Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Genetic Complementation Test”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1993

Schistosoma mansoni: genetic complementation analysis shows that two independent hycanthone/oxamniquine-resistant strains are mutated in the same gene.

Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide whether they are both mutated in the same gene with respect to drug-sensitive schistosomes. One of the two strains was isolated in the laboratory, while the other one originated from a treated uncured patient and was subsequently drug selected in the laboratory. The approach consisted in a genetic complementation test performed essentially by crossing the two strains and assessing resistance in the progeny. Since no reappearance of drug sensitivity was detected in the progeny, it was concluded that the two strains failed to complement and were therefore mutated in the same gene. This finding suggests that a single step of drug activation operates in sensitive schistosomes and is ineffective in resistant worms.

Animals↗
PubMed1989

Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis.

By using genetic complementation tests with various in vitro-constructed mutants with mutations in the cap region (which is essential for encapsulation in Bacillus anthracis), we identified three cistrons, capB, capC, and capA, in this order of arrangement. Minicell analysis revealed that these cistrons produce proteins of 44, 16, and 46 kilodaltons, respectively. The complete nucleotide sequence of 3,244 base pairs covering the whole cap region was determined and revealed the existence of the three open reading frames of capB (397 amino acid residues; molecular weight, 44,872), capC (149 amino acid residues; molecular weight, 16,522), and capA (411 amino acid residues; molecular weight, 46,420) arranged in the order predicted by complementation tests. These three cistrons were all transcribed in the same direction from promoters unique to each cistron. Judging from the predicted amino acid sequence of the three proteins and from their localization and their sensitivity to various physicochemical treatments, they appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane. Capsular peptides immunologically identical to that of B. anthracis were found in B. subtilis, B. megaterium, and B. licheniformis, but no sequence homologous to the cap region was found in any of these bacilli other than B. anthracis. Using strains of B. anthracis with or without insertional inactivation of the cap region, we found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host.

Amino Acid Sequence↗
PubMed1997

Substitutions of charged amino acid residues conserved in subunit I perturb the redox metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and Cu(B), and serves as a reaction center of the oxidase complex. This work focuses on the functional and structural roles of 14 charged amino acid residues that are conserved in subunit I of the heme-copper terminal oxidases. Substitutions of Lys55, Tyr173, Asp188, Asp256, Arg481, and Arg482 by neutral amino acid residues did not affect the catalytic activity and spectroscopic properties of the cytoplasmic membranes. In contrast, genetic complementation tests indicated that replacements of Arg80, Asp135, Arg257, Glu286, Tyr288, Lys362, Asp407, and Glu540 resulted in nonfunctional enzymes. The R80Q mutation caused loss of a diagnostic peak for low-spin heme b in the 77 K redox difference spectrum. The K362Q, D407N, and E540Q mutations affected the CO-binding by the heme-copper binuclear center. The D135N, R257Q, E286Q, and Y288F mutations specifically eliminated the Cu(B) center from the oxidase complex, whereas the E286D mutant did not show significant perturbations on the redox metal centers even though it was still inactive. Based on these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved charged amino acid residues in subunit I of the heme-copper terminal oxidases.

Amino Acid Sequence↗
PubMed2000

Function of RRM domains of Drosophila melanogaster ELAV: Rnp1 mutations and rrm domain replacements with ELAV family proteins and SXL.

Members of the ELAV family of proteins contain three RNA recognition motifs (RRMs), which are highly conserved. ELAV, a Drosophila melanogaster member of this family, provides a vital function and exhibits a predominantly nuclear localization. To investigate if the RNA-binding property of each of the ELAV RRMs is required for ELAV's in vivo function, amino acid residues critical in RNA binding for each RRM were individually mutated. A stringent genetic complementation test revealed that when the mutant protein was the sole source of ELAV, RNA-binding ability of each RRM was essential to ELAV function. To assess the degree to which each domain was specific for ELAV function and which domains perhaps performed a function common to related ELAV proteins, we substituted an ELAV RRM with the corresponding RRM from RBP9, the D. melanogaster protein most homologous to ELAV; HuD, a human ELAV family protein; and SXL, which, although evolutionarily related, is not an ELAV family member. This analysis revealed that RRM3 replacements were fully functional, but RRM1 and RRM2 replacements were largely nonfunctional. Under less stringent conditions RRM1 and RRM2 replacements from SXL and RRM1 replacement from RBP9 were able to provide supplemental function in the presence of a mutant hypomorphic ELAV protein.

Amino Acid Motifs↗
PubMed1979

The uncA gene codes for the alpha-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains.

Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.

Adenosine Triphosphatases↗
PubMed1983

Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex.

Recessive lethal mutations in the T/t-complex of the mouse characteristically show defective genetic complementation, even when they affect very different stages of embryogenesis and are known to be nonallelic. To address the question of their genetic or functional relationship, we have applied the cis-trans test, using several well defined recombinant t-chromosomes that carry two or more lethal mutations, and others that are devoid of specific lethals. We show here that the defective complementation that occurs between different t-lethals is a specific result of the trans configuration; thus these genes, which may map as much as 15 cM apart, constitute a functional unit. Some speculations are presented to interpret this enigma in terms of DNA plasticity.

Alleles↗
PubMed2006

Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme.

In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.

Amino Acid Sequence↗
PubMed1977

Mutations causing rapid development of Dictyostelium discoideum.

A mutation affecting the speed of slime mold development has been genetically analyzed. Strain FR17 carries a recessive mutation on linkage group IV. A selection procedure for isolating more mutants of this type has been developed and new mutations have been tested for complementation. The aberrant morphology of these strains can be partially corrected by development in the presence of glucose.

Dictyostelium↗
PubMed2002

Isolation and characterization of novel mutations of the Broad-Complex, a key regulatory gene of ecdysone induction in Drosophila melanogaster.

Seven new alleles of the Broad-Complex gene of Drosophila melanogaster, which encodes a family of four zinc finger protein isoforms BR-C Z1, Z2, Z3 and Z4, were generated by transposase-induced mobilization of a P[Zw] element inserted in either the first intron downstream from the P165 promoter or the exon encoding the Z2-specific zinc finger domain. They were characterized by genetic complementation tests, molecular mapping and cytogenetic analysis of their effect on ecdysone-induced puffing and BR-C proteins binding to polytene chromosomes. Four mutations that correspond to three overlapping deletions and one tandem insertion of the P[Zw] element are located in the intron. They provide evidence that regulatory elements essential for a correct expression of the BR-C Z2 and BR-C Z3 transcripts are located within the intron downstream from the P165 promoter. Three mutations correspond to internal deletions of the locus and exhibit a complete loss of all BR-C(+) genetic functions in the complementation and cytogenetic tests. They thus provide well characterized new amorphic reference alleles of the BR-C gene. The precise cytogenetic location of more than 300 binding sites of BR-C proteins on larval salivary gland polytene chromosomes was determined by immunostaining using specific antibodies. Sites were found in big ecdysone inducible puffs, constitutively active small puffs as well as interbands. A complete list of the major sites on all four salivary gland polytene chromosomes of BR-C(+) larvae is presented.

Animals↗
PubMedDate not supplied

Niemann-Pick C disease: use of denaturing high performance liquid chromatography for the detection of NPC1 and NPC2 genetic variations and impact on management of patients and families.

Niemann-Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. While the corresponding proteins appear essential for proper cellular cholesterol trafficking, their precise function and relationship are still unclear. Mutational analysis of patients, useful for the study of structure/function relationships, is especially valuable for proper management of affected families. Correlations have been found between genotypes and the severity of the neurological outcome of the patients, and molecular genetics constitutes the optimal approach for prenatal diagnosis. However, mutation detection in NPC disease is a challenge. The NPC1 gene, affected in >95% of the families, is large in size (approximately 50 kb), and the already known disease-causing mutations and numerous polymorphisms are scattered over 25 exons. Furthermore, detection of NPC2 patients by complex genetic complementation tests is unpractical. In the present study, we describe a rapid and reliable strategy for detecting NPC genetic variations using DHPLC analysis. Conditions of analysis were optimized for all the NPC1 and NPC2 30 exons and validated using 38 previously genotyped patients. These conditions were then applied to screen a panel of 35 genetically uncharacterized, unrelated NPC patients. Pathogenic mutations were identified in 68/70 alleles. Among the mutations identified, 29 were novel, including two of the NPC2 gene. We conclude that DHPLC is a rapid, low-cost, highly accurate, and efficient technique for the detection of NPC genetic variants.

Base Sequence↗
PubMed1992

Arginine and proline genes of Aspergillus niger.

Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. The arg genes belong to six different linkage groups and the pro genes to two. One arg-mutant could be complemented by transformation with the A. nidulans argB+ gene, and this A. niger gene thus appeared to be homologous to the A. nidulans argB. We isolated an A. niger strain with the argB gene tightly linked with the nicA1 marker. This strain is very suitable as acceptor for transformation with an argB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using the nicA1 marker gene.

Arginine↗
PubMed1983

Genetic analysis of temperature-sensitive mutants of HSV-1: the combined use of complementation and physical mapping for cistron assignment.

To date, mutations in mutants representing 19 of the 33 recognized HSV-1 complementation groups have been mapped. The physical map locations of mutations in 10 ts mutants of HSV-1 strain KOS representing 8 of the 19 complementation groups are reported herein. The mutations in three mutants were found to lie between coordinates 0.086 and 0.194--two of these were mapped finely to between coordinates 0.095 and 0.108--and in seven mutants, between 0.301 and 0.448. The mutation in 1 of the 10 mutants, tsQ26, was mapped finely to a sequence between 500 and 1000 base pairs to the left of the 3' end of the TK gene (0.301-0.304). The availability of physical mapping data has (1) confirmed the usefulness of the complementation test as a means of identifying viral gene functions, (2) facilitated the rapid assignment of mutants to new and recognized cistrons, and (3) prompted a reevaluation of previously ambiguous complementation for mutants in 2 complementation groups. Thus, the 10 mutants whose ts mutations were mapped in this study had been assigned previously to 8 complementation groups based on the assumption that complementation indices of 2 or greater signified that 2 mutants were in different genes. Combined with physical mapping data, however, the results of complementation tests now indicate that indices between 2 and 10 may reflect either inter- or intragenic complementation. Thus, the 10 mutants have now been assigned to 7 complementation groups. Although physical mapping data have confirmed the results of previous complementation tests for 6 of 8 groups analyzed, reevaluation of complementation data in the light of physical mapping data has resulted in a more precise genetic definition of the locus for viral DNA polymerase and of a locus (represented by mutants in complementation group 1-10) which maps in the left hand portion of UL.

DNA Restriction Enzymes↗
PubMed1988

Relationships among genes and gene products of bacteriophage BF23.

Twenty-five gene products of bacteriophage BF23 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functions were studied in relation to type I and II genes classified by means of genetic complementation tests. All the type I mutants were defective in the synthesis of a tail protein, L3. In addition, 4 type I gene products, L5 (gp21), L7 (gp20), L8 (gp29), and L9 (gp25), were identified as constituents of tails (gp21 denotes that a protein is a product of gene 21). Three type IIb mutants in genes 10, 14, and 19 diminished substantially the production of late proteins, including tail and head proteins, and the two other type IIb mutants in genes 1 and 2 were defective in the synthesis of both early and late proteins. Of 14 type IIa mutants, at least 6 were defective in phage DNA synthesis and 2 were defective in the synthesis of head proteins. The defect in the head donor activities of type IIa mutants in extract complementation tests was due to the failure of the formation of mature heads containing DNA. The above results support directly the results of the genetic characterization of BF23 genes.

Autoradiography↗
PubMed1988

Regulation of Qa-1 expression and determinant modification by an H-2D-linked gene, Qdm.

We examined the regulation of cell surface expression of the Qa-1 alloantigens using a panel of monoclonal anti-Qa-1 cytotoxic T-lymphocyte (mCTL) lines. In contrast to previous reports of tissue-specific expression, we found that Qa-1 was widely expressed, resembling the prototypical class I H-2K/D molecules. We further found that an H-2D-linked gene, which we termed Qdm for Qa-1 determinant modifier, controlled expression of certain CTL-defined Qa-1 antigenic determinants. H-2Dk homozygous haplotypes expressed a recessive allele of the modifier, Qdmk, whereas all other H-2 haplotypes tested expressed a dominant allele, Qdm+. The Qdm+ allele regulated in trans Qa-1 epitope expression from a Qdmk chromosome, modifying expression of particular CTL-defined Qa-1 antigenic determinants rather than affecting levels of cell surface expression. Mechanisms of Qdm function may include either a novel protein modification system or an unprecedented case of antigen recognition restricted by a nonclassical major histocompatibility complex molecule.

Animals↗
PubMed1971

Histidinol dehydrogenase (his D) mutants of Salmonella typhimurium.

A multidisciplinary analysis has been applied to over 150 hisD mutants of Salmonella typhimurium in a study of gene-enzyme relationship. The mutants were examined for production of immunologically cross-reacting material by using antibody to purified histidinol dehydrogenase, and for genetic complementation by using a set of F' factors bearing Escherichia coli hisD complementing mutants. Classifications as to missense, nonsense, frameshift, or deletion mutant are proposed on the basis of mutagenesis and suppression tests. For the suppression tests the mutants were examined both by a simultaneous suppression technique and by testing for response to E. coli F' factors bearing a recessive lethal amber and a recessive lethal ochre suppressor. The data are interpreted in relation to the position of the mutations in the recombination and complementation maps and in relation to the known composition of histidinol dehydrogenase. The gene hisD appears to be single cistron for the production of a single biosynthetic polypeptide.

Acridines↗
PubMed2005

Evolution of mutational robustness in an RNA virus.

Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage phi6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.

Bacteriophage phi 6↗