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PubMed1998

Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus.

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol-redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine-activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti-leishmanial agents for chemotherapeutic use.

Animals↗
PubMed2005

Dealing with allelic dropout when reporting the evidential value in DNA relatedness analysis.

A method is suggested that allows the use of loci that have shown allelic dropout in kinship analysis as used for disaster victim identification (DVI) and missing person work (MP). This approach uses an extension of a previously published approach to modelling allelic dropout. This method may salvage some information in cases where allelic dropout is hindering DVI or MP work particularly in reconciliations involving a large number of bodies and pedigrees. It should not replace the pursuit of more complete DNA profiles by the normal rework process for such samples.

Alleles↗
PubMed1996

Gene targeting of CFTR DNA in CF epithelial cells.

A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wt homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell patch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.

Cell Line↗
PubMed2004

Whole exon 5 and intron 5 replaced by RHCE in DVa(Hus).

The DVa(Hus) was previously investigated through cDNA analysis, which revealed an RHD-CE(5)-D hybrid allele. However, the 5' and 3' breakpoints remain unknown. In this article, gene recombinations between the RHD and RHCE alleles were investigated by a combination approach of a sequence-specific primer PCR (PCR-SSP) and an RHD full-length coding region sequencing method on two Chinese subjects with weak D phenotypes. The hybrid Rhesus box of each individual was also investigated through an established PCR-based method. As a result, two partial D phenotypes, DVa(Hus) and DVI type III, were identified, each carrying one hybrid RHD-CE-D allele. The two samples were also serotyped with Rh phontypes of DccEe and DCcee, respectively. Other sequencing analyses of the DVaHus sample showed that the sequence of intron 4 is identical with RHD, whereas the whole sequence of exon 5 and intron 5 is identical with RHCE except for seven polymorphisms in the intron 5. We may concluded that in the case of this Chinese DVa(Hus), the whole exon 5 and complete intron 5 of a total segment of 1801 nucleotides were replaced by RHCE suggesting that the breakpoints of the replaced region are the 5' end of the exon 5 and the 3' end of the intron 5.

Alleles↗
PubMed2003

Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation.

The alpha-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228-Leu248) to intensive random mutagenesis and screened multiply substituted alleles for receptor function. The 91 partially functional mutant alleles that were recovered contained 96 unique amino acid substitutions. Every position in this region can be replaced with at least two other types of amino acids without a significant effect on function. The tolerance for nonconservative substitutions indicates that activation of the G protein by ligand-bound receptors involves multiple intramolecular interactions that do not strongly depend on particular sequence elements. Many of the functional mutant alleles exhibit greater than normal levels of signaling, consistent with an inhibitory role for the third intracellular loop. Removal of increasing numbers of positively charged residues from the loop by site-directed mutagenesis causes a progressive loss of signaling function, indicating that the overall net charge of the loop is important for receptor function. Introduction of negatively charged residues also leads to a reduced level of signaling. The defects in signaling caused by substitution of charged amino acids are not caused by changes in the abundance of receptors at the cell surface.

Amino Acid Sequence↗
PubMed2002

Isolation, characterization, and family study of DTI, a novel partial D phenotype affecting the fourth external loop of D polypeptides.

BACKGROUND: The Rh system is the most polymorphic of the blood group systems and is of major importance in transfusion medicine. The partial D phenotypes lack one or more of the D epitopes. These variants appear to have arisen through hybrid RhD-CE-D or by spontaneous point mutations in RhD. The serologic findings and the molecular characterization of a novel partial D phenotype, termed DTI, are presented here. STUDY DESIGN AND METHODS: RBCs from the DTI proband and RBCs from individuals with other partial D phenotypes were tested with MoAbs against 16 D epi- topes, according to the recommendations of the 4th ISBT Workshop on MoAbs (Rh Section 1A). A full-length cDNA encoding DTI and introns 4 and 5 of RhD were isolated and analyzed by DNA sequencing. A family study of the DTI allele was carried out using PCR-RFLP and long-range PCR methods. RESULTS: Analysis of RBCs from the proband revealed that the DTI phenotype lacks epitopes D1, D2.1 (partial), D2.2, D5, D6 (partial), and D8. The DTI polypeptide exhibits seven amino acid substitutions in the D polypeptide: F223V, A226P, E233Q, V238M, V245L, G263R, and K267M. The genomic organization of DTI showed that the replacement of RhD with RhCE was located in intron 4, and the replacement of RhCE with RhD was located in intron 5. Family studies revealed that the DTI allele was inherited maternally, whereas the RhD- allele was inherited paternally in the proband. CONCLUSION: The serologic data provide the first molecular characterization of DTI, a previously unknown partial D phenotype. This phenotype affected the D polypeptide within the fourth external loop, resulting in a new RhD-CE (entire exon 5)-D hybrid gene. It is worth noting that P226, encoded by exon 5, is derived from E of RhCE in the DTI polypeptide. Family studies demonstrated that DTI was associated with a cDTIE haplotype.

Alleles↗
PubMed2000

Estrogen receptor alpha gene polymorphisms and endometrial cancer risk.

Since the estrogen receptor alpha (ER) is an important mediator of hormonal responses such as proliferation in estrogen-sensitive tissues, we hypothesized that polymorphisms in the ER gene could be functional and associated with endometrial cancer risk. We performed a population-based case-control study in Sweden, focusing on restriction fragment length polymorphisms for XbaI and PvuII and an upstream TA repeat polymorphism. In the main analysis, 154 cases and 205 controls who never used hormone replacement therapy took part and we calculated age-adjusted and multivariate odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression. The XbaI X allele appeared to confer a reduced risk for endometrial cancer. The multivariate OR for the XX genotype was 0.52 (95% CI 0.21-1.29) compared to the xx genotype and there were suggestions of decreasing risk with increasing number of X alleles (P for trend = 0.07). The PvuII PP genotype was also associated with a non-significantly decreased risk for endometrial cancer (multivariate OR 0.70, 95% CI 0.34-1.44) compared with the pp genotype (P for trend = 0.43). The multivariate OR for two short TA (<19 repeats) alleles versus two long alleles was 1.54 (95% CI 0. 73-3.27) and there were suggestions of increasing risk with increasing number of short alleles (P for trend = 0.26). We observed the same pattern of results in an expanded group of subjects, which included women who had used hormone replacement (in total 288 cases and 392 controls). Our data suggest that variants of the ER gene may be associated with an altered risk of endometrial cancer.

Aged↗
PubMed2004

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange.

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.

Animals↗
PubMed1997

The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex.

The structural and functional organization of the Cct complex was addressed by genetic analyses of subunit interactions and catalytic cooperativity among five of the eight different essential subunits, Cct1p-Cct8p, in the yeast Saccharomyces cerevisiae. The cct1-1, cct2-3, and cct3-1 alleles, containing mutations at the conserved putative ATP-binding motif, GDGTT, are cold-sensitive, whereas single and multiple replacements of the corresponding motif in Cct6p are well tolerated by the cell. We demonstrated herein that cct6-3 (L19S), but not the parolog cct1-5 (R26I), specifically suppresses the cct1-1, cct2-3, and cct3-1 alleles, and that this suppression can be modulated by mutations in a putative phosphorylation motif, RXS, and the putative ATP-binding pocket of Cct6p. Our results suggest that the Cct ring is comprised of a single hetero-oligomer containing eight subunits of differential functional hierarchy, in which catalytic cooperativity of ATP-binding/hydrolysis takes place in a sequential manner different from the concerted cooperativity proposed for GroEL.

Amino Acid Sequence↗
PubMed1998

Analysis of the human LRPAP1 gene coding for the lipoprotein receptor-associated protein: identification of 22 polymorphisms and one mutation.

The lipoprotein receptor-associated protein (RAP) is considered a chaperone protein for the lipoprotein receptor-related protein (LRP) and for the other members of the LDL receptor family. Genetic analysis is anticipated to help in delineating groups or individuals with potential defects or problems in this regard. A combined amplification/sequencing strategy was developed to analyze the human LRPAP1 gene for polymorphisms and mutations. The LRPAP1 gene was amplified from genomic DNA in four long-range PCR amplicons, 2.4 to 7.6 kb in size. Three amplicons were finally used as templates with 14 sequencing primers to obtain the sequence of the eight exons and large portions of adjacent introns from individual DNA. This strategy, applied to sequence the LRPAP1 gene of 14 unrelated, normal individuals revealed, in total, 23 distinct mutations and polymorphisms, mostly intronic substitutions and deletions. In this small group 1 expressed mutation was encountered on one allele in 2 unrelated individuals: a G to A transition results in the replacement of valine by methionine in exon 7 at position 311 of the human RAP precursor protein.

Amino Acid Substitution↗
PubMed1995

Amino acid replacements leading to temperature-sensitive defects of the NS1 protein of influenza A virus.

The nonstructural (NS) genes of two influenza virus temperature-sensitive (ts) reassortants have been sequenced and compared with the corresponding wild type sequences. Ts 412 has a single base substitution (G100-->A) leading to an amino acid replacement (Arg 25-->Lys) in the NS1 protein. Ts 451 also has a single base substitution (U273-->C) leading to an amino acid replacement (Ser 83-->Pro) in the NS1 protein. In ts 412 infected cells at the nonpermissive temperature very little M1 and HA mRNA and proteins are synthesized, suggesting that NS1 is involved in a transcriptional regulation process. The ts mutation in ts 451 could be extragenically suppressed by replacement of the PB1 and/or PA protein genes of the mutant by the allelic genes of PR8. Both observations suggest that NS1 cooperates with the polymerase complex.

Influenza A virus↗
PubMed1994

Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.

Defects in the gene encoding the endothelin-B receptor produce aganglionic megacolon and pigmentary disorders in mice and humans. We report that a targeted disruption of the mouse endothelin-3 ligand (EDN3) gene produces a similar recessive phenotype of megacolon and coat color spotting. A natural recessive mutation that results in the same developmental defects in mice, lethal spotting (ls), failed to complement the targeted EDN3 allele. The ls mice carry a point mutation of the EDN3 gene, which replaces the Arg residue at the C-terminus of the inactive intermediate big EDN3 with a Trp residue. This mutation prevents the proteolytic activation of big EDN3 by ECE-1. These findings indicate that interaction of EDN3 with the endothelin-B receptor is essential in the development of neural crest-derived cell lineages. We postulate that defects in the human EDN3 gene may cause Hirschsprung's disease.

Amino Acid Sequence↗
PubMed1992

Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism.

The extensive polymorphism of the classic class I antigens has been well described. In contrast, the nonclassic HLA antigens are distinguished by their low polymorphism. We examine here the HLA polymorphism of the HLA-E locus by examining the DNA sequence of cDNA from nine ethnically diverse individuals. From this analysis, we show that there is no polymorphism in the regions including exon 1 and from exon 4 to exon 8, the 3' untranslated exon. In exons 2 and 3, there are two base substitutions, one of which is at a replacement site and the other silent. The replacement substitution changes an arginine to a glycine at position 107, defining two alleles at the HLA-E locus. Using the PCR on exon 3 from genomic DNA and hybridization with oligonucleotide probes, we have examined 90 HLA-typed individuals to determine the relative frequency of the two alleles in the population and their association with the classical antigens. This analysis showed that these two alleles were present at nearly equal frequencies in the population. Surprisingly, both alleles were found in an essentially random association with all but one HLA-A and -B haplotype. The single exception was to the A1-B8 haplotype, which appeared to be linked to only one of the two alleles. One implication of this random association is that these HLA-E alleles may have existed before most of the HLA-A and B polymorphism. Thus, selection has maintained the HLA-E locus essentially unaltered during a time when considerable polymorphism was being selected for at the HLA-A and -B loci. This finding may also have important consequences in an unrelated bone marrow transplant, where it is predicted that 37% of HLA-A and -B matched donors are mismatched at the HLA-E locus.

Alleles↗
PubMed1999

Accurate determination of the number of CAG repeats in the Huntington disease gene using a sequence-specific internal DNA standard.

We have developed a sequence-specific internal DNA size standard for the accurate determination of the number of CAG repeats in the Huntington disease (HD) gene by cloning key fragments (between 15 and 64 CAG repeats) of the HD gene. These fragments, pooled to produce a sequence-specific DNA ladder, enabled us to observe the true number of CAG repeats directly, with no need for calculations. Comparison of the calculated numbers of CAG repeats in the HD gene using this sequence-specific DNA standard with a commercially available standard (GENESCAN-500 TAMRA) showed that the latter underestimated the number of CAG repeats by three when analyzed by capillary electrophoresis on the ABI 310 Genetic Analyzer (POP4 polymer). In contrast, the use of the same standard overestimated the number of CAG repeats by one when the samples were analyzed by denaturing polyacrylamide electrophoresis on ABI 377 DNA Sequencer (6% denaturing polyacrylamide gel). This suggests that our sequence-specific standard provides greater accuracy for the determination of the true number of CAG repeats in the HD gene than commercially available standards. The sequence-specific standard can be radioactively labeled and successfully replace conventional DNA size standards when analyzing polymerase chain reaction (PCR)-amplified HD alleles by denaturing polyacrylamide electrophoresis.

Alleles↗
PubMed1998

Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase.

Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease. Many H. pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen-insensitive NADPH nitroreductase activity. The underlying gene (called 'rdxA') was identified in several steps: transformation of Mtz-susceptible (MtzS) H. pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing. We also found that (i) E. coli (normally MtzR) was rendered MtzS by a functional H. pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzR H. pylori rendered it MtzS; and (iii) replacement of rdxA in MtzS H. pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype. The 630 bp rdxA genes of five pairs of H. pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced. In each case, the paired rdxA genes differed from one another by one to three base substitutions. Typical rdxA genes from unrelated isolates differ by 5% in DNA sequence. Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR. Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs. 1 or 2 in CNRs) and isoelectric point (pI=7.99 vs. 5.4-5.6), which might account for its reduction of low redox drugs such as Mtz. We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections. H. pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.

Amino Acid Sequence↗
PubMed1998

Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus.

The hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a two-component regulatory system that influences capsule expression and virulence.

Amino Acid Sequence↗
PubMed2003

A nonconserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins.

A toxic l-proline analogue, l-azetidine-2-carboxylic acid (AZC), causes misfolding of the proteins into which it is incorporated competitively with l-proline, thereby inhibiting the growth of the cells. AZC enters budding yeast Saccharomyces cerevisiae cells primarily through the general amino acid permease Gap1, not through the proline-specific permease Put4. We isolated an AZC-hypersensitive mutant that cannot grow even at low concentrations of AZC because of the accumulation of intracellular AZC. By screening through a yeast genomic library, the mutant was found to carry an allele of RSP5 encoding an E3 ubiquitin ligase. A single amino acid change replacing Ala (GCA) at position 401 with Glu (GAA) showed that Ala-401 in the third WW domain (a protein interaction module) is not conserved in the domain. The addition of NH4+ to yeast cells growing on l-proline induced rapid ubiquitination, endocytosis, and vacuolar degradation of the plasma membrane protein Gap1. However, immunoblot and permease assays indicated that Gap1 in the rsp5 mutant remained stable and active on the plasma membrane probably with no ubiquitination, leading to AZC accumulation and hypersensitivity. The rsp5 mutants also showed hypersensitivity to various stresses (toxic amino acid analogues, high temperature in a rich medium, and oxidative treatments) and defects in spore growth. These results suggest that Rsp5 is involved in selective degradation of abnormal proteins and specific proteins for spore growth, in addition to nitrogen-regulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was considered to have an important role in the ubiquitination of targeted proteins.

Alanine↗
PubMed2024

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Animals↗