, but also the native sequences from which these peptides had been derived. These findings have implications not only for tumor immunity, but also for regulation of B cell dependent autoimmune responses.

Animals↗
PubMed1991

Safety and immunogenicity of a recombinant sporozoite malaria vaccine against Plasmodium vivax.

A recombinant Plasmodium vivax circumsporozoite (CS) antigen representing approximately 70% of the CS protein was expressed in yeast and adsorbed onto aluminum hydroxide for use as a malaria vaccine. In a study of safety and immunogenicity, 30 volunteers were divided into four groups of 5, 5, 10, and 10 individuals, and inoculated intramuscularly with 50, 100, 200, or 400 micrograms of vaccine, respectively. Primary vaccinations were followed by two booster immunizations at six weeks and six months. Overall, the vaccine was well tolerated. Following the third vaccination, one volunteer developed acute hepatitis of uncertain etiology that resolved without sequelae. All volunteers in the 400-micrograms group, and six of 10 in the 200-micrograms group generated IgG against P. vivax CS protein, as determined by Western blot using recombinant CS protein. However, the magnitude of the antibody response measured by indirect immunofluorescence of intact sporozoites or enzyme-linked immunosorbent assay against the recombinant protein was low, and responses could not be boosted. Antigen-driven replication studies using peripheral blood lymphocytes failed to detect proliferative responses specific to peptide sequences represented in the recombinant vaccine, except in one volunteer. Minimal humoral and cell-mediated immune responses developed in most recipients who received this recombinant CS vaccine.

Adult↗
PubMed2001

DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2-acetylaminofluorene adduct.

The NarI sequence represents a strong mutation hot spot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF), a strong chemical carcinogen. Only when bound to the third (underlined) guanine (5'-GGCGCC-->GGCC) can AAF trigger frameshift mutations, suggesting the involvement of a slipped replication intermediate with a two-nucleotide bulge. While base substitutions induced by UV light or abasic sites require DNA polymerase V (Pol V; umuDC), the AAF-induced -2 frameshift pathway requires DNA polymerase II, the polB gene product. Interestingly, error-free bypass of the G-AAF adduct requires Pol V. The genes encoding both Pol II and Pol V are induced by the SOS regulon, a co-ordinated cellular response to environmental stress. A given lesion, G-AAF, can thus be bypassed by two SOS-controlled DNA polymerases (II and V), generating mutagenic (-2 frameshifts) and error-free replication products respectively. Therefore both Pol II and Pol V can compete for the blocked replication intermediate in the vicinity of the lesion and engage in replication by transiently replacing the replicative DNA Pol III. Our data suggest that, in order to cope with the large diversity of existing DNA lesions, cells use a single or a combination of translesional DNA polymerases to achieve translesion synthesis.

2-Acetylaminofluorene↗
PubMed1993

A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant.

The splotch (Sp) mouse mutant displays defects in neural tube closure in the form of exencephaly and spina bifida. Recently, mutations in the Pax-3 gene have been described in the radiation-induced Spr and Sp2H alleles. This led us to examine the integrity of the Pax-3 gene and its cellular mRNA transcript in the original, spontaneously arising Sp allele. A complex mutation in the Pax-3 gene including an A-->T transversion at the invariant 3' AG splice acceptor of intron 3 was identified in the Sp/Sp mutant. This genomic mutation abrogates the normal splicing of intron 3, resulting in the generation of four aberrantly spliced mRNA transcripts. Two of these Pax-3 transcripts make use of cryptic 3' splice sites within the downstream exon, generating small deletions which disrupt the reading frame of the transcripts. A third aberrant splicing event results in the deletion of exon 4, while a fourth retains intron 3. These aberrantly spliced mRNA transcripts are not expected to result in functional Pax-3 proteins and are thus responsible for the phenotype observed in the Sp mouse mutant.

Alternative Splicing↗
PubMed2000

Cloning and cerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms.

We cloned the full length guinea pig H3 receptor cDNA using RT-PCR amplification with primers from the human receptor and templates from brain areas. Evidence was obtained for two isoforms, designated H3L and H3S, differing by a 30 amino acid stretch within the third cytosolic loop, presumably generated by alternative splicing. In situ hybridization using a selective cRNA probe showed the gene transcripts to be highly expressed in discrete neuronal populations, e.g. pyramidal cells in the cerebral cortex or cerebellar Purkinje cells, in some instances already known to express other histamine receptor subtypes.

Amino Acid Sequence↗
PubMed1996

Ectopic expression of the maize homeobox genes ZmHox1a or ZmHox1b causes pleiotropic alterations in the vegetative and floral development of transgenic tobacco.

The ZmHox1a and ZmHox1b (for Zea mays homeobox) genes map on chromosomes 8 and 6, respectively. Both homeobox genes encode proteins that show 91% similarity and are transcribed simultaneously in meristematic and proliferating cells of the maize plant. To gain insight into the biological function of these genes, both open reading frames were expressed in tobacco, under the control of the cauliflower mosaic virus 35S promoter. The resulting transgenic ZmHox1a or ZmHox1b plants showed identical phenotypic alterations that fall into three classes: size reduction, formation of adventitious shoots, and homeotic floral transformations. Approximately 30% of the ZmHox1-expressing plants grew to only one-third of the wild-type size, and most axillary buds gave rise to lateral shoots. Flower abnormalities included formation of petaloid stamens and development of secondary flowers within the primary gynoecium. Therefore, the ectopic expression of the maize ZmHox1 homeobox gene products affects the vegetative as well as the reproductive phase of tobacco plants. All phenotypic alterations were transmitted to the next generation.

Amino Acid Sequence↗
PubMed1981

Analysis of globin gene structure in patients with beta thalassemia by restriction endonuclease mapping.

Twenty-six DNA samples from individuals either heterozygous or homozygous for beta thalassemia were analyzed by restriction endonuclease digestion, agarose gel electrophoresis, and Southern blot analysis to define DNA fragments containing portions or all of the beta globin gene. A total of twenty-seven genes affected by a beta thalassemia mutation and twenty-seven genes affected by a beta thalassemia mutation and twenty-two normal beta globin genes were examined in Italian, Greek, or Asian individuals. With all four restriction endonucleases used, the fragments generated from DNA of thalassemic individuals were identical to those found in DNA from normal. Thus, gross rearrangement or deletion within the genomic region containing the beta globin gene is not characteristic of mutations which cause a thalassemia. A third patient homozygous for pancellular hereditary persistence of fetal hemoglobin was shown to have complete deletion of the delta and beta globin genes.

Base Sequence↗
PubMed1994

Chromosomally encoded cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Proteus vulgaris RO104 strain produces a chromosomally encoded beta-lactamase that confers resistance to various beta-lactam antibiotics including methoxyimino third-generation cephalosporins. The beta-lactamase hydrolyzes first- and second-generation cephalosporins efficiently and cefotaxime to a lesser extent. Catalytic activity is inhibited by low concentrations of clavulanic acid and sulbactam. By its broad-spectrum substrate profile, beta-lactamase of Proteus vulgaris RO104 belongs to the group 2e defined by Bush. The protein purified to homogeneity by a four-step procedure was characterized by a pI of 8.31 and a specific activity of 1200 U/mg. The beta-lactamase was digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino-acid sequence determinations of the resulting peptides allowed the alignment of the 271 amino-acid residues of the protein which did not contain any cysteine residue. From amino-acid sequence comparisons, Proteus vulgaris RO104 beta-lactamase was found to share about 68% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca D488 and E23004. Therefore, the cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Amino Acid Sequence↗
PubMed1999

Familial dilated cardiomyopathy locus maps to chromosome 2q31.

BACKGROUND: Inherited gene defects are an important cause of dilated cardiomyopathy. Although the chromosome locations of some defects and 1 disease gene (actin) have been identified, the genetic etiologies of most cases of familial dilated cardiomyopathy remain unknown. METHODS AND RESULTS: We clinically evaluated 3 generations of a kindred with autosomal dominant transmission of dilated cardiomyopathy. Nine surviving and affected individuals had early-onset disease (ventricular chamber dilation during the teenage years and congestive heart failure during the third decade of life). The disease was nonpenetrant in 2 obligate carriers. To identify the causal gene defect, linkage studies were performed. A new dilated cardiomyopathy locus was identified on chromosome 2 between loci GCG and D2S72 (maximum logarithm of odds [LOD] score=4.86 at theta=0). Because the massive gene encoding titin, a cytoskeletal muscle protein, resides in this disease interval, sequences encoding 900 amino acid residues of the cardiac-specific (N2-B) domain were analyzed. Five sequence variants were identified, but none segregated with disease in this family. CONCLUSIONS: A dilated cardiomyopathy locus (designated CMD1G) is located on chromosome 2q31 and causes early-onset congestive heart failure. Although titin remains an intriguing candidate gene for this disorder, a disease-causing mutation is not present in its cardiac-specific N2-B domain.

Adolescent↗
PubMed2000

Genetic, geographical and temporal variation of porcine reproductive and respiratory syndrome virus in Illinois.

Porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene sequences were generated by RT-PCR from 55 field isolates collected in Illinois and eastern Iowa. Spatial and temporal patterns of genetic variation in the virus were examined on a local geographical scale in order to test the hypothesis that the genetic similarity of PRRSV isolates (measured as their percentage pairwise ORF5 nucleotide similarity) was positively correlated with their geographical proximity. Levels of genetic variability in the Illinois/eastern Iowa PRRSV sample were similar to levels of variability seen across broader geographical regions within North America. The genetic similarity of isolates did not correlate with their geographical distance. These results imply that the movement of PRRSV onto farms does not generally occur via distance-limited processes such as wind or wildlife vectors, but more typically occurs via the long-distance transport of animals or semen. Genetic distances between PRRSV isolates collected from the same farms at different times increased as the time separating the collection events increased. This result implies rapid movement of new genetic types of PRRSV into and out of farms. PRRSV ORF5 displayed a pattern of third-codon-position diversity bias that was not evident in a geographically comparable sample of pseudorabies virus (a swine alphaherpesvirus) gC gene sequences. This result provides evidence that PRRSV ORF5 is experiencing stabilizing selection against structural novelty. Despite high genetic variability at all geographical levels, PRRSV ORF5 nevertheless contained potentially antigenic regions that were invariant at the amino acid level. These regions should make effective vaccine targets if they prove to be immunogenic.

Animals↗
PubMed1994

TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis.

The catalytic properties of six "natural" mutants of the TEM-1 beta-lactamase have been studied in detail, with special emphasis on their activity versus third-generation cephalosporins. On the basis of the recently determined high-resolution structure of the wild-type enzyme, and of the substrates' structures optimized by the AMI quantum chemistry method, we have attempted to explain the influences of the mutations on the substrate profiles of the enzymes. Some of the kinetic results have thus received a satisfactory, semi-quantitative interpretation, especially in the case of single mutations. Analysis of the double mutants proved more hazardous. Extending the comparison to some other class A beta-lactamases showed that similar properties could result from different sequences, supplying an interesting example of convergent evolution within a generally diverging family.

Aztreonam↗
PubMed1991

RNA editing of sorghum mitochondrial atp6 transcripts changes 15 amino acids and generates a carboxy-terminus identical to yeast.

Sequencing of sorghum mitochondrial atp6 cDNA clones revealed 19 C-to-U transcript editing events within a 756 bp-conserved core gene; three were silent and 16 resulted in 15 amino acid changes. Only one edit, which was silent, was found in the 381 bp amino-extension to the core gene. Eleven of the 15 changed amino acids were identical with or else represented conservative changes compared to yeast atp6. Editing of a CAA codon to TAA truncates the carboxy-terminus to a position identical to that of yeast. The frequency of editing at sites which change amino acids was very high in contrast to partial editing at silent, third base, sites.

Base Sequence↗