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Results for “Genetic Complementation Test”

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At least 199 records · Page 11Linked to original sources
PubMed1997

Neurotoxicity of intracerebral injection of a replication-defective adenoviral vector in a semipermissive species (cotton rat).

The neurotoxicity of an adenoviral vector (Adv.RSVtk) carrying the gene for herpes simplex virus thymidine kinase (HSVtk) was tested in the cotton rat, a semipermissive host. Adv.RSVtk was injected intracerebrally in cotton rats at a dose of 5.0 x 10(6) or 7.5 x 10(7) p.f.u. No signs of illness were observed. Histological inspection at 12 and 28 days after injection showed inflammation of the ependyma and choroid plexus and at the injection site. No demyelination, viral inclusions, cerebral edema, necrosis, cavities or vascular necrosis were seen in the brains. There was no significant difference between animals injected with 5.0 x 10(6) or 7.5 x 10(7) p.f.u., nor was there a difference between animals analyzed at 12 or 28 days after vector injection. This inflammation was similar in animals that had been preimmunized with wild-type virus and in animals that had been treated with ganciclovir. No histopathology, was observed in the lungs of the animals and no replication-competent virus was detected. These experiments indicate that Adv.RSVtk has limited neurotoxicity which would not prohibit its use in a limited phase I clinical trial in humans that have malignant tumors of the central nervous system.

Animals↗
PubMed1974

Mitochondrial genetics. VII. Allelism and mapping studies of ribosomal mutants resistant to chloramphenicol, erythromycin and spiramycin in S. cerevisiae.

We have isolated 15 spontaneous mutants resistant to one or several antibiotics like chloramphenicol, erythromycin and spiramycin. We have shown by several criteria that all of them result from mutations localized in the mitochondrial DNA. The mutations have been mapped by allelism tests and by two- and three-factor crosses involving various configurations of resistant and sensitive alleles associated in cis or in trans with the mitochondrial locus omega which governs the polarity of genetic recombination. A general mapping procedure based on results of heterosexual (omega(+)x omega(-)) crosses and applicable to mutations localized in the polar segment is described and shown to be more resolving than that based on results of homosexual crosses. Mutations fall into three loci which are all linked and map in the following order: omega-R(I)-R(II)-R(III). The first locus is very tightly linked with omega while the second is less linked to the first. Mutations of similar resistance phenotype can belong to different loci and different phenotypes to the same locus. Mutations confer antibiotic resistance on isolated mitochondrial ribosomes and delineate a ribosomal segment of the mitochondrial DNA. Homo- and hetero-sexual crosses between mutants of the ribosomal segment and those belonging to the genetically unlinked ATPase locus, O(I), have been performed in various allele configurations. The polarity of recombination between R(I), R(II), R(III) and O(I) decreases as a function of the distance of the R locus from the omega locus rather than as a function of the distance of the R locus from the O(I) locus.

Adenosine Triphosphatases↗
PubMed2005

cblE type of homocystinuria due to methionine synthase reductase deficiency: functional correction by minigene expression.

The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Removal of an intron with unique 3' branch site creates an amino-terminal protein sequence directing the scERV1 gene product to mitochondria.

The yeast scERV1 gene is of special interest because it has a dual function in mitochondrial biogenesis and in the regulation of the cell cycle. The recent discovery that the yeast scERV1 gene has a structural and functional human homologue initiated a detailed comparison of the genes and their structures. In addition the homologous ALR (augmenter of liver regeneration) genes from rat and mouse have just been identified and it has been found that the mammalian proteins have a specific function in liver regeneration and in spermatogenesis. It now turns out that the organization of the 5' regions of these genes is much more complicated than expected. The latest research has discovered an additional intron in the 5' region of the mouse gene and possible amino-terminal extensions of the reading frames. In this work, reinvestigation of the 5' region of the yeast gene identifies a putative intron with an unusual 3' branch site. It is shown that a small intron of 83 nucleotides is present in this genomic region. Analysis of cDNA clones demonstrates that the intron is correctly removed from the messenger RNA and that therefore the unusual 3' branch site is probably functional. Furthermore, studies with antibodies directed against recombinant scERV1 protein demonstrate that the gene product is associated with mitochondria, in agreement with its involvement in mitochondrial biogenesis. Complementation experiments with mutants and different 5' deletions of the gene identify the corresponding promotor for transcription and the start codon for translation.

Amino Acid Sequence↗
PubMed1998

A highly conserved intraspecies homolog of the Saccharomyces cerevisiae elongation factor-3 encoded by the HEF3 gene.

A paralog (intraspecies homolog) of the Saccharomyces cerevisiae YEF3 gene, encoding elongation factor-3, has been sequenced in the course of the yeast genome project, and identified by database searching; this gene has been designated HEF3. Bioinformatic and Northern blot analysis indicate that the HEF3 gene is not expressed during vegetative growth. Deletion of the HEF3 gene reveals no growth defects, nor any defects in mating or sporulation. A high copy 2 mu clone of HEF3 was constructed, and was shown to be unable to complement a null allele of yef3. Finally, an in vitro assay for ribosome-stimulated ATPase activity was performed with isogenic HEF3 and delta hef3 strains; no difference in biochemical activity could be detected in these strains. From these results, we conclude that the HEF3 gene does not encode a functional homolog of YEF3.

Adenosine Triphosphatases↗
PubMed1999

Analysis of genetic interactions between DHH1, SSD1 and ELM1 indicates their involvement in cellular morphology determination in Saccharomyces cerevisiae.

The DHH1 gene of Saccharomyces cerevisiae belongs to a family of genes that encode highly conserved DEAD-box proteins commonly present in various eukaryotic organisms. Its precise function in yeast has not yet been well documented. To investigate its role in vivo, we constructed a DHH1 disruptant, characterized it genetically and searched for genes the mutations in which would cause synthetic lethality in combination with the DHH1 disruption. CDC28, ELM1 and SSD1 were thus found to be such candidates and we subsequently analysed their interactions. Mutations in ELM1 were previously reported to result in the elongation of cells. We confirmed this phenotype and observed in addition elongated bud formation in an Elm1p overproducing strain. Also, Elm1p fused with the green fluorescent protein (GFP) was found to be localized at the bud neck. These and other observations seem to suggest that Elm1p plays a role during cytokinesis in S. cerevisiae. The phenotypes of strains harbouring either delta dhh1 delta elm1 or ssd1-d delta elm1 were very similar to each other, showing abnormal cellular morphology and defects in cytokinesis and mitosis. Furthermore, DHH1 and SSD1 could functionally complement each other in the ade2 red colour pigment formation, hypersensitivity to SDS, growth on synthetic media and at high temperature. A triple mutant, delta dhh1 ssd1-d delta elm1, apparently had very fragile cell walls and could grow only in a medium supplemented with 1 M sorbitol.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

Identification of a putative DEAD-box RNA helicase and a zinc-finger protein in Candida albicans by functional complementation of the S. cerevisiae rok1 mutation.

We identified two novel genes, CHR1 and CSR1, of the fungal pathogen Candida albicans, by functional complementation of the Saccharomyces cerevisiae rok1 mutation. The Rok1 protein is a member of the DEAD protein family of ATP-dependent RNA helicases. ROK1 is required for cell cycle progression and also for rRNA processing. The CHR1 gene product of 578 amino acids is highly homologous to the Rok1 protein (54% identity) and is considered to be a putative DEAD-box RNA helicase. We predict that the CSR1 gene encodes a 73 kDa protein of 612 amino acids with five zinc-finger motifs at the C-terminal region. CHR1 or CSR1 on a high-copy number plasmid showed a slow-growth phenotype in a condition where the ROK1 expression is turned on from the GAL1 promoter. This result is consistent with the lethality caused by the ROK1 overexpression. We conclude that CHR1 encodes a functional homologue of Rok1 protein and CSR1 is a heterologous suppressor of the rok1 mutation.

Amino Acid Sequence↗
PubMed1997

1-NGFR receptor is a new flow cytometric tool for rapid cell cycle-correlated gene therapy complementation studies in viable cells.

Control of successful genetic complementation of cellular defects in heterogenous cell populations requires biochemical selection markers or cell analytical specifiers. With available gene therapy technologies, only a fraction of a cell population is transfected or transduced. We applied and optimized a novel dual-laser flow cytometric technique to analyze immediately the genetic complementation of cells dysregulated in cell proliferation and DNA repair. A novel bicistronic retrovirus carrying the normal Fanconi anemia gene (group C) and the cell surface marker gene l-NGFR was constructed to analyze the normalization of the G2-phase cell cycle defect in DNA-repair-deficient FA(C) lymphoblastoid cells after transduction. Using a dual-laser multiparameter technology, we 1) analyzed the cell-cycle distribution of viable/dead cells using Hoechst 33342 (with ultraviolet light), 2) identified the genetically complemented cells by FITC antibody labeling of the novel l-NGFR surface marker (488 nm), and 3) recorded mitomycin C-induced cell death in nontransduced and transduced cells by propidium iodide. Artificial l-NGFR expression is high and similar in ontogenetically and phylogenetically different cell populations. With this novel l-NGFR marker technology, the success of gene therapy of cell-cycle dysregulation in even small subpopulations of cells can be recorded within 48 h. In addition to improved cell analysis, l-NGFR surface marker expression can also be used for rapid generation of pure cell populations by column cell separation technologies.

Antibiotics, Antineoplastic↗
PubMed1977

Specific neuroanatomical changes in chemosensory mutants of the nematode Caenorhabditis elegans.

Eight of nineteen chemotactic mutants of the nematode Caenorhabditis elegans have morphological defects in the sensory endings of neurons at the tip of the head. The mutants were obtained as worms swimming away from attractant or found amongst male potency mutants or mutants exhibiting erratic behavior. The nineteen mutants fall into at least twelve complementation groups. Mutants E1034 and E1035, alleles of che-1, show morphological alterations in the sensory endings of amphidial neurons and inner labial type 2 neurons, both prospective chemosensory neurons. Both mutants contain non-complementing ts sterile mutations linked to the chemosensory mutation. E1066 shows abnormalities in all the sheath cells associated with the sensory neurons and in the bundling pattern of the amphidial neurons. E1126 is structurally abnormal only in the sensory endings of inner labial type 2 neurons, supporting a chemosensory role for these neurons. E1033 (che-2) and E1124 (che-3) cause defects in the ciliary structure of all but one type of ciliated sensory neuron in the head. E1062 is grossly defective in head structure and the structure of the male copulatory organ, suggesting these opposite ends of the nematode rich in sensory structures share gene functions in embryogenesis. Our study illustrates the possibilities for genetic dissection of the development of a small set of nerves in a simple organism.

Animals↗
PubMed2004

Complementation of melanocyte development in SOX10 mutant neural crest using lineage-directed gene transfer.

An in vitro gene complementation approach has been developed to dissect gene function and regulation in neural crest (NC) development and disease. The approach uses the avian RCAS virus to express genes in NC cells derived from transgenic mice expressing the RCAS receptor TVA, under the control of defined promoter elements. Constructs for creating TVA transgenic mice were developed using site-specific recombination GATEWAY (GW), compatible vectors that can also be used to facilitate analysis of genomic fragments for transcriptional regulatory elements. By using these GW vectors to facilitate cloning, transgenic mouse lines were generated that express TVA in SOX10-expressing NC stem cells under the control of the Pax3 promoter. The Pax3-tv-a transgene was bred onto a Sox10-deficient background, and the feasibility of complementing genetic NC defects was demonstrated by infecting the Pax3-tv-a cells with an RCAS-Sox10 expression virus, thereby rescuing melanocyte development of Sox10-deficient NC cells. This system will be useful for assessing genetic hierarchies in NC development. Developmental Dynamics 229:54-62, 2004.

Animals↗
PubMed1976

Genetic control of the immune response to the radom linear terpolymer of L-glutamic acid, L-lysine and L-leucine (GLleu) by complementing Ir genes.

Three levels of humoral responsiveness (none, high or marginal) were noted following secondary immunization with random linear terpolymer of L-glutamic acid, L-lysine and L-leucine (GLleu). These immune responses were under dual H-2-linked Ir gene control. Gene complementation was observed in appropriate F1 offspring between two nonresponder strains and in selected intra-I region recombinants derived from crossing over between nonresponder parental haplotypes. One of the Ir-GLleu genes, beta, was localized within the I-A or I-B subregions. The other Ir-GLleu locus, termed alpha, was mapped in the I-C or S subregions. Differences in the strain distribution of immune responsiveness to GLleu and related terpolymer GLphe (which is also under complementing Ir gene control) are primarily attributable to differences among the Ir-GLleu and Ir-GLphe beta alleles. A correlation exists between the presence of the Ir-GLleu alpha(¿allele and the gene coding for specificity Ia.7.

Alleles↗
PubMed1992

Characterization of a complex chromosomal rearrangement maps the locus for in vitro complementation of xeroderma pigmentosum group D to human chromosome band 19q13.

Microcell-mediated chromosome transfer (MMCT) is a powerful genetic technique that permits the transfer of a single chromosome from one mammalian cell to another. The utility of MMCT for gene mapping strategies is critically dependent on the careful characterization of the chromosomes being transferred. We have recently reported the identification of a single rearranged human chromosome, designated Tneo, which corrects the UV sensitivity and excision repair defect of cells of xeroderma pigmentosum genetic complementation group D (XP-D) in culture (Flejter WL et al., Proc Natl Acad Sci USA 89:261-265, 1992). Additionally, those studies demonstrated a role for the excision repair cross-complementing 2 (ERCC2) gene in the observed phenotypic correction. We now report the results of detailed conventional and molecular cytogenetic characterization of the complementing Tneo chromosome. This analysis revealed a complex rearrangement involving material from human chromosomes 16, 17, and 19. Characterization of deletions of Tneo which retained or lost XP-D complementing ability mapped the gene responsible for phenotypic correction to a small region of the terminal q-arm of this chromosome. This region includes the previously described human DNA repair gene cluster located in the region 19q13.2-q13.3, a result consistent with the notion that the in vitro correction of XP-D cells by the Tneo chromosome is rendered by the ERCC2 locus. The data illustrate the potential value of detailed cytogenetic characterization of a human chromosome present in a somatic cell hybrid, even when that material involves complex rearrangements.

Cell Line↗
PubMed1992

Human RAG2, like RAG1, is on chromosome 11 band p13 and therefore not linked to ataxia telangiectasia complementation groups.

Ataxia telangiectasia (A-T) is an inherited, recessive, cancer-prone disease with associated immunodeficiency and chromosome abnormalities involving TCR loci. The latter phenomena implicate errors of the enzyme(s) responsible for assembly of antigen receptor genes (recombinase) in disease pathogenesis. Here we report the location of a human recombination activating gene (RAG2), in addition to RAG1, on chromosome 11, band p13, thereby formally demonstrating linkage of these genes in humans and showing that they are not linked to the known locus responsible for the A-T syndrome.

Ataxia Telangiectasia↗
PubMed2003

Alpha complementation in the Cre recombinase enzyme.

The Cre-loxP system is increasingly exploited for spatial and temporal gene inactivation. Here we present a novel approach to achieve this goal of selective gene inactivation. Following the model of alpha complementation in the beta-galactosidase enzyme, where the enzyme is split into independent polypeptides which are able to associate and maintain the enzymatic activity, we divided the Cre recombinase into two independent polypeptides (one containing the NH(2) terminus (alpha) and a second one containing the COOH-terminus (beta)). Individually, the two polypeptides have no detectable activity. However, when coexpressed the polypeptides are able to associate, giving rise to Cre enzymatic activity, which optimally is as high as 30% of that seen with wildtype Cre recombinase in vitro. We present this strategy as a modification of the traditional Cre-loxP system, which could be used to obtain a highly specific recombination pattern by expressing the two halves under the control of separate promoters.

Amino Acid Sequence↗