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PubMed1999

The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.

Targeted mutagenesis was used to produce two mutations in the murine hemochromatosis gene (Hfe) locus. The first mutation deletes a large portion of the coding sequence, generating a null allele. The second mutation introduces a missense mutation (C282Y) into the Hfe locus, but otherwise leaves the gene intact. This mutation is identical to the disease-causing mutation in patients with hereditary hemochromatosis. Mice carrying each of the two mutations were bred and analyzed. Homozygosity for either mutation results in postnatal iron loading. The effects of the null mutation are more severe than the effects of the C282Y mutation. Mice heterozygous for either mutation accumulate more iron than normal controls. Interestingly, although liver iron stores are greatly increased, splenic iron is decreased. We conclude that the C282Y mutation does not result in a null allele.

Alleles↗

PubMed2000

Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver.

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previously described in mammalian tissues. We report here the biochemical characterization of NTPDases in the pig liver. Optimum pH of catalysis is more acidic for this enzyme than for NTPDases (neutral or alkaline pH) found in other mammalian tissues. It is less sensitive to bile salts than the bovine spleen NTPDase. Calculated Km values for ATP and ADP (31 and 21 microM, respectively) are slightly higher than those reported for the latter enzyme. Electrophoretograms of these enzymes also show different migration patterns. Western blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimated molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A second antibody, generated against a different sequence of NTPDase I, does not recognize the liver enzyme, thereby indicating some differences in primary structure. Immunolocalization produced a strong signal on hepatocytes, epithelial cells of the bile duct system, and vascular cells. Immunoreactivity was variable among hepatocytes of different lobules and among hepatocytes within a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein.

Acid Anhydride Hydrolases↗

PubMed1990

Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation.

Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.

Amino Acid Sequence↗

PubMed1999

Cutis laxa arising from frameshift mutations in exon 30 of the elastin gene (ELN).

Congenital cutis laxa, a rare syndrome with marked skin laxity and pulmonary and cardiovascular compromise, is due to defective elastic fiber formation. In several cases, skin fibroblast tropoelastin production is markedly reduced yet reversed in vitro by transforming growth factor-beta treatment. We previously showed that this reversal was due to elastin mRNA stabilization in one cell strain, and here this behavior was confirmed in skin fibroblasts from two generations of a second family. cDNA sequencing and heteroduplex analysis of elastin gene transcripts from three fibroblast strains in two kindreds now identify two frameshift mutations (2012DeltaG and 2039DeltaC) in elastin gene exon 30, thus leading to missense C termini. No other mutations were present in the ELN cDNA sequences of all three affected individuals. Transcripts from both alleles in each kindred were unstable and responsive to transforming growth factor-beta. Exons 22, 23, 26A, and 32 were always absent. Since exon 30 underwent alternative splicing in fibroblasts, we speculate that a differential splicing pattern could conceivably lead to phenotypic rescue. These two dominant-acting, apparently de novo mutations in the elastin gene appear to be responsible for qualitative and quantitative defects in elastin, resulting in the cutis laxa phenotype.

Alleles↗

PubMed2001

Database verification studies of SWISS-PROT and GenBank.

PROBLEM STATEMENT: We have studied the relationships among SWISS-PROT, TrEMBL, and GenBank with two goals. First is to determine whether users can reliably identify those proteins in SWISS-PROT whose functions were determined experimentally, as opposed to proteins whose functions were predicted computationally. If this information was present in reasonable quantities, it would allow researchers to decrease the propagation of incorrect function predictions during sequence annotation, and to assemble training sets for developing the next generation of sequence-analysis algorithms. Second is to assess the consistency between translated GenBank sequences and sequences in SWISS-PROT and TrEMBL. RESULTS: (1) Contrary to claims by the SWISS-PROT authors, we conclude that SWISS-PROT does not identify a significant number of experimentally characterized proteins. (2) SWISS-PROT is more incomplete than we expected in that version 38.0 from July 1999 lacks many proteins from the full genomes of important organisms that were sequenced years earlier. (3) Even if we combine SWISS-PROT and TrEMBL, some sequences from the full genomes are missing from the combined dataset. (4) In many cases, translated GenBank genes do not exactly match the corresponding SWISS-PROT sequences, for reasons that include missing or removed methionines, differing translation start positions, individual amino-acid differences, and inclusion of sequence data from multiple sequencing projects. For example, results show that for Escherichia coli, 80.6% of the proteins in the GenBank entry for the complete genome have identical sequence matches with SWISS-PROT/TrEMBL sequences, 13.4% have exact substring matches, and matches for 4.1% can be found using BLAST search; the remaining 2.0% of E.coli protein sequences (most of which are ORFs) have no clear matches to SWISS-PROT/TrEMBL. Although many of these differences can be explained by the complexity of the DB, and by the curation processes used to create it, the scale of the differences is notable.

Algorithms↗

PubMed1997

Use of a hammerhead ribozyme with cationic liposomes to reduce leukocyte type 12-lipoxygenase expression in vascular smooth muscle.

Chemically synthesized hammerhead-type ribozymes targeted against the porcine leukocyte-type 12-lipoxygenase (LO) have been developed and studied. One chimeric ribozyme consists of DNA in the non-enzymatic portions, and RNA in the enzymatic core as well as two phosphorothioate internucleotide linkages at 3' terminus. The second ribozyme consists of ribonucleotide sequences generated by in vitro transcription. In this chapter we describe methodologies to first analyze the ribozyme catalytic activity in vitro by studying cleavage of target RNA in vitro. The subsequent sections will describe how to target the catalytic ribozyme and deliver it to porcine vascular smooth muscle cells (PVSMC) by a liposome-mediated method. Finally ways to evaluate its activity to inhibit expression of the 12-LO mRNA will be presented. These results demonstrate the feasibility of using ribozymes as novel candidates for therapeutic agents to block specific gene expression in vascular cells.

Animals↗

PubMed2001

An algorithm for statistical alignment of sequences related by a binary tree.

An algorithm is presented that allows the calculation of the probability of a set of sequences related by a binary tree that has evolved according to the Thorne-Kishino-Felsenstein model (1991) for a fixed set of parameters. There are two ideas underlying this algorithm. Firstly, a markov chain is defined that generates ancestral sequences and their alignment at two neighboring nodes in a tree. Secondly, a stochastic walk on the binary tree, that defines a markov chain generating ancestral sequences and their alignment at the internal nodes in the tree is described. The running time of this algorithm is O(l2 kappa), where l is the geometric average of the sequence lengths and kappa the number of sequences--leaves at the binary tree. This could be improved to O(l kappa).

Algorithms↗

PubMedDate not supplied

Transposition of Tn554 does not generate a target duplication.

Transposable elements from prokaryotic and eukaryotic organisms are discrete DNA segments bounded by inverted or directly repeated sequences that insert into non-homologous DNA in a reaction that is independent of the general recombination functions of the host. The mechanisms proposed generally involve a staggered double-stranded scission of the target DNA, ligation to the nicked ends of the transposable element, and replication of the element, resulting in the generation of a directly repeated oligonucleotide target sequence flanking the new copy of the element. Most transposons have a relatively low degree of target site specificity coupled with a low insertion frequency. Tn554, a Staphylococcus aureus transposon which specifies resistances to erythromycin and spectinomycin, displays an unusually high degree of insertion specificity. Tn554 transposes with high efficiency to a unique ('primary') site in the S. aureus chromosome and only rarely (less than 10(-6) per transductant) to other, secondary sites. We report here the nucleotide sequences surrounding the junctions of Tn554 in three independent 'primary' insertions and two 'secondary' insertions of the transposon. Two unusual features are revealed: first, the termini of Tn554 contain neither inverted nor directly repeated sequences. Second, transposition of Tn554 does not generate the short direct repeats of the target DNA that are characteristic of other transposable elements. These results suggest that the mechanism of Tn554 insertion may be significantly different from that of other transposons.

Base Sequence↗

PubMed1996

An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA.

The export of Escherichia coli hemolysin across the cytoplasmic and the outer membranes requires the COOH-terminal signal sequence of HlyA, the two specific translocator proteins HlyB and HlyD, and the outer membrane protein TolC. We have developed an export cloning system that is composed of two vectors: one in which the fusion of the desired gene with the 3'-end of hlyA is generated, and a second in which the sequences containing the fusion are combined with the accessory genes hlyB and hlyD, thereby reconstructing the natural organization of the hly locus. In the second vector the fusion and the accessory genes are flanked by Notl sites, allowing subcloning of the whole cluster into a variety of minitransposons to achieve the stable integration of the constructs into the chromosome of Gram-negative bacteria. Since some applications may require the production of transcriptional fusions, an alternative version of the system provides the efficient translation initiation region of T7 phage gene 10 upstream of the fusion protein coding sequence. The usefulness of the system was assessed by constructing a fusion between the gene encoding the B subunit of Shiga-like toxin lle and the 3'-end of hlyA. An attenuated Salmonella typhimurium vaccine strain harboring the resulting construct, either in multicopy or monocopy, efficiently expressed and exported the chimeric protein. We anticipate that this system will lead to a higher stability of the engineered function and permit a faithful monitoring of the export of the recombinant peptide under physiologic single-copy conditions.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2001

Selection of enantioselective acyl transfer catalysts from a pooled peptide library through a fluorescence-based activity assay: an approach to kinetic resolution of secondary alcohols of broad structural scope.

An assay employing a fluorescently labeled split and pool peptide library has been applied to the discovery of a new class of octapeptide catalysts for the kinetic resolution of secondary alcohols. A highly diverse library of peptide-based catalysts was synthesized on solid-phase synthesis beads such that each individual bead was co-functionalized with (i) a uniform loading of a pH-sensitive fluorophore and (ii) a unique peptide-based catalyst. The library was then screened for activity in acylation reactions employing (+/-)-sec-phenylethanol as the substrate and acetic anhydride as the acylation agent. From the most active catalysts, a lead peptide (4) was identified that provides a selectivity-factor (k(rel)) of 8.2 upon resynthesis and evaluation under homogeneous conditions. A "directed" second-generation split and pool peptide library was synthesized such that the new peptide sequences in the library were biased toward the lead structure. Random samples of the second generation library were screened in single bead assays that revealed several new peptide-based catalysts that afford improved selectivities in kinetic resolutions. Peptide catalyst 13 proves effective for the kinetic resolution of sec-phenylethanol (k(rel) = 20), as well as eight other secondary alcohols of a broad substrate scope (k(rel) = 4 to >50).

Acylation↗

PubMed1999

A method for computational combinatorial peptide design of inhibitors of Ras protein.

A computational combinatorial approach is proposed for the design of a peptide inhibitor of Ras protein. The procedure involves three steps. First, a 'Multiple Copy Simultaneous Search' identifies the location of specific functional groups on the Ras surface. This search method allowed us to identify an important binding surface consisting of two beta strands (residues 5-8 and 52-56), in addition to the well known Ras effector loop and switch II region. The two beta strands had not previously been reported to be involved in Ras-Raf interaction. Second, after constructing the peptide inhibitor chain based on the location of N-methylacetamide (NMA) minima, functional groups are selected and connected to the main chain Calpha atom. This step generates a number of possible peptides with different sequences on the Ras surface. Third, potential inhibitors are designed based on a sequence alignment of the peptides generated in the second step. This computational approach reproduces the conserved pattern of hydrophobic, hydrophilic and charged amino acids identified from the Ras effectors. The advantages and limitations of this approach are discussed.

Acetamides↗

PubMed1997

Cephalosporin substrate specificity determinants of TEM-1 beta-lactamase.

beta-lactamase is a bacterial enzyme that catalyzes the hydrolysis of beta-lactam antibiotics such as penicillins and cephalosporins. TEM-1 beta-lactamase is a prevalent beta-lactamase found in Gram-negative bacteria and is capable of hydrolyzing both penicillins and cephalosporins, except for the extended-spectrum cephalosporins. To identify the sequence determinants in the active site for a given antibiotic substrate, random libraries were constructed that each contain all possible amino acid combinations for the designated region of TEM-1 beta-lactamase. To establish the determinants of substrate specificity for cephalosporins versus those for penicillins, these active site libraries have been screened for mutants with high levels of activity for the second generation cephalosporin cephaloridine. Based on the sequence results, substitutions of W165S, A237T, and E240C were identified as cephalosporin-specific. Kinetic analysis of these mutants was done to determine whether each is capable of distinguishing between the two classes of antibiotics. Both the A237T and E240C substitutions, alone or in combination, exhibited increased cephalosporinase activity and decreased penicillinase activity relative to the wild-type enzyme. A sequence comparison between functional mutants selected for cephaloridine hydrolytic activity and functional mutants previously selected for ampicillin hydrolytic activity suggests that TEM-1 beta-lactamase has greater restrictions in maintaining cephalosporinase activity versus maintaining penicillinase activity.

Amino Acid Sequence↗

PubMed1992

Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.

Amino Acid Sequence↗

PubMed2005

DNA recognition and recognition control of alpha-peptide ribonucleic acids by external factors.

Peptide ribonucleic acid (PRNA) is one of artificial nucleic acids, which enables us to control its nucleobase orientation and recognition behavior by external factors through the synergetic effects of reversible borate ester formation and intramolecular hydrogen bond formation. In this study, a series of novel alpha-PRNA oligomers, possessing alternative alpha-PRNA/basic amino acid sequences, were newly designed, synthesized, and evaluated as the second-generation PRNA. As expected, these alpha-PRNAs indeed formed highly stable sequence-specific complexes with the complementary DNAs, for which both the conventional hydrogen-bonding interactions between the complementary nucleobase pairs and the electrostatic interactions between the basic amino acid's ammonium cation and the DNA's phosphate anion on the backbone are jointly responsible.

Buffers↗

PubMed2002

Backbone modification alters the efficacy of antisense oligonucleotides directed against mRNA encoding either TGF-alpha or EGFR in the treatment of prostate cancer cell lines.

Antisense oligonucleotides (oligos) directed against mRNA-encoding, transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGFR), have been shown to significantly inhibit in vitro and in vivo growth of prostate tumor models. Recently, second generation oligos have been employed with identical base sequences, but containing backbome modifications that enhance stability, solubility and circulatory patterns. Using relatively low concentrations of oligos, we compared the efficacy of the first generation phosphorothioated oligos against TGF-alpha (MR1) and EGFR (MR2) with second generation oligos containing completely phosphorothioated backbones and different patterns of 2'-methoxyethyl (2'-MOE) backbone modifications, while retaining the original designated base sequence using, the LNCaP and PC-3 prostate cancer cell lines, respectively. All experiments were conducted in vitro with lipofectin to enhance oligo entry. Under these conditions, using oligo concentrations between 0.83 and 3.32 microM for LNCaP cells treated with oligos directed against TGF-alpha only the first generation MR1 had inhibitory activity. When treated with oligos directed against EGFR, none of the oligos had inhibitory activity and they behaved similarly. Using the PC-3 cell line and treatment directed against TGF-alpha with oligo concentrations between 0.42 and 3.32 microM, first generation MR1 and second generation 5005 behaved similarly with no notable effect, while second generation 5007 produced dramatic growth stimulation. When PC-3 cells were treated with oligos directed against EGFR, second generation 5006 and 5008 had similar and apparently dose-dependent inhibition. We conclude that backbone modifications influence oligo efficacy and may result in either enhanced or diminished activity. Because of their activity against the hormone insensitive PC-3 cells, the 5006 and 5008 compounds warrant additional study at greater concentrations and also merit in vivo testing.

ErbB Receptors↗

PubMed2000

Synthesis of the C29-C44 portion of spongistatin 1 (altohyrtin A).

Two synthetic approaches to the C29-C44 portion of spongistatin 1 (altohyrtin A) have been developed. The key step of the first approach relies on the Claisen rearrangement of glucal 18 to provide ester 20a. This intermediate was advanced to silyl enol ether 30, which was coupled under Mukaiyama aldol conditions with aldehyde 3. Cyclization of this aldol adduct completed our first synthesis of the C29-C44 portion of spongistatin 1, requiring 25 total steps and occurring in 2.4% yield over the longest linear sequence (21 steps). We have also developed a second-generation approach based on the C-glycosidation of glucal 43. Through equilibration of the corresponding C-glycosides 49a/b and 50a/b the desired C-glycoside (50a) was obtained in good yield. Aldol condensation of this ketone provided cyclization precursor 67, which undergoes acid-catalyzed ketalization to close the E-ring of the spongistatins. An oxidation/reduction protocol was employed to set the C37 stereocenter. Protection of the C37 carbonol and selective unmasking of the C44 carbonol completed our second generation synthesis. This approach requires 27 steps and occurred in 13.2% yield over the longest linear sequence (18 steps).

Antineoplastic Agents↗

PubMed2006

Incrimination of Anopheles (Nyssorhynchus) rangeli and An. (Nys.) oswaldoi as natural vectors of Plasmodium vivax in Southern Colombia.

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

Animals↗

PubMed1991

Automated amplification and sequencing of human mitochondrial DNA.

Part of the human mitochondrial D-loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M13-21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single-stranded DNA was generated. This was then sequenced directly by the diodeoxy chain termination method using dye-labelled universal sequencing primers in conjunction with a fluorescence-based DNA sequencer. This enabled a 403-base-pair hypervariable segment of the D-loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.

Amino Acid Sequence↗