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Results for “Genetic Complementation Test”

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PubMed1992

Development of a specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora: a review.

In recognition of the need for a more comprehensive data base for genetic risk assessment of human exposure to mutagenic agents in the environment, a model system was developed for specific-locus studies in Neurospora crassa. This lower eukaryotic organism permits the utilization of microbial techniques for recovery of large numbers of specific-locus mutations at two closely linked loci as well as their subsequent genetic analysis. In particular, this assay makes possible exploratory experiments with different environmental mutagens to obtain data on a wide variety of experimental conditions. Such data make it possible to study induction kinetics and mutational spectra in a manner that is not as yet feasible in higher eukaryotic organisms. The adenine-3 (ad-3) specific-locus assay was modeled after the 2-gene, morphological specific-locus assay in the dilute-short-ear region of the mouse, and it also detects forward-mutations at two closely linked loci, namely, ad-3A and ad-3B. Because ad-3 mutations are recovered by a direct method, based on the accumulation of a reddish-purple pigment in the vacuoles of the mycelium rather than their requirement for adenine, this system is both a morphological and biochemical specific-locus assay. The use of the ad-3 assay system in experiments with different environmental mutagens has provided precise dose-response curves not only for inactivation, but also the overall induction of ad-3 mutations. Genetic characterization of these ad-3 mutations by a series of 3 rapid and simple genetic tests permits the identification of 18 subclasses of gene/point mutations, and 12 subclasses of multilocus deletion mutations. These subclasses also include 3 different classes of multiple-locus mutations with separate sites of recessive lethal damage either in the immediately adjacent regions or elsewhere in the genome. In summary, this specific-locus assay provides a capability that is unique among eukaryotic organisms for the recovery and analysis of genetic damage at 2 closely linked loci.

Adenine↗

PubMed1993

Complementation assay of primer protein: gene expression systems of plasmid vectors support the infection of suppressor sensitive mutant phages M2 and phi 29.

Two different expression systems of genes of primer proteins (pE for phage M2, and p3 for phi 29) were constructed to study the protein primed DNA replication of Bacillus phages M2 and phi 29. In one system, expression of the genes was under the control of the inducible spac promoter, whereas in the other system, the expression was under the control of the constitutive promoter in plasmid pUB110. Complementation tests in vivo were performed between the primer proteins expressed by these systems and mutant phages having suppressor sensitive mutations in the genes of the primer proteins. The phages M2 susE and phi 29 sus3 were complemented by pE and p3 expressed by the systems, respectively. However, the complementation and apparent phage DNA synthesis were not detected in the combinations between susE of phage M2 and p3 of phage phi 29, and vice versa. Although pE and p3 proteins exhibited structurally and functionally similar characteristics, these proteins showed species specificity in the protein primed DNA replication of bacteriophages M2 and phi 29.

Bacillus Phages↗

PubMed1970

Studies on the in vitro assembly of bacteriophage phi 80 and phi 80-lambda hybrids.

Suppressor-sensitive (sus) mutants of bacteriophage phi80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative phi80 (the tail specifying cluster being distal). In vitro cross complementation between phi80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind phi80 tails to form viable phage, the union of phi80 heads and lambda tails was very much less efficient. Deletion mapping of the phi80 sus mutants, using both phi80 and i(phi80)h(lambda) deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.

Coliphages↗

PubMed1977

Frequencies of twelve ascus-types and arrangement of three genes from tetrad data.

Equations expressing the theoretical frequencies of twelve ascus-types in the tetrad analysis of a triply heterozygous diploid are described. Using these equations, a mapping procedure for a gene X, is proposed. The procedure requires that two genes, X and Y, of the same phenotype be heterozygous and that the map position of Y be known, and that another standard gene, Z, show an independent phenotype from X and Y. This procedure does not require the laborious allelism test of the segregants to determine the allelic 2:2 segregation in tetrads for the X and Y genes, which is indispensable for mapping by the conventional procedure. The exact placement of the X gene on a chromosome is possible by the chi2 minimization procedure in comparison with the expected frequencies of the six ascus-types or four spore-types deduced from the twelve expected ascus-types to give the optimal fit with the observed data.

Chromosome Mapping↗

PubMed1976

Isolation and characterization of mutations creating high-efficiency transcription initiation signals within the trp operon of Escherichia coli.

Two different mutational events generate promoter-active deoxyribonucleic acid sequences within the trp operon of Escherichia coli, probably through single base-pair changes. The mutations, obtained after ethyl methane sulfonate mutagenesis by selecting for elevated lac gene expression in a trp-lac fusion, are cis dominant and trans recessive with respect to their effects on the synthesis of downstream enzymes. One of the mutants (trpD11), obtained repeatedly under the selective conditions employed, prevents the formation of active phosphoribosyltransferase. The second mutation, trp3B, has no effects on any trp enzyme. By deletion mapping, trpD11 was localized near the operator-distal end of trpD, outside the segment of deoxyribonucleic acid that contains the low-efficiency internal promoter trpP2. Reversion to prototrophy of trpD11 was greatly stimulated by 2-aminopurine and ethyl methane sulfonate. Tests with suppressors indicated that trpD11 is a UAA (ochre) nonsense mutation. Under repression conditions, strains harboring either lesion in a normal trp operon synthesize the tryptophan biosynthetic enzymes in a noncoordinate fashion. The products of the operator-distal structural genes trpC, trpB, and trpA are formed at rates approximately 15-fold higher than those of wild type. The enzymes encoded by operator-proximal genes trpE and trpD are low or not detectable. Under derepression conditions, coordinate expression of the operon was observed.

Alleles↗

PubMed1988

Molecular characterization of a spontaneously generated new allele at a VNTR locus: no exchange of flanking DNA sequence.

Variable-number tandem-repeat (VNTR) DNA markers are contributing new power to human genetic studies because their hypervariable nature allows individualization at the DNA level. The practical value of VNTR markers has been well established for genetic linkage mapping, forensic biology, paternity testing, and monitoring of bone marrow transplants. A popular hypothesis attributes generation of variability at VNTR loci to unequal exchange between homologous chromosomes at meiosis. Contrary to the prediction of this hypothesis, we report here the finding that a newly generated VNTR allele is parental for closely spaced flanking markers; the new allele was generated by loss of one repeat unit, without exchange of flanking DNA sequences. These results are consistent with sister chromatid exchange and polymerase slippage or deletion, as well as with some models for gene conversion.

Alleles↗

PubMed1983

[Chinese hamster cells mutant for the hypoxanthine-guanine phosphoribosyltransferase locus. IV. The biochemical characteristics of the hypoxanthine-guanine phosphoribosyltransferase of hybrid clones obtained by intragenic complementation].

A biochemical study of hypoxanthine-guanine-phosphoribosyltransferase (HPRT) has been carried out in hybrid clones of Chinese hamster cells obtained in complementation experiments. A wide range of biochemical characteristics made it possible to identify a hybrid form of HPRT differing from the enzyme of parental clones in virtually every hybrid tested. The presence of hybrid HPRT was detected by the changed kinetic properties of temperature sensitivity and electrophoretic mobility compared to the enzyme in mutant cells. Since HPRT consists of identical subunits, the hybrid nature of the enzyme in cells obtained through hybridization of HPRT-mutant clones may be regarded as evidence for intragenic complementation. None of the hybrid clones contained an enzyme with the normal properties. Groups of hybrids with similar biochemical characteristics of HPRT can be obtained, if one of the mutant partners involved in hybridization belongs to one and the same complementation group; the major characteristics of hybrid HPRT are then determined by the partner having the higher level of enzyme activity. The series of studies of intragenic complementation in the HPRT gene is summarized.

Animals↗

PubMed1996

Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytes. II. Induced chromosome loss and dominant visible mutations.

The rates of X-ray induced loss of chromosome 19 in mouse oocytes were investigated in 2 experiments using a genetic complementation test. After 1 Gy of acute X-rays to immediately preovulatory stage oocytes, chromosome 19 loss was estimated to have occurred in 1.68% of cells. In comparison, after 4 Gy of acute X-rays to dictyate stage oocytes, the rate was estimated at 1.18%. The slightly higher rate of chromosome loss in the former cell stage after a smaller dose of radiation reflects the known increased radiosensitivity of mouse oocytes in the period shortly before ovulation. Comparison of the observations here for chromosome 19 with published data for chromosome 1 suggests that chromosome length is one of the principal factors in determining the initial rate of induced loss in mouse oocytes. Ten dominant visible mutations were recovered among 1674 offspring following irradiation of preovulatory oocytes, and 8 in 2025 offspring after treatment of dictyate cells. Nine dominant mutations were karyotyped, 5 of these were found to be associated with a visible chromosome rearrangement. The data obtained in the present study show that radiation-induced chromosome anomalies in female germ cells are not all filtered out by prenatal embryonic death but that a proportion has the potential to contribute to the genetic burden of the next generation.

Animals↗

PubMed2000

Complementation of physiological and behavioral defects by a slowpoke Ca(2+) -activated K(+) channel transgene.

The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo(4) mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types.

Action Potentials↗

PubMed1999

N-ethyl-N-nitrosourea mutagenesis of a 6- to 11-cM subregion of the Fah-Hbb interval of mouse chromosome 7: Completed testing of 4557 gametes and deletion mapping and complementation analysis of 31 mutations.

An interval of mouse chromosome (Chr) 7 surrounding the albino (Tyr; c) locus, and corresponding to a long 6- to 11-cM Tyr deletion, has been the target of a large-scale mutagenesis screen with the chemical supermutagen N-ethyl-N-nitrosourea (ENU). A segment of Chr 7, from a mutagenized genome bred from ENU-treated males, was made hemizygous opposite the long deletion for recognition and recovery of new recessive mutations that map within the albino deletion complex. Over 6000 pedigrees were analyzed, and 4557 of these were completely tested for mutations specifying both lethal and gross visible phenotypes. Thirty-one nonclustered mutations were identified and assigned to 10 complementation groups by pairwise trans-complementation crosses. Deletion-mapping analyses, using the extensive series of radiation-induced Tyr deletions, placed the loci defined by each of these complementation groups into defined intervals of the Tyr-region deletion map, which facilitates the identification of each locus on physical and transcription maps of the region. These mutations identified seven new loci and provided new ENU-induced alleles at three previously defined loci. Interestingly, no mutations were recovered that recapitulated three phenotypes defined by analysis of homozygous or partially complementing albino deletions. On the basis of our experience with this screen, we discuss a number of issues (e.g., locus mutability, failure to saturate, number of gametes to screen, allelic series) of concern when application of chemical mutagenesis screens to megabase regions of the mouse genome is considered.

Animals↗

PubMed1997

Positive control of the two-component RcsC/B signal transduction network by DjlA: a member of the DnaJ family of molecular chaperones in Escherichia coli.

The membrane-anchored DjIA protein represents the third member of the DnaJ 'J-domain' family of Escherichia coli that includes DnaJ and CbpA. DjIA possesses a J-domain at its extreme C-terminus but shares no additional homology with DnaJ. Our genetic analysis suggests that DjIA acts in concert with the RcsB/C two-component signal transduction system to augment induction of the cps (capsular polysaccharide) operon and synthesis of colanic acid mucoid capsule. The DjIA J-domain is essential for the observed stimulation of this pathway as deletion, or introduction of the mutation H233Q, within the highly conserved HPD tripeptide abolished all inducing activity. Deletion of the transmembrane anchor sequence also abolished all inducing activity. djIA is not an essential gene under all conditions tested, nor is it essential for mucoid capsule biosynthesis; however, strong overexpression leads to rapid loss of cell viability suggesting that the gene is normally tightly regulated. Northern analysis revealed that djIA message was extremely unstable but could be induced or stabilized in response to cold shock. The activation of the cps operon by DjIA is dependent upon both DnaK(Hsp70) and GrpE, and therefore we propose a role for DjIA, together with this chaperone machine, as a novel regulator of a two-component histidine kinase signal transduction pathway.

Amino Acid Sequence↗

PubMed1993

A yeast expression system for human galactose-1-phosphate uridylyltransferase.

Galactose-1-phosphate uridylyltransferase (GALT) (UTP: alpha-D-hexose-1-phosphate uridylyltransferase, EC 2.7.7.10) is an essential enzyme of the Leloir pathway of galactose metabolism. Mutations in human GALT are associated with the potentially lethal disorder galactosemia, which affects 1 in 30,000-60,000 live-born infants. Although a number of base substitutions have been identified in the GALT alleles of galactosemia patients, the detailed biochemical impact of these mutations on GALT enzymatic activity remains obscure. Similarly, little is known about the sequence/structure/function relationships for wild-type human GALT. As a first step toward addressing these questions, we have developed a yeast-based expression system for the human enzyme. The wild-type human GALT coding sequence has been introduced into a strain of Saccharomyces cerevisiae that carries a disruption of the GALT-encoding GAL7 gene and, therefore, expresses no endogenous GALT. Transformants were tested for restoration of GALT activity both indirectly, by cell growth on galactose, and directly, by analysis of enzyme activity in cell extracts. The results of both tests were striking; wild-type human GALT functioned in yeast almost as well as the endogenous enzyme. In contrast, cells transformed with either human or yeast GALT sequences engineered to carry a common human GALT mutation, Q188R (changing Gln188 to Arg), exhibited essentially no detectable GALT activity and failed to grow on galactose. Lymphoblasts from patients homozygous for the Q188R mutation similarly exhibited essentially no detectable GALT activity in parallel assays. The results reported here establish the utility of the yeast-based expression system for human GALT and set the stage for more detailed studies of this important enzyme and its role in galactosemia.

Base Sequence↗

PubMed2001

Human immunodeficiency virus type 1 VPU protein affects Sindbis virus glycoprotein processing and enhances membrane permeabilization.

The human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that forms oligomeric structures in membranes. Expression of vpu using Sindbis virus (SV) as a vector leads to permeabilization of plasma membrane to hydrophilic molecules and impaired maturation of wild type SV glycoproteins in BHK cells. The 6K protein is a membrane protein encoded in the SV genome that facilitates budding of virus particles and regulates transport of viral glycoproteins through the secretory pathway. Some of these functions were assayed with a SV mutant containing a partially deleted 6K gene. Transfection of BHK cells with pSVDelta6K vector rendered defective SVDelta6K virus, which had lower membrane permeabilization, impaired glycoprotein processing, and deficient virion budding. Replacement of 6K function by HIV-1 Vpu in SVDelta6K was tested by cloning the vpu gene under a duplicated late promoter (pSVDelta6KVpu). The presence of the vpu gene in the 6K-deleted virus enhances membrane permeability, modifies glycoprotein precursor processing, and facilitates infectious virus particle production. Restoration of infectivity of 6K-deleted SV by Vpu was evidenced by increased PFU production and cytopathic effect on infected cells. The modification of SVDelta6K glycoprotein maturation by Vpu was reflected in augmented processing of B precursor and impairment of PE2 cleavage. Taken together, our data support the notion that HIV-1 Vpu and SV 6K proteins share some analogous functions.

Animals↗

PubMed1998

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle.

Animals↗

PubMed1998

A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex.

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98-63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98-63 spc110-221 cells is caused by the failure of Spc98-63p to interact with Spc110-221p. In contrast, the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

Calmodulin-Binding Proteins↗

PubMed1991

Limits of the distal inversion in the t complex of the house mouse: evidence from linkage disequilibria.

The suppression of crossing-over and the consequent linkage disequilibrium of genetic markers within the t complex of the house mouse is caused by two large and two short inversions. The inversions encompass a region that is some 15 centiMorgans (cM) long in the homologous wild-type chromosome. The limits of the proximal inversions are reasonably well-defined, those of the distal inversions much less so. We have recently obtained seven new DNA markers (D17Tu) which in wild-type chromosomes map into the region presumably involved in the distal inversions of the t chromosomes. To find out whether the corresponding loci do indeed reside within the inversions, we have determined their variability among 26 complete and 12 partial t haplotypes. In addition, we also tested the same collection of t haplotypes for their variability at five D17Leh, Hba-ps4, Pim-1, and Crya-1 loci. The results suggest that the distal end of the most distal inversion lies between the loci D17Leh467 and D17Tu26. The proximal end of the large distal inversion was mapped to the region between the D17Tu43 and Hba-ps4 loci, but this assignment is rather ambiguous. The loci Pim-1, Crya-1, and the H-2 complex, which have been mapped between the Hba-ps4 and Grr within the large distal inversion, behave as if they recombine from time to time with their wild-type homologs.

Animals↗

PubMed1992

Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast.

We have constructed a galactose-inducible expression library by cloning yeast cDNAs unidirectionally under control of the GAL1 promoter in a centromeric shuttle vector. Eleven independent libraries were made each with an average size of about 1 x 10(6) clones, about 50 times larger than the reported mRNA population in a yeast cell. From this library, LEU2 and HIS3 cDNAs were recovered at a frequency of about 1 in 10(4) and in 12 out of 13 cases these were expressed in a galactose-dependent manner. Sequence analysis of leu2 and his3 complementing cDNAs indicates that they contain all the coding sequence and much of the 5' untranslated region. To test the utility of the library for the identification of genes whose overexpression confers a specific phenotype, we screened 25,000 yeast transformants for lethality on galactose. Among 15 clones that showed galactose inducible lethality were cDNAs encoding structural proteins, including ACT1 (actin), TUB2 (beta-tubulin) and ABP1 (actin-binding protein 1), and genes in signal transduction pathways, including TPK1 (a cAMP-dependent protein kinase) and GLC7 (type 1 protein phosphatase). cDNAs overexpressing NHPB (nonhistone protein B) and NSR1 (nuclear sequence recognition protein) were also found to be lethal. Among these, ACT1 was isolated four times, and NSR1 three times. The useful features of this library for cDNA cloning in yeast by complementation, and for the identification of genes whose over-expression confers specific phenotypes, are discussed.

Base Sequence↗

PubMed1996

Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

Amino Acid Sequence↗