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PubMed2001

Genomic structure of the gene for mouse germ-cell nuclear factor (GCNF). II. Comparison with the genomic structure of the human GCNF gene.

BACKGROUND: Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus. RESULTS: The genomic structures of the mouse and human GCNF genes are highly conserved. The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon. Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon. CONCLUSION: By homology with the mouse gene, 11 GCNF coding exons can be defined on human chromosome 9. All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon. Although the genomic sequence is highly conserved, the analysis suggests that alternative splicing generates a higher complexity of human GCNF isoforms compared with the situation in the mouse.

Alternative Splicing↗
PubMed1997

Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV.

Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Ambler's class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

Amino Acid Sequence↗
PubMed1988

Molecular studies of a rare DR2/LD-5a/DQw3 HLA class II haplotype. Multiple genetic mechanisms in the generation of polymorphic HLA class II genes.

A cDNA library was constructed from a homozygous B lymphoblastoid cell line (REM) obtained from an individual of a long isolated American Indian tribe, the Warao. The REM cell line expresses serologically defined determinants, DR2 and DQw3, and the T lymphocyte-defined (Dw/LD) specificity, LD-5a. T cells can recognize differences between FJO (a DR2/DQw1 cell line that expresses the Dw specificity MN2) and REM for both DR and DQ molecules. cNDA clones encoding the polymorphic DR beta 1-, DR beta 2-, DQ beta-, and DQ alpha-chains were sequenced and compared with other DR and DQ gene sequences. The DR beta 1-sequence of REM is identical to the DR beta 1-sequence of FJO; the DR beta 2-sequence is also identical to that of FJO except for one amino acid difference at position 67 in the polymorphic first domain (Leu in REM, Phe in FJO) due to a single point mutation. The DQ beta-sequence is identical to that of DR4/DQw3 haplotype; the DQ alpha is different from the DQ alpha of DR4/DQw3 haplotype and identical to the DQ alpha of both the DR3/DQw2 haplotype of a Raji cell line and the DR5/DQw3 haplotype in deduced amino acid sequence. Taken together, these findings suggest that: 1) a single amino acid difference (position 67) in the third hypervariable region of the first domain of the DR beta 2-chain in the DR2 haplotype is apparently sufficient for stimulating T cell responses; 2) the DQw3 serologic specificity may be defined mainly by the DQ beta-rather than DQ alpha-chain; and 3) multiple genetic events have probably occurred to generate the rarely found REM (DR2/LD-5a/DQw3) haplotype.

Amino Acid Sequence↗
PubMed1992

A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold.

Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue-type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross-reactivity between receptors and their various ligands. To examine receptor-ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin-like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C-terminal half of the third immunoglobulin-like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor-ligand specificity and generating receptor diversity.

3T3 Cells↗
PubMed1994

Behavioral effects of enantiomers of dizocilpine under two "counting" procedures in rats.

Stereoisomers of the N-methyl-D-aspartate antagonist dizocilpine (MK-801) were studied to determine whether behavioral effects on complex operants depend on reinforcement loss accompanying behavioral disruption. Rats earned food pellets if the run of consecutive left-lever presses preceding a trial-terminating right-lever press approximated a target of 12. A percentile schedule reinforced any run closer to the target than two-thirds of the runs on the most recent 24 trials. Once the sequence was learned, half the subjects were shifted to a procedure that yoked reinforcement for each length run to the probability that length generated pellets during asymptototic percentile performance. Although these two procedures generate similar control run and reinforcement distributions, disrupting behavior reduced reinforcement probability far more under the yoked than the percentile procedure. Despite this difference in drug-induced reinforcement loss, both enantiomers produced similar dose-related decreases in run length and response rate under both procedures, with the (-) isomer approximately one log unit less potent than the (+) isomer. The absence of differential effects under these procedures diminishes the likelihood that reinforcement loss contributes to dizocilpine's effects, indirectly bolstering claims that dizocilpine directly affects learning.

Animals↗
PubMed1996

The ribonucleotide reductase system of Lactococcus lactis. Characterization of an NrdEF enzyme and a new electron transport protein.

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD- mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.

Amino Acid Sequence↗
PubMed1997

Performance of basic ventricular tachycardia detection algorithms in implantable cardioverter defibrillators: implications for device programming.

Around 20% of patients with third generation implantable cardioverter defibrillators receive inappropriate therapy, usually triggered by atrial fibrillation. This is because the criteria used for ventricular tachycardia detection by current implantable cardioverter defibrillators are based on the analysis of a sequence of RR intervals and may be inappropriately satisfied by supraventricular tachyarrhythmias. Algorithms for ventricular tachycardia detection were challenged against the full RR interval sequences from 482 spontaneous episodes of atrial fibrillation and 260 spontaneous episodes of ventricular tachycardia to determine their ability to discriminate between the arrhythmias. The sensitivities and specificities of the algorithms were calculated over a wide range of programmable parameters. For a given window length and detection interval, the most stringent algorithms, that required all beats to be classified as "fast", were more specific than those allowing a proportion of "normal" intervals, even after adjustment for differing sensitivity. These differences were less marked for faster tachycardias. Specificity increased with the detection window length to a limit of approximately 18 beats. We conclude that ventricular tachycardia is detected with the highest specificity if all beats in an analyzed sequence are required to be "fast," even after lengthening of the tachycardia detection interval to maintain sensitivity. Further improvement in algorithm performance may require the incorporation of criteria such as tachycardia onset and stability.

Algorithms↗
PubMed1984

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

Animals↗
PubMed1989

Identification of a heat-shock pseudogene from Caenorhabditis elegans.

While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5' end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.

Amino Acid Sequence↗
PubMed2001

[From heparin to synthetic antithrombotic drugs].

Low molecular weight heparins (LMWH) have become the reference drugs for the prevention and treatment of venous thromboembolism. Among the newer antithrombotic agents, the pentasaccharide is the most advanced and promising alternative of low molecular weight heparins. The pentasaccharide is a synthetic and specific inhibitor of activated coagulation factor X (factor Xa). The pentasaccharide is identical to the active sequence of heparin which is present in about a third of commercially available heparin mixtures. The pentasaccharide binds specifically to antithrombin III (ATIII) thus potentiating its inhibitory activity on factor Xa. As a result, thrombin generation and the formation of fibrin and thus of thrombi is inhibited. After a large phase II dose finding clinical trial, four large clinical trials with pentasaccharide in the prevention of venous thromboembolism in hip or knee surgery have been conducted in Europe and North America. The results of these trials show that pentasaccharide prevents venous thromboembolism to a larger extent than enoxaparine with an overall risk reduction of 50% and no significant increase in bleeding. Among newer antithrombotic drugs, an oral, direct thrombin inhibitor, ximelagatran, has been developed and evaluated in the prophylaxis of venous thromboembolism in hip or knee surgery in comparison with enoxaparine with promising RESULTS. Oral direct thrombin inhibitors have the advantage of predictable dose response with the possibility of oral administration without laboratory monitoring.

Anticoagulants↗
PubMed1999

Identification of virulence genes of Helicobacter pylori by random insertion mutagenesis.

The complete genome of the gram-negative bacterial pathogen Helicobacter pylori, an important etiological agent of gastroduodenal disease in humans, has recently been published. This sequence revealed that the putative products of roughly one-third of the open reading frames (ORFs) have no significant homology to any known proteins. To be able to analyze the functions of all ORFs, we constructed an integration plasmid for H. pylori and used it to generate a random mutant library in this organism. This integration plasmid, designated pBCalpha3, integrated randomly into the chromosome of H. pylori. To test the capacity of this library to identify virulence genes, subsets of this library were screened for urease-negative mutants and for nonmotile mutants. Three urease-negative mutants in a subset of 1,251 mutants (0.25%) and 5 nonmotile mutants in a subset of 180 mutants (2.7%) were identified. Analysis of the disrupted ORFs in the urease-negative mutants revealed that two had disruptions of genes of the urease locus, ureB and ureI, and the third had a disruption of a unrelated gene; a homologue of deaD, which encodes an RNA helicase. Analysis of the disrupted ORFs in the nonmotile mutants revealed one ORF encoding a homologue of the paralyzed flagellar protein, previously shown to be involved in motility in Campylobacter jejuni. The other four ORFs have not been implicated in motility before. Based on these data, we concluded that we have generated a random insertion library in H. pylori that allows for the functional identification of genes in H. pylori.

Base Sequence↗
PubMed2001

Computer simulations aimed at structure prediction of supersecondary motifs in proteins.

It is well established that protein structures are more conserved than protein sequences. One-third of all known protein structures can be classified into ten protein folds, which themselves are composed mainly of alpha-helical hairpin, beta hairpin, and betaalphabeta supersecondary structural elements. In this study, we explore the ability of a recent Monte Carlo-based procedure to generate the 3D structures of eight polypeptides that correspond to units of supersecondary structure and three-stranded antiparallel beta sheet. Starting from extended or misfolded compact conformations, all Monte Carlo simulations show significant success in predicting the native topology using a simplified chain representation and an energy model optimized on other structures. Preliminary results on model peptides from nucleotide binding proteins suggest that this simple protein folding model can help clarify the relation between sequence and topology.

Amino Acid Motifs↗
PubMed2006

A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Chromosome Mapping↗
PubMed2006

Recent developments in decision-analytic modelling for economic evaluation.

The past few years have seen rapid changes in the methods of decision-analytic modelling of healthcare programmes for the purposes of economic evaluation. This paper focuses on four developments in modelling that have emerged over the past few years or have become more widely used. First, no one optimal method for extrapolating outcomes from clinical trials has yet been established. Modellers may draw from a set of varied assumptions about survival extrapolation that encompass a range of possibilities from highly optimistic to extremely cautious. Secondly, the practicality and appeal of microsimulation as a method for analysing healthcare decision problems has increased dramatically with the speed of computing technology. Individual instantiations of a system are generated by using a random process to draw from probability distributions a large number of times (also known as Monte Carlo or probabilistic simulation). Microsimulation is moving in new directions, such as discrete-event simulations that simulate sequences of events by drawing directly from probability distributions of event times; this approach is now being broadly applied to model situations where populations of patients interact with healthcare delivery systems. Microsimulation modelling of transmission systems at the population level is also rapidly developing. Thirdly, model calibration is emerging as a new tool that may offer health scientists a means of generating important fundamental knowledge about disease processes. Model calibration allows evidence synthesis in which observations on observable quantities are used to draw inferences about unobservable quantities. The methodology of model calibration has advanced considerably, drawing on theories of numerical analysis and mathematical programming such as gradient methods, intelligent grid search algorithms, and many more. As a fourth issue, an area of extraordinary activity is in the use of transmission models to analyse interventions for infectious diseases, including population-wide effects of vaccination. Transmission models use differential equations to simulate, deterministically for the most part, transitions among infection-related health states. Only recently have modelling methodologies been combined so that cost-effectiveness analyses can consider explicitly not only the patient-level benefits of interventions but also the secondary benefits through transmission dynamics. Advances in technology allow more realistic and complex healthcare models to be simulated more rapidly. However, decision makers will not readily accept results from models unless they can understand them intuitively and explain them to others in relatively simple terms. The challenge for the next generation of modellers is not only to harness the power available from these newly accessible methods, but also to extract from the new generation of models the insights that will have the power to influence decision makers.

Clinical Trials as Topic↗
PubMed2001

Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro.

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.

Amino Acid Sequence↗
PubMed1999

A fourth isoform of endothelin-converting enzyme (ECE-1) is generated from an additional promoter molecular cloning and characterization.

Human endothelin-converting enzyme (ECE-1) has been shown to exist as three isoforms (ECE-1a, ECE-1b and ECE-1c) diverging in their N-terminal sequence and displaying different patterns of subcellular localization. We report here the cloning of ECE-1d, a novel isoform of 767 amino acids, which is generated from the same gene via the existence of an additional promoter located upstream from the third exon of the ECE-1 gene. ECE-1d converting activity is comparable to that of the other three isoenzymes. In contrast to ECE-1b, ECE-1d is expressed at the cell surface, although less strongly than ECE-1a. We have also shown, by identifying ECE-1b and ECE-1d in rat, that the ECE-1 diversity is conserved between human and rodent, suggesting its physiological relevance. The mRNA levels of the four isoforms were assessed in the two species in various cell types, revealing some differences. In particular, the ECE-1a isoform, strongly expressed at the plasma membrane, was found to be highly expressed in primary cultures of endothelial cells but absent from primary cultures of smooth muscle cells.

Amino Acid Sequence↗
PubMed2002

Cross-linking of a DNA conjugate tethering a cis-bifunctional platinated complex to a target DNA duplex.

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a ligand for complexation with K2PtCl4. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. Cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand or cases of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA; conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.

Cross-Linking Reagents↗
PubMed1988

Two genes encoding gas vacuole proteins in Halobacterium halobium.

The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G + C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.

Amino Acid Sequence↗