Triallelic complementation and hybrid enzyme formation in Chlamydomonas reinhardi

Summary In Chlamydomonas, the arg-7 cistron (linkage group I) is the structural gene for the multimeric (probably pentameric) enzyme argininosuccinate lyase. Most of the alleles of the cistron were previously shown to complement in some pair combinations, giving rise to phenotypically wild-type diploids.By crossing diploid (mt^-) and haploid (mt⁺) cells bearing different markers of auxotrophy, seven different presumptive triploid strains, phenotypically wild-type, were isolated. Each strain had 3 different arg-7 alleles or 2 mutant alleles associated with a wild one.The isolates were cytologically and biochemically analyzed: it could be concluded that they were triploid or ar least trisomic for the linkage group I.The specific activity and the thermosensitivity of the lyase were compared in the different triploids and in the diploids bearing two of the three corresponding arg-7 alleles. In most cases, the enzyme formed by triallelic complementation was more active and more heat resistant than the enzyme formed by diallelic complementation. These results can be interpreted by assuming that hybrid enzyme is formed by interaction between the products of the three different alleles. They provide a molecular basis for explaining the increased vigor often found in polyploids.     

Allelic complementation between arg-7 mutants in Chlamydomonas reinhardi

In Chlamydomonas reinhardi, the arg-7 locus is the structural gene for the enzyme argininosuccinate lyase (ASL) which catalyzes the last reaction in arginine biosynthesisOut of the nine ASL^--mutants so far investigated, seven were shown to fall within three distinct groups of complementation.The activity of ASL produced in diploids formed by complementation is much lower than the activity of ASL in the wild-type strain.The study of the heat sensitivity of the enzymes seems to indicate that in every diploid only one enzymic variety is formed which in most cases displays a much greater sensitivity to heat than the enzyme formed by the wild-type strain.These results are discussed in relation to the hypothesis according to which the arg-7 gene in Chlamydomonas reinhardi corresponds to one cistron and the ASL enzyme is composed of at least two identical polypeptide chains.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique     

The actual biochemical block in the arg-2 mutant of Chlamydomonas reinhardi

Arg-2, one of the first arginine-requiring mutants isolated in Chlamydomonas reinhardi, has long been regarded as lacking the enzyme argininosuccinate synthetase. In view of various discrepancies found in the literature, the position of this mutant has been reviewed. The results show that arg-2 has a normal argininosuccinate synthetase activity but lacks argininosuccinate lyase. This finding is in agreement with the results of recombination and complementation analysis, which indicate that arg-2 is included in the arg-7 cistron.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

Mechanisms for intragenic complementation at the human argininosuccinate lyase locus.

Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.     

Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli

Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library. The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals. Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase. This homology suggests a similar mechanism for catalysis. Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion. This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.     

Functional Complementation in Yeast Allows Molecular Characterization of Missense Argininosuccinate Lyase Mutations

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL^null strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, “compound heterozygous” yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.Argininosuccinic aciduria (ASAuria, MIM 207900)³ is an autosomal recessive disorder of the urea cycle caused by mutations of the ASL gene (hASL, MIM 608310), encoding argininosuccinate lyase (ASL; EC 4.3.2.1.) (1). This enzyme is ubiquitously expressed and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. ASL belongs to a superfamily of hydrolases that includes adenylosuccinate lyase and fumarase, which share a homotetrameric structure and a similar catalytic mechanism. The tetrameric structure of ASL accounts for the phenomenon of intragenic complementation. This particular situation occurs when a multimeric protein is formed from subunits produced by differently mutated alleles of the same gene. On complementation, a partially functional hybrid protein is produced from the two distinct types of mutant subunits, neither of which individually has appreciable enzymatic activity (2).ASL participates to the urea cycle, and in humans it is essential for ammonia detoxification, whereas in lower organisms it is required for the biosynthesis of arginine. Saccharomyces cerevisiae strains harboring a deletion of the homolog of human ASL (ARG4) cannot grow on media lacking arginine (3).ASAuria is characterized by accumulation of argininosuccinic acid (ASA) in body fluids, and severe hyperammonaemia. The disease displays clinical heterogeneity with two main clinical phenotypes: the acute/neonatal onset form, with symptoms rapidly progressing to deep coma, apnea, and death (1), and the subacute/late onset type, which is diagnosed in infancy or childhood (4). Such patients may present simply with mental retardation or an epileptic disorder. In both types the diagnosis is established unambiguously by measuring plasma levels of ammonia (not always elevated in the late onset form), ASA, and its anhydrides by plasma amino acids assay (1). Over 40 mutations of the ASL gene have been reported, both amino acid substitutions and truncating variants, which are scattered throughout the gene (5, 6).We have previously reported the identification of novel mutations of the ASL gene in a cohort of Italian patients (7). In this study we employed a yeast model to validate the pathogenicity of missense ASL mutations found in our cohort, to study the effects of different allelic combinations, and to establish possible genotype-phenotype correlations.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary 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-1xpab-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.     

Complementation experiments between conditional lethal mutants of bacteriophage ?X174

Summary Complementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage X174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.     

Genetics of Aspergillus flavus: linkage of aflatoxin mutants

Eight aflatoxin (afl) mutants of Aspergillus flavus were induced with N-methyl-N'-nitro-N-nitrosoguanidine. Heterozygous diploids formed between afl mutants and tester strains revealed that each afl mutant was recessive. Haploids selected from these heterozygous diploids indicated the linkage of all eight afl mutants to markers on group VII. These include previously mapped arg-7 (arginine), leu (leucine), dominant afl-1, and nor which accumulates norsolorinic acid that is visible as an orange-red pigment. Diploid complementation tests indicated that all but two afl mutants were nonallelic. Diploids homozygous for nor, resulting from crossing-over, were isolated and used to map new afl genes.     

Genetic studies of the pyrimidine permeases from Saccharomyces cerevisiae: lack of intragenic complementation.

Summary A search for intragenic complementation of mutants of the cytosine and uracil permeases of Saccharomyces cerevisiae was made. Among numerous diploïd pairs of mutants of the cytosine permease gene no complementation was found. Similarly negative results were obtained with pairs of mutants of the uracil permease. The significance of these results is discussed.     

Argininosuccinate lyase deficiency: evidence for heterogeneous structural gene mutations by immunoblotting.

Argininosuccinate lyase (AS lyase) deficiency is an inborn error of the urea cycle with extensive clinical and genetic heterogeneity. We investigated the biochemical basis of the enzyme defect and the genetic heterogeneity in this disorder using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of fibroblast extracts. The AS lyase monomer in control fibroblasts was present in two bands of approximately 51 and approximately 49 Kd. Each of 28 mutant strains had some cross-reactive material (CRM) of the lower (approximately 49 Kd) MW, in quantities ranging from trace to substantial levels. The approximately 51 Kd band was found in only six mutants with near-normal amounts of AS lyase CRM or high residual enzyme activity. The residual AS lyase enzyme activity in a mutant did not necessarily reflect the amount of the 49-51 Kd monomer in that strain. In contrast, there was a strong general correlation between the quantity of 49-51 Kd CRM in a mutant and the frequency of complementation by that mutant. In addition to the CRM of normal molecular weight (MW) (49-51 Kd), the majority of mutants (but not controls) had significant CRM present in one to five bands of MW less than 49 Kd. The immunoprecipitation of at least one of these low MW bands was inhibited by purified human AS lyase. Mutants indistinguishable by clinical, enzymatic, or complementation analysis have been shown to be heterogeneous in their content of AS lyase CRM, greatly extending the number of distinct mutant alleles identified at this locus. These data demonstrate that multiple unique mutations in the structural gene coding for the monomer cause AS lyase deficiency and that the AS lyase monomers made by these mutants may be unstable. Integration of these findings with enzymatic and complementation data has indicated the functional domain of the AS lyase monomer likely to be altered in certain mutants.     

An analysis of xanthine dehydrogenase negative mutants of the rosy locus in Drosophila melanogaster.

Eighteen alleles of the rosy locus in Drosophila melanogaster were characterized to identify putative nonsense mutants. Seven alleles exhibited no evidence of intragenic complementation, no evidence of immunological complementation, no evidence of immunological cross-reactivity to antibodies elicited by wild type xanthine dehydrogenase (XDH), and of course were completely deficient in measurable XDH activity. It is possible that one or more of these highly negative ry alleles are nonsense mutants. The remaining eleven ry alleles code for XDH molecules that retain some antigenic similarities to the wild type enzyme as assessed by immunoelectrophoresis and six of these eleven were capable of intragenic complementation.     

Process of Infection with Bacteriophage φX174: XXXV. Cistron VIII

Twenty-two new amber and ochre mutants of phiX174 were isolated and classified into complementation groups. Three ochre mutants gave positive complementation tests with reference mutants in the seven previously defined groups and thus represent an eighth cistron. Studies of the physiology of infection in the nonpermissive condition for mutants in cistron VIII yielded the following information. (i) Replicative-form synthesis proceeds at a normal rate, and is turned off at the usual time. (ii) Synthesis of single-stranded deoxyribonucleic acid (DNA) is delayed until nearly 40 min after infection (in the absence of lysis), at which time a slow synthesis of infectious phage particles commences. The synthesis of infectious particles at late times is interpreted as a consequence of "leakage," and indicates that the cistron VIII product is required in very small quantities. (iii) During the normal period of single-strand synthesis, most of the replicative-form DNA is found in a form with properties similar to those of the transient intermediates of single-strand DNA synthesized during normal infection.     

New arginine-requiring mutants in Chlamydomonas reinhardtii

Twelve arginine-requiring mutants of the unicellular green alga Chlamydomonas reinhardtii previously isolated in our laboratory were investigated to find new blocks in the biosynthetic pathway of arginine. In addition to the already described mutants lacking acetylglutamyl phosphate reductase (arg 1), ornithine carbamoyltransferase (arg4) and argininosuccinate lyase (arg7), three new types of mutants were found lacking acetylornithine aminotransferase (arg9-1, arg9-2), acetylornithine glutamate transacetylase (arg10) and argininosuccinate synthetase (arg8-1, arg8-2, arg8-3) respectively. The genetic analysis of these new mutants showed that arg9 and arg8 are unlinked to the other arginine markers and that arg10 probably carries a chromosomal mutation inducing a very high lethality of meiotic products.Abbreviations WT wild-type - mt mating-type - SP spore plating - ZP zygote plating     

Amino acid sequence of rat argininosuccinate lyase deduced from cDNA

Argininosuccinate lyase [EC 4.3.2.1] is an enzyme of the urea cycle in the liver of ureotelic animals. The enzymes of the urea cycle, including argininosuccinate lyase, are regulated developmentally and in response to dietary and hormonal changes, in a coordinated manner. The nucleotide sequence of rat argininosuccinate lyase cDNA, which was isolated previously (Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimula, S., & Mori, M. (1986) Biochem. Int. 13, 433-438), was determined. The cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,549), a 5'-untranslated sequence of 150 bp, and a 3'-untranslated sequence of 41 bp. The amino acid composition of rat liver argininosuccinate lyase predicted from the cDNA sequence is in close agreement with that determined on the purified enzyme. The predicted amino acid sequences of the human and yeast enzymes along the entire sequences (94 and 39%, respectively), except for a region of 66 residues of the human enzyme near the COOH terminus. However, the sequence of this region of the human enzyme predicted from another reading frame of the human enzyme cDNA is homologous with the corresponding sequences of the rat and yeast enzymes. Therefore, the human sequence should be re-examined. Lysine-51, the putative binding site for argininosuccinate, and the flanking sequences are highly conserved among the rat, steer, human, and yeast enzymes.     

An enzymatic method for the assay of serum argininosuccinate lyase

A rapid enzymatic method was developed for the assay of serum argininosuccinate lyase (ASAL: EC 4.3.2.1) which is a useful marker enzyme for diagnosis of parenchymal liver diseases. Fumarate, liberated from argininosuccinate in the lyase-mediated reaction, was converted to pyruvate via L-malate by the actions of fumarase and malic enzyme in the presence of NADP+. The NADPH formed was then oxidized with a diaphorase-resazurin system to give a highly fluorescent resorufin. All the enzymatic reactions proceeded continuously in 0.1 M Tris-HCl buffer (pH 7.5) and allowed direct assay of ASAL in serum by monitoring the increase in the fluorescence intensity due to resorufin. The method is rapid and sensitive; only 50 microliter of serum is required. This method was used to detect increases in the activities in sera from patients with liver diseases.     

Partial characterization of 5-fluoropyrimidine-resistant mutants of Neurospora crassa

Summary The primary lesion in a number of 5-fluoropyrimidine resistant mutants of Neurospora crassa has been identified. ud-1 mutants, previously designated fdu-2, are deficient in nucleoside uptake and show extensive intragenic complementation. uc-4 mutants lack uracil phosphoribosyl transferase with no complementation between 23 alleles. udk mutants lack uridine kinase activity. fdu-2 mutants affect the repression of the first two de novo pyrimidine biosynthetic enzymes, have no detectable uridine kinase activity and show decreased uridine uptake. Accordingly, fdu-2 may be involved in the regulation of pyrimidine uptake, salvage and de novo synthesis.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Intragenic mapping of the Neurospora pyrimidine-3 locus by functional deletions

Summary On the basis of complementation between polar and non-polar pyrimidine-3 mutants of Neurospora crassa, a physical linear order of the polar mutants within the locus is derived. The basis of this derivation is that a polar mutant is an intragenic functional deletion extending from the site of the mutation to the translationally distal end of the locus.Supported by Public Health Service Research Grant AI-01462.     

Control of argininosuccinate synthetase by arginine in human lymphoblasts

Human lymphoblasts in long-term culture have the enzyme activities necessary to convert citrulline to arginine: argininosuccinate synthetase and argininosuccinate lyase. Upon transfer from arginine-supplemented to citrulline-supplemented medium, lymphoblasts exhibit a lag period before resuming exponential growth. During this lag the specific activity of argininosuccinate synthetase increases an average of 60-fold. Argininosuccinate lyase activity remains unchanged. If normal lymphoblasts are starved in arginine-deficient medium without citrulline or if argininosuccinate lyase--deficient lymphoblasts are transferred to citrulline-containing medium, argininosuccinate synthetase activity increases linearly for several days and reaches even higher levels. Cycloheximide blocks the increase in enzyme activity. Cells grown in citrulline medium and pulse labeled with 35S-methionine incorporate more 35S-methionine into argininosuccinate synthetase protein than cells grown in arginine; the rate of disappearance of radioactively labeled enzyme is the same in citrulline- and arginine-grown cells. Arginine or a closely related metabolite thus appears to repress the synthesis of argininosuccinate synthetase of human lymphoblasts in culture.