SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
We report here the homology of different genes with an 18-nucleotide sequence element derived from a conserved region of the homoeo boxes. Possible evolutionary relationships are discussed.
The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40phox. While the first two factors are indispensable for the activity, p40phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40phox revealed that it plays a critical role in the binding to p67phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.
Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.
Explore the source record for details and available documents.
A unique sequence pattern, designated the GD/GNH signature, was shown to be conserved in a wide variety of phosphoesterases. The enzymes containing this signature cleave phosphoester bonds in such different substrates as (1) phosphoserine and phosphothreonine in polypeptides; (2) bis(5'-nucleosidyl)-tetraphosphates; (3) nucleoside 5' phosphates; (4) 2',3'-cyclic nucleotide phosphates; (5) polynucleotides; (6) 2'-5' phosphodiesters in RNA (intron) lariats; (7) sphingomyelin; and (7) various phosphomonoesters. Two conserved acidic amino acid residues and a conserved histidine residue may be directly involved in phosphoester bond cleavage.
Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).
Previous loss-of-heterozygosity studies in endometrial carcinoma mapped a putative tumor suppressor gene to 10q25.3-26.1. An analysis of genomic sequences for the deletion interval showed several expressed sequence tags and the homeodomain gene EMX2, a homologue of Drosophila melanogaster empty spiracles. Expression studies showed that EMX2 transcripts are abundant in the adult uterus and that message levels seem to be inversely correlated with endometrial proliferation. EMX2 RNA was more abundant in quiescent postmenopausal endometrium than in premenopausal endometrium. We found decreased EMX2 expression in a subset of primary endometrial tumors, and four of six endometrial cancer cell lines investigated failed to express EMX2. The predicted protein showed extensive amino acid conservation with EMX2 sequences from several vertebrates. There was also considerable evolutionary conservation in the 3' untranslated region. To examine the potential function of EMX2 in endometrial tumorigenesis, we investigated 20 primary tumors and 6 endometrial cancer cell lines for mutations. Two primary tumors had mutations. Inactivation or reduced expression of EMX2 in cancers, coupled with increased expression in the quiescent endometrium, indicate that this homeodomain gene is involved in maintenance of the differentiated state.
The guanine nucleotide-exchange factor eIF2B mediates the exchange of GDP bound to translation initiation factor eIF2 for GTP. This exchange process is a key regulatory step for the control of translation initiation in eukaryotic organisms. To improve our understanding of the structure, function and regulation of eIF2B, we have obtained and sequenced cDNA species encoding all of its five subunits. Here we report the sequences of eIF2B beta and delta from rat. This paper focuses on sequence similarities between the alpha, beta and delta subunits of mammalian eIF2B. Earlier work showed that the amino acid sequences of the corresponding subunits of eIF2B in the yeast Saccharomyces cerevisiae (GCN3, GCD7 and GCD2) exhibit considerable similarity. We demonstrate that this is also true for the mammalian subunits. Moreover, alignment of the eIF2B alpha, beta and delta sequences from mammals and yeast, along with the sequence of the putative eIF2B alpha subunit from Caenorhabditis elegans and eIF2B delta from Schizosaccharomyces pombe shows that a large number of residues are identical or conserved between the C-terminal regions of all these sequences. This strong sequence conservation points to the likely functional importance of these residues. The implications of this are discussed in the light of results concerning the functions of the subunits of eIF2B in yeast and mammals. Our results also indicate that the large apparent differences in mobility on SDS/PAGE between eIF2B beta and delta subunits from rat and rabbit are not due to differences in their lengths but reflect differences in amino acid composition. We have also examined the relative expression of mRNA species encoding the alpha, beta, delta and epsilon subunits of eIF2B in a range of rat tissues by Northern blot analysis. As might be expected for mRNA species encoding subunits of a heterotrimeric protein, the ratios of expression levels of these subunits to one another did not vary between the different rat tissues examined (with the possible exception of liver). This represents the first analysis of the levels of expression of mRNA species encoding the different subunits of eIF2B.
Olfactory receptor-like proteins (ORLs) are seven transmembrane domain G protein-coupled receptors. We hypothesize that, in contrast with the hypervariable regions that may interact with a variety of odor ligands, the external and internal segments of the ORLs contain conserved regions that may interact with conserved olfactory binding proteins or direct axon guidance, and with G proteins, respectively. To test this hypothesis, a comprehensive analysis using the multiple expectation maximization for motif elicitation discovery tool was performed in all the full-length ORL clones deposited in the public section of the olfactory receptor database. Ten motifs have been identified that are present in all the olfactory receptors, in the same order, and are not present in other G protein-coupled receptors. These motifs are concentrated either in the extracellular-most or the intracellular-most parts of the receptors. The generality of these motifs was verified by their existence in the partial ORLs and 28 newly identified human receptors. The existence and localization of these motifs, suggest that they may be involved in the interactions of the receptors with their upstream and downstream signaling partners. In addition, the motifs present an additional to overall homology criterion for ORL family definition.
A Polymerase chain reaction was developed to amplify the entire open reading frame of flaB, the gene encoding the endoflagellin subunit protein. The 852 bp amplified products from 23 serovars of the genus Leptospira were subjected to restriction endonuclease analysis and the profiles correlated well with phylogenetic relationships between these serovars. The flaB deoxynucleotide sequences of L. hardjo-bovis, L. hardjo-prajitno and L. grippotyphosa were determined. The deduced primary amino acid sequences of each were highly conserved with only three amino acid residue differences observed. The deoxynucleotide sequences showed genetic drift with alternative bases in the third position of codons. The PCR product derived by amplification of flaB from L. grippotyphosa was cloned into the expression vector pGEX-2T and a recombinant FlaB fusion protein made. As predicted from the deduced amino acid sequences, the recombinant FlaB cross-reacted with heterologous antiserum derived from a rabbit infected with L. hardjo-bovis.
The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the alpha and beta subunits of the ATP synthase. Various mutations were introduced in the alpha Lys-175 and alpha Thr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169-176) of the Escherichia coli ATP synthase alpha subunit. Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme. The entire enzyme assembly was lost by alpha Lys-175-->Phe (or Trp) or alpha Thr-176-->Phe (or Tyr) mutation. Other mutants had similar (alpha His-175, alpha Ser-175, alpha Gly-175, alpha Ser-176, and alpha His-176 mutants) or lower (alpha Ala-176, alpha Cys-176, alpha Leu-176, and alpha Val-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the alpha Ser-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as alpha Gly-175 and alpha His-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the alpha Lys-175 and alpha Thr-176 residues are not absolutely essential for catalysis, but that they, or possibly the entire conserved sequence, are located in the key domain for the subunit-subunit interactions essential for enzyme stability and steady state activity.(ABSTRACT TRUNCATED AT 250 WORDS)
The antigen-specific receptors of T and B lymphocytes are distinct, though structurally related, molecules. During development, lymphoid cells assemble functional variable (V) region genes for each receptor chain from separate multimember gene families by somatic DNA rearrangements of individual germ-line segments. Transcription may play a role in regulating the tissue and stage specificity of these rearrangements by controlling the accessibility of germ-line loci to the recombinational machinery. Immunoglobulin V-region genes are transcribed from tissue-specific promoters that have been well characterized. We report here the characterization of 14 T-cell receptor beta-chain V-region gene promoters. Sequence analysis indicates that these promoters do not contain the conserved octamer that is located upstream of all immunoglobulin genes. However, a unique decanucleotide sequence, not present in immunoglobulin genes, is conserved in the promoter region of murine and human V beta genes. We identify this sequence as a potential regulatory element, based on its position, conservation, and sequence homology to sites known to bind transcription-activating factors. The possibility that the distinct structures of immunoglobulin and T-cell receptor gene promoters may contribute to the tissue-specific rearrangement and expression of receptor gene families is discussed.
Eleven of thirteen infectious bursal disease viruses (IBDV) were shown to be similar following polymerase chain reaction (PCR) amplification of two regions (293 bp and 651 bp cDNA fragments) in genomic segment B and restriction enzyme analysis of the PCR products. Two IBDV strains differed from the eleven viruses when examined by BstEII digestion of the 293 bp PCR product, but all thirteen viruses were similar using the 651 bp cDNA fragment and digestion with AvaI. Because segment B encodes replicating enzyme(s), the results suggest that its sequences may be highly conserved among IBDV isolates.
Exon 18 of the muscle myosin heavy chain gene (Mhc) of Drosophila melanogaster is excluded from larval transcripts but included in most adult transcripts. To identify cis-acting elements regulating this alternative RNA splicing, we sequenced the 3' end of Mhc from the distantly related species D. virilis. Three noncoding regions are conserved: (1) the nonconsensus splice junctions at either end of exon 18; (2) exon 18 itself; and (3) a 30-nucleotide, pyrimidine-rich sequence located about 40 nt upstream of the 3' splice site of exon 18. We generated transgenic flies expressing Mhc mini-genes designed to test the function of these regions. Improvement of both splice sites of adult-specific exon 18 toward the consensus sequence switches the splicing pattern to include exon 18 in all larval transcripts. Thus nonconsensus splice junctions are critical to stage-specific exclusion of this exon. Deletion of nearly all of exon 18 does not affect stage-specific utilization. However, splicing of transcripts lacking the conserved pyrimidine sequence is severely disrupted in adults. Disruption is not rescued by insertion of a different polypyrimidine tract, suggesting that the conserved pyrimidine-rich sequence interacts with tissue-specific splicing factors to activate utilization of the poor splice sites of exon 18 in adult muscle.
Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes. We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes. We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT. Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures. We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA.
Transcriptional regulation of vertebrate Hox genes involves enhancer sequences located either inside or outside the gene clusters. In the mouse Hoxd complex, for example, series of contiguous genes are coordinately controlled by regulatory sequences located at remote distances. However, in different cellular contexts, Hox genes may have to be insulated from undesirable external regulatory influences to prevent ectopic gene activation, a situation that would likely be detrimental to the developing embryo. We show the presence of an insulator activity, at one extremity of the Hoxd complex, that is composed of at least two distinct DNA elements, one of which is conserved throughout vertebrate species. However, deletion of this element on its own did not detectably affect Hoxd gene expression, unless another DNA fragment located nearby was removed in cis. These results suggest that insulation of this important gene cluster relies, at least in part, upon a sequence-specific mechanism that displays some redundancy.
Computer-assisted analysis of amino acid sequences using methods for database screening with individual sequences and with multiple alignment blocks reveals a complex multidomain organization of yeast proteins GCD6 and GCD1, and mammalian homolog of GCD6-subunits of the eukaryotic translation initiation factor eIF-2B involved in GDP/GTP exchange on eIF-2. It is shown that these proteins contain a putative nucleotide-binding domain related to a variety of nucleotidyltransferases, most of which are involved in nucleoside diphosphate-sugar formation in bacteria. Three conserved motifs, one of which appears to be a variant of the phosphate-binding site (P-loop) and another that may be considered a specific version of the Mg(2+)-binding site of NTP-utilizing enzymes, were identified in the nucleotidyltransferase-related domain. Together with the third unique motif adjacent to the the P-loop, these motifs comprise the signature of a new superfamily of nucleotide-binding domains. A domain consisting of hexapeptide amino acid repeats with a periodic distribution of bulky hydrophobic residues (isoleucine patch), which previously have been identified in bacterial acetyltransferases, is located toward the C-terminus from the nucleotidyltransferase-related domain. Finally, at the very C-termini of GCD6, eIF-2B epsilon, and two other eukaryotic translation initiation factors, eIF-4 gamma and eIF-5, there is a previously undetected, conserved domain. It is hypothesized that the nucleotidyltransferase-related domain is directly involved in the GDP/GTP exchange, whereas the C-terminal conserved domain may be involved in the interaction of eIF-2B, eIF-4 gamma, and eIF-5 with eIF-2.
We have identified a novel Drosophila protein, DBP80, that exhibits significant similarity to mouse mDEAD5, yeast TIF1/2, and mammalian eIF-4A. DBP80 is a member of a subclass of DEAD-box proteins that contains a distinct domain, PX(I/R)ILLKR(E/D)EETLEGIKQ(F/Y)(F/Y), in addition to the seven canonical helicase domains.