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PubMed1998

Gene discovery through expressed sequence Tag sequencing in Trypanosoma cruzi.

Analysis of expressed sequence tags (ESTs) constitutes a useful approach for gene identification that, in the case of human pathogens, might result in the identification of new targets for chemotherapy and vaccine development. As part of the Trypanosoma cruzi genome project, we have partially sequenced the 5' ends of 1, 949 clones to generate ESTs. The clones were randomly selected from a normalized CL Brener epimastigote cDNA library. A total of 14.6% of the clones were homologous to previously identified T. cruzi genes, while 18.4% had significant matches to genes from other organisms in the database. A total of 67% of the ESTs had no matches in the database, and thus, some of them might be T. cruzi-specific genes. Functional groups of those sequences with matches in the database were constructed according to their putative biological functions. The two largest categories were protein synthesis (23.3%) and cell surface molecules (10.8%). The information reported in this paper should be useful for researchers in the field to analyze genes and proteins of their own interest.

Animals↗
PubMed1988

DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA: evidence for evolutionary divergence of intradiol dioxygenases by acquisition of DNA sequence repetitions.

The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.

Acinetobacter↗
PubMed1994

Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences.

The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains. The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins. Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E. coli strain (A30). The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF. It was organized in two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin. AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins. No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin. Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common ancestor plasmid. This region contains an integrase-like gene (int). Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate from a recombinant plasmid to the E. coli chromosome. This translocation event occurred via IS1-specific recombination mediated by a recA-independent mechanism.

Adhesins, Escherichia coli↗
PubMed2003

Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination.

Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.

Alleles↗
PubMed1992

U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation.

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

3T3 Cells↗
PubMed1988

Human laminin B2 chain. Comparison of the complete amino acid sequence with the B1 chain reveals variability in sequence homology between different structural domains.

The complete amino acid sequence of the human laminin B2 chain has been determined by sequencing of cDNA clones. The six overlapping clones studied cover approximately 7.5 kilobases of which 5312 nucleotides were sequenced from the 5' end. The open reading frame codes for a 33-residue signal peptide and a 1576-residue B2 chain proper, which is 189 residues less than in the highly homologous B1 chain (Pikkarainen, T., Eddy, R., Fukushima, Y., Byers, M., Shows, T., Pihlajaniemi, T., Saraste, M., and Tryggvason, K. (1987) J. Biol. Chem. 262, 10454-10462). Computer analysis revealed that the B2 chain consists of distinct domains that contain helical structures, cysteine-rich repeats, and globular regions, as does the B1 chain. However, domain alpha and domain beta of the B1 chain have no counterpart in B2, and the number of cysteine-rich repeats is 12, or 1 less than in the B1 chain. The degree of homology between the two chains is highest in the cysteine repeat-containing domains III and V where 40% of the residues match. However, results demonstrate that the B1 and B2 chains of laminin are highly homologous proteins that are probably the products of related genes.

Amino Acid Sequence↗
PubMed1988

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens.

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).

Amino Acid Sequence↗
PubMed1995

The 5' flanking sequences of two S alleles in Lycopersicon peruvianum are highly heterologous but contain short blocks of homologous sequences.

To characterize the molecular structure of the S alleles in Lycopersicon peruvianum, we isolated two genomic fragments that corresponded to cDNAs for S11 and S12 RNases from a genomic library of a plant with the S11S12 genotype. The coding regions and the flanking regions in both fragments were sequenced. A single short intron was found in each gene for RNase, and it was located in the previously identified HV2 region [Chung et al. (1994) Plant Mol. Biol. 26: 757]. The site of initiation of transcription of the gene for S11 RNase was determined by a primer extension analysis and a putative TATA box was identified. Three short sequences that were highly homologous in the two S alleles were found within 360 bp upstream of the ATG initiation codon. It is suggested that these sequences might be involved in the regulation of transcription required for the appropriate-level or tissue-specific expression of the S genes in L. peruvianum.

Alleles↗
PubMed1995

[Sequencing of polyhedrin gene of Leucania separata multiple capsid nuclear polyhedrosis virus by silver sequencing system].

The genome of LsNPV was digested with EcoRV and cloned into pBluescript. With the partial polyhedrin gene of Autographa californica as a probe, positive recombinants were screened by colony in situ hybridization, further characterized by sequencing, and designated as pLsEV and pLsPH5. The intact 741 bp polyhedrin gene of LsNPV is located on the two recombinants revealed by Silver Sequencing System, and shares 80.0% and 97.0% nucleotide identity of the coding sequence, 87.8% and 97.5% amino acid identity with AcMNPV and MbMNPV respectively. Glu is the richest amino acid, and Gln and Trp the poorest amino acid in the polyhedrin of LsNPV. The codons ended with pyrimidine were higher in the codon usage. There was a signal-peptide-like hydrophobic region of 26 amino acids in the N end of polyhedrin.

Amino Acid Sequence↗
PubMed2000

Comparative sequence analysis (CSA): a new sequence-based method for the identification and characterization of mutations in DNA.

Direct sequencing analysis is largely used to confirm and characterize mutations previously detected by more rapid tests. We have developed a method-Comparative Sequence Analysis (CSA)-that simplifies the analysis of sequencing data facilitating its use as a first screen for mutation detection. Sequence data were split into their component electrophoretograms and the use of a size standard enabled equivalent traces from different individuals to be overlaid. This allowed simple and rapid visual analysis of the results. Using this technique in a blind study, we tested 576 samples for mutations in the Von Hippel-Lindau tumor suppressor gene, VHL. We were able to identify and characterize all 78 known mutations present within the sample set (100% sensitivity and specificity).

DNA Mutational Analysis↗
PubMed1992

Differential hydration of homopurine sequences relative to alternating purine/pyrimidine sequences.

The minor groove ligand distamycin A has been used to probe the relative hydration of the minor groove of eight synthetic polynucleotides of known sequence and composition. A combination of densimetric, calorimetric, and temperature-dependent spectroscopic techniques have been used to obtain complete thermodynamic profiles (delta Gzero, delta Hzero, delta Szero, and delta Vzero) for the association of distamycin A to all polymer duplexes. In 10 mM phosphate buffer, pH 7, binding of the drug to each of the polymeric duplexes resulted in characteristic negative changes in both the volume and enthalpy. Although the binding constants were found to be identical for pairs of isomer polynucleotides having identical compositions but different sequences, the values of delta Hzero, delta Szero, and delta Vzero of each such pair were remarkably different. The entropy changes were found to roughly parallel the volume changes; no such trend was seen between delta Hzero and delta Vzero. The data support the hypothesis that the volume changes observed for these systems reflect the coulombic-hydration contribution to the entropy. The heteropolymer duplexes generated much larger exothermic contributions, less favorable entropies and larger volume contractions than did the corresponding homopolymer duplexes of identical composition, and strongly suggest that polynucleotides with homopurine sequences are more hydrated than polynucleotides with alternating purine/pyrimidine sequences. In addition, it was found that duplexes containing guanine sharply reduced the affinity for the drug, also lowering the exothermicity but raising the entropy. This may be explained by the presence of an amino group in the minor groove that prevents hydrogen bonding. Substitution of the guanine with inosine reversed this trend in the thermodynamic properties. Furthermore, substitution of poly(dA) for poly(rA) in a duplex produced a similar reduction in the affinity, while raising the exothermic contribution and greatly reducing the favorable entropy effect in agreement with an apparent increase in the hydration state.

Base Sequence↗
PubMed1992

Light regulatory sequences are located within the 5' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.

Chromosome Deletion↗
PubMed1984

Structural homologies between the amino acid sequence of Clostridium pasteurianum MoFe protein and the DNA sequences of nifD and K genes of phylogenetically diverse bacteria.

The complete amino acid sequence of the larger (alpha-) subunit and about 70% of the total sequence of the smaller (beta-) subunit of the MoFe protein from Clostridium pasteurianum was determined by analyses of peptides derived from BrCN cleavage and by digestions with trypsin, staphylococcal protease and lysylendo-peptidase of the separated subunits. The alpha-subunit has 529 amino acid residues, giving an Mr value of 58 774. This is the first complete sequence for the alpha-subunit of an isolated MoFe protein. In comparing the sequences of both subunits to those from other sources, 5 out of 9 cysteines in the alpha-subunit and 3 out of 6 in the beta-subunit are invariant, thus suggesting a function as ligands to FeS and MoFeS clusters in the MoFe protein. All of these cysteines are located in the amino terminal halves of both subunits.

Amino Acid Sequence↗
PubMed1984

Amino acid sequence of human factor D of the complement system. Similarity in sequence between factor D and proteases of non-plasma origin.

The amino acid sequence of human factor D is proposed from the analysis of the peptides produced by treatment of the factor D with cyanogen bromide, iodosobenzoic acid, trypsin and V-8 protease. Comparison of the proposed sequence with the sequences of other serine esterases indicated that factor D, although it is a plasma serine esterase, is more closely related to certain proteases not found in the plasma than to other plasma serine esterases of the complement system. For example, 36% and 32% identity in amino acid sequence was found on comparison of factor D with elastase and group-specific protease, respectively. Whereas only 27% and 18% identity was observed between factor D and the other complement serine esterases, Clr and factor B, respectively.

Amino Acid Sequence↗
PubMed1986

Energetics of interaction of oligopeptide (lac 53-57) with DNA base sequences and origin of sequence-specific recognition.

Computer model building with a dynamic energy minimization procedure is used here to study the interaction of a pentapeptide sequence from the lac repressor headpiece (lac 53-57) with different base sequences of DNA. The peptide fragment for this purpose was considered in the classical beta-antiparallel as well as the beta-associated conformation. The model of its interaction with DNA was optimised for various binding positions and base sequences. Partitioning of energy is analysed for different dielectric constant values and the main contributing factors to sequence-specific binding are discussed.

Base Sequence↗
PubMed1984

Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast.

We examined the controversial question concerning DNA sequences required for replication in Xenopus eggs. First we used yeast to isolate ARS elements from the Xenopus genome. They show a striking sequence homology with the yeast ARS consensus sequence. The cloning vector and the ARS-containing plasmids replicate equally after injection into Xenopus eggs. Second, we compared a wide range of DNA templates from procaryotes and eucaryotes. All DNA molecules tested replicate as monomeric molecules, and the efficiency is proportional to their size for templates between 4 and 12 kb. Third, we re-examined two reports of replication origins from the Xenopus genome. In both cases, the vector and the recombinant molecules replicate equally under all conditions tested. The apparent lack of sequence specificity for replication in Xenopus eggs does not prevent the injected molecule from being under cellular temporal control of replication. These results are compared with those from yeast.

Animals↗
PubMed1987

Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I.

A DNA sequence (HOT1) from the repeated ribosomal RNA gene cluster of Saccharomyces cerevisiae can stimulate genetic exchange when inserted at novel locations in the yeast genome. Localization of the sequences required for HOT1 activity demonstrates that two noncontiguous fragments of DNA are required for the stimulation of recombination. One of these fragments contains the transcription initiation site for the major 35S ribosomal RNA precursor. The other contains an enhancer of RNA polymerase I transcription. We suggest that transcription by RNA polymerase I initiating in the inserted rDNA and proceeding through the adjacent sequences is responsible for the stimulation of exchange. Consistent with this interpretation, insertion of the putative termination site for RNA polymerase I transcription between HOT1 and the adjacent recombining DNA abolishes the recombination stimulation. Transcription through both copies of the homologous recombining sequences appears to be necessary for enhanced exchange.

Base Sequence↗
PubMed1985

Amino acid sequence studies of human C4b-binding protein: N-terminal sequence analysis and alignment of the fragments produced by limited proteolysis with chymotrypsin and the peptides produced by cyanogen bromide treatment.

Treatment of human C4b-binding protein (C4BP) with cyanogen bromide gave five major peptides and limited proteolysis with chymotrypsin yielded two fragments. The yields, apparent mol. wts and N-terminal amino acid sequences of these peptides and fragments indicates that in dissociating conditions, after reduction of disulphide bonds, C4BP is composed of only one type of polypeptide chain of approx. 70,000 mol. wt. The amino acid sequence data obtained, which accounts for over 55% of the total sequence, allows an alignment of the cyanogen bromide peptides. In addition the amino acid sequence data indicates that the 70,000-dalton polypeptide chain of C4BP contains nine internal homology regions, each 60 amino acids long, which would account for 540 of the expected 600 amino acids in C4BP. Similar internal homology regions are found within the Ba region of factor B [Morley and Campbell, EMBO J. 3, 153-157 (1984)] and it is of interest that the regions found in C4BP are homologous to those found in Ba.

Amino Acid Sequence↗