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Results for “Genetic Complementation Test”

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At least 19 recordsLinked to original sources
PubMed2006

Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without cleft palate in A/WySn mice, as confirmed by a genetic complementation test.

BACKGROUND: Nonsyndromic cleft lip (CL) with or without cleft palate (CLP) is a common human birth defect with complex genetic etiology. One of the unidentified genes maps to chromosome 17q21. A mouse strain, A/WySn, has CLP with complex genetic etiology that models the human defect, and 1 of its causative genes, clf1, maps to a region homologous to human 17q21. Extensive studies of the candidate region pointed to a novel insertion of an IAP transposon 3' from the gene Wnt9b as the clf1 mutation. Independently a recessive knockout mutation of Wnt9b (Wnt9b-) was reported to cause a lethal syndrome that includes some CLP. METHODS: A standard genetic test of allelism between clf1 and the Wnt9b- mutation was done. A total of 83 F1 embryos at gestation day 14 (GD 14) from Wnt9b-/+ males crossed with A/WySn females, and 79 BC1 GD 14 embryos from F1 Wnt9b-/clf1 males back-crossed to A/WySn females were observed for CL. Embryo genotypes at clf1 and Wnt9b were obtained from DNA markers. Genotypes for a second unlinked modifier locus from A/WySn, clf2, were similarly obtained. RESULTS: The compound mutant embryos (Wnt9b-/clf1) had high frequencies of CL: 27% in the F1 and 63% in the BC1. The clf2 modifier gene was found to have 3 alleles segregating in this study and to strongly influence the penetrance of CL in the compound mutant. CONCLUSIONS: The noncomplementation of clf1 and Wnt9b- confirms that clf1 is a mutation of the Wnt9b gene. The homologous human WNT9B gene and 3' conserved noncoding region should be examined for a role in human nonsyndromic CLP.

Animals↗
PubMed1977

Studies on the biosynthetic pathway of human P erythrocyte antigen using genetic complementation tests between fibroblasts from rare p and Pk phenotype donors.

Using the indirect immunofluorescence technique, P antigen is not detected on skin fibroblasts from Pk1, Pk2 or p donors. P antigen is reexpressed on polykaryon cells obtained by fusion of Pk x p fibroblasts. On the contrary, P antigen is not expressed on polykaryon cells from Pk x Pk or p x p fusion. All these data suggest that in Pk and p fibroblasts, at least two distinct genes are modified, one in the p phenotype, and the other in the Pk phenotype. The two Pk phenotypes studied seem to be homogeneous from a genetic point of view, as do the five p phenotypes.

Animals↗
PubMed1984

Mini-Mu-duction as a test for genetic complementation in Escherichia coli.

In mini-Mu-duction, segments of host DNA bracketed between two copies of an internally deleted Mu phage (a mini-Mu) can be packaged within Mu phage particles. Upon infection of a second host strain, the DNA injected by these particles can insert into the chromosomal DNA in a reaction catalyzed by the phage A gene product (transposase), which is independent of homologous recombination. This results in a partially diploid host strain in which the duplicated host DNA is bracketed by two copies of the mini-Mu phage (Faelen et al., Mol. Gen. Genet. 176:191-197, 1979). The frequency of mini-Mu-duction reported previously was low (10(-8) to 10(-9) per recipient cell) thus limiting its use to rather stable mutational lesions. I have increased the frequency of mini-Mu-duction 10- to 100-fold by use of a helper phage lacking the kil gene and by UV irradiation of the phage stocks. I have also shown that mini-Mu-duction is a reliable complementation assay in rec+ as well as recA recipient strains. This genetic complementation test does not require prior gene localization and (due to the extended host range of phage Mu) should be applicable to many enterobacterial species.

Ampicillin↗
PubMed1989

X-ray-induced specific locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. III. Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions by means of complementation tests.

Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions was made by means of complementation tests on all possible pairwise combinations of 50 X-ray-induced irreparable adenine-3 mutants (designated ad-3IR). All mutants were induced in either heterokaryon 11 or heterokaryon 12 of Neurospora crassa, 2-component heterokaryons heterozygous for mutants at the 3 closely linked loci ad-3A and ad-3B and nic-2 (nicotinamide-requiring) located about 5.0 map units distal to ad-3B. The complementation tests involved mutants of the following genotypes: 15 ad-3A, 27 ad-3B, 7 ad-3A ad-3B nic-2 and 1 ad-3B nic-2. To facilitate mapping, 5 additional strains (each consisting of a gene/point mutation at the ad-3A or ad-3B locus and a separate site of closely linked recessive lethal damage in the immediately adjacent regions [designated ad-3R + RLCL]) were also included. The data from these complementation tests showed that the majority (46/50) of X-ray-induced irreparable ad-3 mutants mapped as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B. The remaining mutants (4/50) were found to result from a series of closely linked, but separate, mutations (designated multilocus mutations) of the type ad-3IR + RLCL, different from those found in previous studies (de Serres, 1968; de Serres and Brockman, 1968). The data from the present complementation tests have expanded the process of genetic fine structure mapping of the ad-3 and immediately adjacent regions (de Serres, 1968) and defined the presence of the following 11 genetic loci: (a) 4 loci (with either known [i.e. col-1t] or unknown [i.e. unknA]) function proximal to ad-3A: unknA, unknB, col-1t, and col-2t, (b) 4 loci in the 'X' region: unknC, unknD, unknE, and unknF, (c) 2 loci distal to ad-3B: unknG, col-3t, and (d) 1 locus distal to nic-2: unknH.

Adenine↗
PubMed1987

Functional expression of a cloned herpes simplex virus type 1 DNA polymerase gene.

A vector which expresses the herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene was constructed by ligating two separately cloned HSV DNA restriction fragments into an intermediate plasmid and then mobilizing the intact polymerase gene-encoding sequence into a pSV2 derivative. The expression vector (pD7) contains a functional simian virus 40 replication origin and early enhancer-promoter upstream from the HSV DNA polymerase-encoding sequence. COS-1 cells transfected with pD7 contained an RNA species, shown by Northern blot analysis to hybridize specifically with an HSV DNA pol probe and to be the same size (4.3 kilobases) as the pol mRNA found in HSV-1-infected COS-1 cells. A genetic complementation test was used to establish that pD7 expresses a functional pol gene product. COS-1 cells transfected with pD7 were able to partially complement the growth defect of an HSV-1 (KOS) temperature-sensitive mutant, tsC7, in the DNA polymerase gene at the nonpermissive temperature.

Animals↗
PubMed1994

Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known omega 3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a delta 12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized omega 6 desaturase corresponding to the Arabidopsis fad2 gene.

Amino Acid Sequence↗
PubMed1982

Microinjection of cytoplasm as a test of complementation in Paramecium.

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.

Action Potentials↗
PubMed1978

The control region of the F sex factor DNA transfer cistrons: restriction mapping and DNA cloning.

A restriction endonuclease map of EcoRI fragment f6 of F sex factor DNA was constructed and aligned with pre-existing physical and genetic maps. Results of genetic complementation tests and analysis of proteins synthesized in minicells from PstI and BglII sub-fragment clones, or from a specific BglII fragment deletion, have allowed mapping of the locations of the origin of DNA transfer and many of the transfer genes known to lie on f6. The proteins detected account for 78% of the coding capacity of fragment f6.

Chromosome Mapping↗
PubMed1996

Physical and genetic mapping of chromosome 9S in maize using mutations with terminal deficiencies.

Deletion mapping was employed to determine the physical order of five morphological variants, pyd1, yg2, wd1, v28 and u31, with respect to restriction fragment length polymorphism (RFLP) markers located at the distal end of chromosome 9S in maize. The genetic materials used were a series of terminal-deficiency mutants, newly derived with McCLintock's original stocks developed in the 1940s, via breakage-fusion-bridge cycles. A combined physical map and genetic map has been constructed based on data gathered from both genetic complementation tests and RFLP analysis. The location of v31 in relation to RFLP markers was further determined by interval mapping. The physical distance between the healed telomeric end and the most distal RFLP marker in two terminal-deficiency lines was established by using pulsed field gel electrophoresis and verified by Bal31 digestion. The results from this study set a foundation for studies on the mechanism of healing of broken chromosome ends in higher plants.

Chromosome Mapping↗
PubMed1997

Assignment and functional roles of the cyoABCDE gene products required for the Escherichia coli bo-type quinol oxidase.

Cytochrome bo from Escherichia coli belongs to the heme-copper terminal oxidase superfamily and functions as a redox-driven proton pump. In the present study, we examined the functional roles of the cyoABCDE genes, which encode cytochrome bo. We expressed the cyoABCDE genes in minicells using pTTQ18 derivatives and identified subunits II, I, III, and IV of the oxidase complex and heme O synthase as polypeptides with molecular weights of 33,500, 75,000, 20,500, 12,000, and 28,000, respectively. The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just tightly enough for the incorporation of heme O into the oxidase complex. To facilitate functional analysis of the gene products, we developed a single copy expression vector pHNF2, a derivative of the F-sex factor. Genetic complementation tests showed that deletions in each gene resulted in nonfunctional enzymes. Western blotting analysis indicated that the expression levels of subunits I and II were not affected by the deletions in the other cyo gene products. However, spectroscopic analyses of the mutant membranes revealed that all the deletions perturbed or eliminated the redox metal centers in subunit I. Present findings suggest that subunits II, III, and IV of the oxidase complex are required for the assembly of the metal centers in subunit I.

Alkyl and Aryl Transferases↗
PubMed1979

Investigations of the F conjugation gene traI:traI mutants and lambdatraI transducing phages.

A series of traI point and deletion mutants of Flac, and a traM mutant, were characterised. Complementation tests with an amber Flac traI mutant confirmed their genotypes, and in addition all the traI mutants, but not the traM mutant, were complemented by pRS31 (PSC101 traDI) and EDlambda109 (lambdatraI). Judging from the efficiencies of plating of F-specific phages, none of the mutations affected pilus formation. The traI products of F and of the F-like plasmid R1 were interchangeable with each other but not with that of R100, while the traM product of F could not be replaced by those of R1 or of R100. Neither traI nor traM were needed for conjugal transfer of ColE1. Three lambda transducing phages carrying traI were isolated by in vivo or in vitro techniques, and characterised by genetic complementation tests, by analysis of the fragments produced by restriction endonucleases, and by measurement of heteroduplex molecules. The genetic structures together with the sizes and F coordinates, of the transfer regions carried by the phages were thereby determined. Comparison of the proteins synthesised in UV-irradiated cells by one of the lambdatraI phages with those made by a derivative carrying an amber traI mutation, allowed the traI product to be identified as a protein of molecular weight 174,000. In addition, the molecular weights of the traD (84,000), traS (18,000), and traT (25,000) products made by the lambdatraSTD1 phage EDlambda107 were measured. The possible roles of the traI and traM products in conjugation are discussed.

Coliphages↗
PubMed1979

The control region of the F sex factor DNA transfer cistrons: physical mapping by deletion analysis.

A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.

Bacterial Proteins↗
PubMed1975

Ultraviolet-inactivation of conidia from heterokaryons of Neurospora crassa containing uv-sensitive mutations.

The effect of three UV-sensitive mutations of Neurospora crassa, upr-I, uvs-4 and uvs-6, on the ultraviolet-inactivation of conidia from two-component heterokaryons was investigated. In two-component heterokaryons with wild-type sensitivity to radiation inactivation, all three conidial fractions exhibited similar ultraviolet-inactivation curves. Each UV-sensitive mutation studied uniquely modified the ultraviolet-inactivation curves of conidia from two-component heterokaryons. In heterokaryons heterokaryotic for upr-I, the upr-I mutation was recessive and the repair function determined by the wild type allele was functional to some degree in homokaryotic upr-I conidia. All three conidial fractions of heterokaryons containing upr-I in both components showed increased sensitivity to ultraviolet light. The uvs-4 mutation was recessive and resulted in conidia with increased UV-sensitivity only when included in both components of a heterokaryon. Homokaryotic uvs-4 conidia, which arose from heterokaryons containing both uvs-4 and wild-type components, exhibited wild-type survival. Therefore, as with upr-I, there was a carryover the repair capability to conidia which were genetically UV-sensitive. The uvs-6 mutation, when included in one component of a two-component heterokaryon, resulted in increased UV-sensitivity of both heterokaryotic and homokaryotic uvs-6 conidia. When both components contained uvs-6, the UV-sensitivity of all three conidial fractions was increased and all showed similar inactivation curves. Thus, as with upr-I and uvs-4, there was a carryover of the wild-type repair capability to genetically uvs-6 conidia. Heterokaryon tests for complementation between two non-allelic UV-sensitive mutations showed that in heterokaryotic conidia, complete complementation occurred between upr-I and uvs-4.

Cell Nucleus↗
PubMed1990

Extragenic suppressors of mar2(sir3) mutations in Saccharomyces cerevisiae.

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by four trans-acting MAR (or SIR) loci. We have isolated extragenic suppressors of the mar2-1 mutation which, based on genetic complementation tests, define two additional loci involved in regulating the expression of HML and HMR. A strain with the genotype HMLa MAT alpha HMRa mar2-1 is sterile due to the simultaneous expression of a and alpha information. Two mutants exhibiting an alpha phenotype (which may result from the restoration of MAR/SIR repression) were isolated and genetically characterized. The mutations in these strains: (1) are recessive, (2) are capable of suppressing a mar2-deletion mutation, (3) are unlinked to MAT, (4) complement one another as well as the previously identified sum1-1 mutation, and (5) are not new alleles of the known MAR/SIR loci. We designate these new regulatory loci SUM2 and SUM3 (suppressor of mar). Unlike the sum1-1 mutation, suppression by sum2-1 and sum3-1 is mar2-locus specific. Both sum2-1 and sum3-1 affect the expression of a information at the HM loci. Transcript analysis shows a significant reduction in HMLa and HMRa gene transcription in mar2-1 sum2-1 and mar2-1 sum3-1 cells. Furthermore, we have found genetic evidence to suggest that mar2-1 sum2-1 cells exhibit only partial expression of silent alpha information. We conclude that the SUM2 and SUM3 gene products are required for expression of the HM loci and act downstream of the MAR2 (SIR3) gene function. Possible mechanisms for the action of the SUM gene products are discussed.

Alleles↗