Interallelic Complementation Research Articles

Reversion and interallelic complementation at four urease loci in Neurospora crassa

Analysis of heat stability of urease in extracts of 24 revertants, six for each of four ure loci, revealed that at least one revertant for each locus had a heat stability about one-third that of wild type. Similar results were obtained with urease formed by interallelic complementation at the ure-2 and ure-4 loci, but interallelic complementation at the ure-1 and ure-3 loci produced insufficient urease activity for analysis. The data are interpreted to suggest, as a tentative model, a structural function for each of the four ure loci.

A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme

The pyrimidine-3 locus of Neurospora crassa specifies a multienzyme complex comprising pyrimidine-specific carbamoyl phosphate synthase (CPSpyr) and aspartate carbamoyl transferase (ACT). It appears to be divided into a translationally proximal CPS-specific region and a distal ACT-specific region. Levels of complementation for ACT activity between pairs of four pyr-3 CPS+ ACT- mutants showed a range from 12% to 68% of the wild-type level of the enzyme. This is interpreted as interallelic complementation, contradicting certain earlier suggestion of two dissimilar ACT subunits. Proteolysis of an extract from a heterokaryon formed from two of the above CPS+ ACT- alleles (alpha and beta) did not lead to loss of ACT activity, but led to the formation of a fragment with ACT activity with a similar molecular weight (92,000 daltons) to that produced in extracts of wild type strain. The pyr-3 polar mutant 43-174 which is enzymatically CPS+ ACT- and which fails to complement with any other CPS+ ACT- alleles, thus suggesting its location towards the proximal end of the ACT region, has CPS activity associated with a form of 180,000 daltons molecular weight. These findings are used to contruct a model for structure of the native enzyme complex.

Evidence against mutational "hot-spots" at aggregation loci in Dictyostelium discoideum.

Complementation tests were performed on 10 strains of Dictyostelium discoideum which carry developmental mutations representing aggregation loci identified previously in two independent studies. When the 5 aggregation-deficient strains representing loci CGI-5 were fused with the 5 strains carrying mutations at loci ago A-E, all 25 crosses produced aggregation-competent diploids. Complicating factors, such as negative gene interactions and possible interallelic complementation are discussed. The results of this experiment suggest that the 10 aggregation loci identified in the two studies are different and that aggregation loci in D. discoideum are probably not associated with significant mutational "hot-spots".

Resistance to 2-deoxyglucose in yeast: A direct selection of mutants lacking glucose-phosphorylating enzymes

When strains of Saccharomyces cerevisiae carrying a single glucose-phosphorylating enzyme such as hexokinase Pl or hexokinase P2 or glucokinase, are subjected to the selection pressure against the toxic sugar 2-deoxyglucose, the majority of survivors are mutants lacking the respective enzymes. All the 2-deoxyglucose-resistant segregants recovered from backcrosses of these mutants to a wild type strain are glucose-negative and all the sensitive ones are glucose-positive. The hexokinase mutations are located in the same complementation groups as defined by the structural genes of hexokinase P1 and hexokinase P2. No interallelic complementation has been observed either in hexokinase P1 or in hexokinase P2 amongst a total of 4 X 64, and 5 X 60 different combinations of independent mutants at the hxk1 and hxk2 loci respectively. There appears to be neither a common genetic regulator controlling two or more of these glucose-phosphorylating enzymes nor a sugar carrier that can be dispensed with.

Extension of the limits of the XDH structural element in Drosophila melanogaster.

Experiments expanding the array of mutants affecting the xanthine dehydrogenase (XDH) structural element in Drosophila melanogaster are described. These include rosy eye color mutants which exhibit interallelic complementation, and mutants with normal eye color but lowered levels of XDH. Evidence is presented which argues that these are structural alterations in the enzyme. Recombination experiments were performed using these mutants as well as some electrophoretic variants. The two ends of the rosy locus are marked with mutant sites which are clearly structural in nature; the XDH structural element and the rosy null mutant map are completely concordant. A possible procedure to recover control element mutants is described.

A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis.

A series of aggregation-deficient (aggregateless) mutants were isolated in genetically marked haploid strains of the cellular slime mould Dictyostelium discoideum. Diploids were produced from pairs of such haploid mutants by a fusion system based on this organism's parasexual cycle. The diploids were isolated from the haploids by using complementation of non-allelic growth-temperature-sensitive mutations and selection at the restrictive temperature. Complementation between the aggregateless mutations uas then assessed in 419 diploids so formed. The non-complementing aggregateless mutations fell into five complementation groups (ago A, B, C, D, and E) and a dominant aggregation class that allowed little or no aggregation when present in a diploid with any of the other mutations tested or the parental wild type. Complicating factors, including partial dominance, multiple mutations, and possible interallelic conplementation, are discussed. Data on the linkage of the aggregateless mutations was obtained by using recessive drug resistance mutations on three linkage groups to segregate haploids from the diploids. Calculations from our results suggest a genetic complexity of about 50 genes that are specific and essential for aggregation.

Arg-7 mutant X wild-type crosses in Chlamydomonas reinhardi: study of the enzyme produced in diploid strains.

The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL. Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size. The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1 X pab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned. These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.

Genetic and physiological characterization of met15 mutants of Saccharomyces cerevisiae: a selective system for forward and reverse mutations.

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus. -- It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.

Temperature-sensitive mutations of the notch locus in Drosophila melanogaster.

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)Nts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax59d5 is found to be temperature-sensitive, while fag, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fag, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fag, spl, and l(1)N cause defects at specific, non-overlapping times in development. -- We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.

Specificity and genetics of S-adenosylmethionine transport in Saccharomyces cerevisiae.

The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine congruent to S-adenosyl-(5')-3-methyl-thiopropylamine greater than S-adenosylethionine greater than S-inosylmethionine greater than S-adenosylhomocysteins. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-insoyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as group important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.

Phenylalanine hydroxylase activity in liver biopsies from hyperphenylalaninemia heterozygotes: deviation from proportionality with gene dosage.

Liver biopsy samples from the patients with hyperphenylalaninemia have an average of 5 percent of the normal hydroxylase activity. The parents of the patients have between 7.3 percent (excluding the value for one parent) and 10 percent of the normal hepatic hydroxylase activity. An explanation for these findings involves negative interallelic complementation, which involves protein-protein interaction between subunits in a multimeric enzyme. In support of this model is the evidence that rat liver phenylalanine hydroxylase is a multimeric protein composed of two electrophoretically distinguishable subunits.

THE MOLECULAR BASIS FOR ALLELIC COMPLEMENTATION OF ALCOHOL DEHYDROGENASE MUTANTS OF MAIZE

Studies on interallelic complementation with two temperature-sensitive alcohol dehydrogenase mutants in maize are described. The data suggest that the phenomenon results from instability or abnormal maturation of the mutant homodimer rather than correction of configurational defects in mutant heterodimers.

Mutant hamster cells exhibiting a pleiotropic effect on carbohydrate metabolism.

Sixty-seven galactose negative (Gal minus) clones were established from survivors after treatment of Chinese hamster lung (V79) cells with 5-bromodeoxyuridine and black light. The mutational origin of these clonal isolates was inferred on the basis of the previously demonstrated mutagenic action of the combined treatment, persistence of the Gal minus phenotype in cell progeny in the absence of selection, conditional lethality of three isolates, interallelic complementation, and assignment of a homologous human gene to chromosome A2. In contrast to the parental cells, Gal minus mutants could not utilize exogenous galactose, mannose, fructose, galactose 1-phosphate, glucose 1-phosphate, or glucose 6-phosphate. Permeation of galactose into mutant cells appeared unimpaired. In intra- and interspecific cell hybrids, the mutation causing the Gal minus phenotype behaved like a recessive character. The pleiotropic nutritional response was due not to deficiency of any one specific enzyme in the Leloir pathway but to significant reduction in activities of phosphoglucomutase, NADP-dependent isocitrate dehydrogenase, and perhaps other enzymes. In temperature-sensitive Gal minus mutants grown at permissive temperature or in complementing intra- and interspecific cell hybrids, the activities of these enzymes were restored to normal levels, accompanied by a regained ability to use the particular hexoses or hexose monophosphates. We postulate that the change from Gal + to Gal minus phenotype in hamster cells could be due to mutations at a regulatory gene locus, or at a yet unknown locus with enzymic defect that causes secondary metabolic imbalances.

The Genetic Defect in the De Sanctis-Cacchione Syndrome

A family with five children affected with the de Sanctis-Cacchione (DSC) syndrome is presented. Ultraviolet light-induced DNA repair synthesis, as measured by autoradiography as well as by isopyknic centrifugation was studied in cell strains from three affected members and their heterozygote parents. Similar, low amounts of repair synthesis were found in the cells of all patients. In cell strains from the heterozygous parents a reduced repair activity was demonstrated. Complementation studies with cell hybridization techniques were performed using cells of our patients, cells of one of our patients and those of an unrelated DSC patient, and cells of one of our patients and those of a classic xeroderma pigmentosum (XP) patient. The fusion of cells front a classic XP patient with those ot one of our DSC patients resulted in hybrids that had nomal amounts of repair synthesis. The hybrids resulting from the other fusions revealed very low or negligible repair activity. These results demonstrate genetics heterogeneity between DSC and classical XP cases. They strongly suggest either intergenic or interallelic complementation during hybridization between XP and DSC cells.

Functional aspects of fluoroacetate resistance in Coprinus with special reference to acetyl-CoA synthetase deficiency

Mutants of Coprinus lagopus unable to utilise acetate were selected on a glucose medium containing the toxic analogue fluoroacetic acid. The mutants map at three unlinked loci, designated acu-1, acu-11, and acu-12. Mutations at the acu-1 locus lead to loss of acetyl-CoA synthetase activity; the functions of the other two loci were not resolved but were shown not to involve either this enzyme or isocitrate lyase. Mutants at different loci showed normal complementation when combined in pairs in dikaryons or heterozygous diploids showing that there is no localised intranuclear activity of the gene products involved. Tests for interallelic complementation between twenty-five differently isolated acu-1 mutants proved negative, indicating that the acetyl-CoA synthetase activity is carried on a single polypeptide.

Allelic contracomplementation possible mechanism for initiating sympatric speciation.

IN Drosophila melanogaster, certain heterozygous combinations of viable alleles of the sex-linked Abruptex locus are lethal. This new type of allelic interaction has been called contracomplementation1. The phenomenon is equivalent or related to negative complementation, but the term contracomplementation is more descriptive in this specific case. The following heterozygous combinations of viable Abruptex alleles are found to be lethal: Ax9 B2/AxE2, Ax9 B2/Ax16172 (ref. 2), Ax28/Ax7 1d (ref. 1), Ax7 1d/Ax9 B2, and Ax28/Ax16172. Interallelic complementation has been supposed to have certain evolutionary significance; it is an intermediate stage in the evolution of heteromultimeric proteins, such as human haemoglobin, from homomultimeric proteins3. Thus, interallelic complementation may be a stage in the progressive evolution of a population. Contracomplementation as an opposite of complementation should accordingly have an ‘opposite’ evolutionary significance, being an initiation mechanism in splitting one population into two parts, that is an initiation mechanism of sympatric speciation.

Intragenic complementation between temperature-sensitive mutants of gene 42 (dCMP hydroxymethylase) of bacteriophage T4.

Interallelic complementation between certain temperature-sensitive mutants of gene 42 of bacteriophage T4 was demonstrated by measuring the incorporation of labeled thymine into DNA.

Characterization of chemically induced mutations in the ad-1 locus of Schizosaccharomyces pombe

An attempt to assess the frequencies of mutations of the base-pair substitution type and of the addition/deletion type was undertaken in 64 ICR-170-, 28 MNNG- and 50 EMS-induced ad-1 mutant strains of Schizosaccharomyces pombe. By using temperature sensitivity, osmotic remediability, and interallelic complementation, sensitivity to nonsense suppressors and revertibility tests with 2-methoxy- 6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) and N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) as diagnostic criteria to distinguish between the two types of alterations, the following conclusions were reached: ( 1) The mutational alteration in all of the MNNG-induced and in at least 74% of the ethyl methanesulfonate(EMS)-induced mutant strains is of the base-pair substitution type; ( 2) Both types of mutation were found amongst ICR-170-induced strains.

Interallelic complementation in hydrid cells derived from Chinese hamster diploid clones deficient in hypoxanthine-guanine phosphoribosyl-transferase activity

Activity of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) has been demonstrated in the hybrid cells formed from the fusion of clonal cells from four Chinese hamster diploid clones, Cl. 250, 252, 253, and 254 cells derived from clonal wild type cells, deficient in this enzyme activity, by the stepwise treatment with 8-azaguanine and 6-thioguanine. The HGPRT-positive cells, characterized by tetraploid karyology, were isolated clonally by the chemical selection (HAT) in 3 hybrid mixtures; Cl. 250 × 254, 252 × 254, and 253 × 254 cells, while none was isolated in another seven hybrid mixtures including 4 homologous mixtures. The enzyme activity was proved by enzymological as well as autoradiographical studies with the use of 3H-hypoxanthine. These results indicate that mutually complementable alterations rather than gene deletions were induced in the 2 cistrons of the locus responsible for the relevant enzyme activity in hamster cells by analog treatment. Interallelic complementation occurred in these hybrid cells.

Genetic analysis of replicating instabilities in yeast

Strains showing ethyl methanesulfonate (EMS)-induced replicating instability were genetically analysed to test whether within a given line, mosaics from different plating generations carry a mutation at the same site within the locus. A forward mutation system involving five loci controlling adenine biosynthesis in Schizosaccharomyces pombe was used. Genetic analysis was carried out by interallelic complementation and intragenic recombination tests. The data showed that EMS-induced instabilities are site-specific in being confined to the same recombination unit. This finding is discussed in relation to the possible mechanism(s) of replicating instabilities after different mutagenic treatments in a variety of biological systems.