Interallelic Complementation Research Articles

A de novo ARIH2 gene mutation was detected in a patient with autism spectrum disorders and intellectual disability

E3 ubiquitin protein ligase encoded by ARIH2 gene catalyses the ubiquitination of target proteins and plays a crucial role in posttranslational modifications across various cellular processes. As prior documented, mutations in genes involved in the ubiquitination process are often associated with autism spectrum disorder (ASD) and/or intellectual disability (ID). In the current study, a de novo heterozygous mutation was identified in the splicing intronic region adjacent to the last exon of the ARIH2 gene using whole exome sequencing (WES). We hypothesize that this mutation, found in an ASD/ID patient, disrupts the protein Ariadne domain which is involved in the autoinhibition of ARIH2 enzyme. Predictive analyses elucidated the implications of the novel mutation in the splicing process and confirmed its autosomal dominant inheritance model. Nevertheless, we cannot exclude the possibility that other genetic factors, undetectable by WES, such as mutations in non-coding regions and polygenic risk in inter-allelic complementation, may contribute to the patient's phenotype. This work aims to suggest potential relationship between the detected mutation in ARIH2 gene and both ASD and ID, even though functional studies combined with new sequencing approaches will be necessary to validate this hypothesis.

Structure–Function Analysis of Interallelic Complementation in ROOTY Transheterozygotes

Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in genes involved in glucosinolate biosynthesis, a metabolically related route that produces defense compounds, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic Arabidopsis (Arabidopsis thaliana) mutants for the essential glucosinolate biosynthetic gene ROOTY (RTY) that exhibit a range of phenotypic defects characteristic of enhanced auxin production. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 cyp79b3, wei2, and wei7 mutations and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY elucidated the relationships between structure and function in the RTY homo- and heterodimers, and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.

Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance

Glutaric Aciduria Type I (GA-I), is an autosomal recessive neurometabolic disease caused by mutations in the GCDH gene that encodes for glutaryl-CoA dehydrogenase (GCDH), a flavoprotein involved in the metabolism of tryptophan, lysine and hydroxylysine. Although over 200 disease mutations have been reported a clear correlation between genotype and phenotype has been difficult to establish. To contribute to a better molecular understanding of GA-I we undertook a detailed molecular study on two GCDH disease-related variants, GCDH-p.Arg227Pro and GCDH-p.Val400Met. Heterozygous patients harbouring these two mutations have increased residual enzymatic activity in relation to homozygous patients with only one of the mutations, suggesting a complementation effect between the two.Combining biochemical, biophysical and structural methods we here establish the effects of these mutations on protein folding, stability and catalytic activity. We show that both variants retain the overall protein fold, but with compromised enzymatic activities. Detailed enzyme kinetic studies reveal that GCDH-p.Arg227Pro has impaired function due to deficient substrate affinity as evidenced by its higher Km, and that the lower activity in GCDH-p.Val400Met results from weaker interactions with its physiological redox partner (electron transfer flavoprotein). Moreover, the GCDH-p.Val400Met variant has a significantly lower thermal stability (ΔTm ≈ 9 °C), and impaired binding of the FAD cofactor in relation to wild-type protein. On these grounds, we provide a rational for the possible interallelic complementation observed in heterozygous patients based on the fact that in GCDH, the low active p.Arg227Pro variant contributes to stabilize the tetramer while the structurally unstable p.Val400Met variant compensates for enzyme activity.

A novel compound heterozygous genotype associated with aromatic amino acid decarboxylase deficiency: Clinical aspects and biochemical studies

Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal neurometabolic disorder caused by a deficit of AADC, a pyridoxal 5′-phosphate (PLP)-dependent enzyme, which catalyzes the synthesis of dopamine and serotonin. While many studies have highlighted the molecular defects of the homozygous pathogenic variants, so far only a study investigated heterozygous variants at protein level. Here, we report a clinical case of one AADC deficiency compound heterozygous patient bearing the A91V mutation and the novel C410G mutation. To elucidate its enzymatic phenotype, the A91V and C410G homodimers were first expressed in Escherichia coli, purified and characterized. Although both apo variants display an unaltered overall tertiary structure, they show a ̴ 20-fold decreased PLP binding affinity. The C410G mutation only causes a ̴ 4-fold decrease of the catalytic efficiency, while the A91V mutation causes a 1300-fold decrease of the kcat/Km, and changes in the holoAADC consisting in a marked alteration of the tertiary structure and the coenzyme microenvironment. Structural analyses of these mutations are in agreement with these data. Unfortunately, the C410G/A91V heterodimer was constructed, expressed and purified in rather modest amount. Anyway, measurements of decarboxylase activity indicate that its putative kcat value is lower than that predicted by averaging the kcat values of the two parental enzymes. This indicates a negative interallelic complementation between the C410G and A91V monomers. Overall, this study allowed to relate the clinical to the enzymatic phenotype of the patient and to extend knowledge in the clinical and molecular pathogenesis of AADC deficiency.

C-terminal oligomerization of podocin mediates interallelic interactions

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3′ mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283–382): principally through the first C-terminal helical region (H1, 283–313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332–348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r2 = 0.68, P = 9.2 × 10−32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.

Heterozygosis in aromatic amino acid decarboxylase deficiency: Evidence for a positive interallelic complementation between R347Q and R358H mutations.

Aromatic amino acid or Dopa decarboxylase (AADC or DDC) is a homodimeric pyridoxal 5'-phosphate (PLP) enzyme responsible for the generation of the neurotransmitters dopamine and serotonin. AADC deficiency is a rare inborn disease caused by mutations of the AADC gene leading to a defect of AADC enzyme and resulting in impaired dopamine and serotonin synthesis. Until now, only the molecular effects of homozygous mutations were analyzed. However, although heterozygous carriers of AADC deficiency were identified, the molecular aspects of their enzymatic phenotypes are not yet investigated. Here, we focus our attention on the R347Q/R358H and R347Q/R160W heterozygous mutations, and report for the first time the isolation and characterization, in the purified recombinant form, of the R347Q/R358H heterodimer and of the R358H homodimer. The results, integrated with those already known of the R347Q homodimeric variant, provide evidence that (i) the R358H mutation strongly reduces the PLP-binding affinity and the catalytic activity, and (ii) a positive interallelic complementation exists between the R347Q and the R358H mutations. Bioinformatics analyses provide the structural basis for these data. Unfortunately, the R347Q/R160W heterodimer was not obtained in a sufficient amount to allow its purification and characterization. Nevertheless, the biochemical features of the R160W homodimer give a contribution to the enzymatic phenotype of the heterozygous R347Q/R160W and suggest the possible relevance of Arg160 in the proper folding of human DDC. © 2018 IUBMB Life, 70(3):215-223, 2018.

Unique properties of multiple tandem copies of the M26 recombination hotspot in mitosis and meiosis in Schizosaccharomyces pombe

The M26 hotspot of the fission yeast Schizosaccharomyces pombe is one of the best-characterized eukaryotic hotspots of recombination. The hotspot requires a seven bp sequence, ATGACGT, that serves as a binding site for the Atf1-Pcr1 transcription factor, which is also required for activity. The M26 hotspot is active in meiosis but not mitosis and is active in some but not all chromosomal contexts and not on a plasmid. A longer palindromic version of M26, ATGACGTCAT, shows significantly greater activity than the seven bp sequence. Here, we tested whether the properties of the seven bp sequence were also true of the longer sequence by placing one, two, or three copies of the sequence into the ade6 gene, where M26 was originally discovered. These constructs were tested for activity when located on a plasmid or on a chromosome in mitosis and meiosis. We found that two copies of the 10bp M26 motif on a chromosome were significantly more active for meiotic recombination than one, but no further increase was observed with three copies. However, three copies of M26 on a chromosome created an Atf1-dependent mitotic recombination hotspot. When located on a plasmid, M26 also appears to behave as a mitotic recombination hotspot; however, this behavior most likely results from Atf1-dependent inter-allelic complementation between the plasmid and chromosomal ade6 alleles.

Co-expression of phenylalanine hydroxylase variants and effects of interallelic complementation on in vitro enzyme activity and genotype-phenotype correlation.

In phenylketonuria (PKU) patients, the combination of two phenylalanine hydroxylase (PAH) alleles is the main determinant of residual enzyme activity in vivo and in vitro. Inconsistencies in genotype-phenotype correlations have been observed in compound heterozygous patients and a particular combination of two PAH alleles may produce a phenotype that is different from the expected one, possibly due to interallelic complementation. A dual eukaryotic vector system with two distinct PAH proteins N-terminally fused to different epitope tags was used to investigate the co-expression of PAH alleles reported in patients with inconsistent phenotypes. PAH variant proteins were transiently co-transfected in COS-7 cells. PAH activity was measured by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), and protein expression was measured by Western blot. Genotypes were compared with predicted PAH activity from the PAH locus-specific database (PAHvdb) and with phenotypes and tetrahydrobiopterin (BH4) responsiveness from more than 10,000 PKU patients (BIOPKU database). Through the expression and co-expression of 17 variant alleles we demonstrated that interallelic interaction could be both positive and negative. The co-expressions of p.[I65T];[R261Q] (19.5% activity; predicted 43.5%) and p.[I65T];[R408W] (15.0% vs. 26.8% activity) are examples of genotypes with negative interallelic interaction. The co-expressions of p.[E178G];[Q232E] (55.0% vs.36.4%) and p.[P384S];[R408W] (56.1% vs. 40.8%) are examples of positive subunit interactions. Inconsistencies of PAH residual enzyme activity in vitro and of PKU patients' phenotypes were observed as well. The PAH activity of p.[R408W];[A300S] is 18.0% of the wild-type activity; however, 88% of patients with this genotype exhibit mild hyperphenylalaninemias (MHPs). The co-expression of two distinct PAH variants revealed possible dominance effects (positive or negative) by one of the variants on residual PAH activity as a result of interallelic complementation.

Interallelic complementation provides functional evidence for cohesin-cohesin interactions on DNA.

The cohesin complex (Mcd1p, Smc1p, Smc3p, and Scc3p) has multiple roles in chromosome architecture, such as promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. The prevailing embrace model for sister chromatid cohesion posits that a single cohesin complex entraps both sister chromatids. We report interallelic complementation between pairs of nonfunctional mcd1 alleles (mcd1-1 and mcd1-Q266) or smc3 alleles (smc3-42 and smc3-K113R). Cells bearing individual mcd1 or smc3 mutant alleles are inviable and defective for both sister chromatid cohesion and condensation. However, cells coexpressing two defective mcd1 or two defective smc3 alleles are viable and have cohesion and condensation. Because cohesin contains only a single copy of Smc3p or Mcd1p, these examples of interallelic complementation must result from interplay or communication between the two defective cohesin complexes, each harboring one of the mutant allele products. Neither mcd1-1p nor smc3-42p is bound to chromosomes when expressed individually at its restrictive temperature. However, their chromosome binding is restored when they are coexpressed with their chromosome-bound interallelic complementing partner. Our results support a mechanism by which multiple cohesin complexes interact on DNA to mediate cohesion and condensation.

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation.

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5′-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.

Molecular Characteristics, Phenotypic Diversity and Genotype-Estimated Therapeutic Responsiveness of Serbian Patients with Phenylketonuria / MOLEKULARNE KARAKTERISTIKE, FENOTIPSKA RAZNOLIKOST I PROCENA ODGOVORA NA TERAPIJU ZASNOVANA NA GENOTIPU KOD SRPSKIH PACIJENATA SA FENILKETONURIJOM

Summary Phenylketonuria (PKU) is a rare, inherited metabolic disease which is transmitted in an autosomal recessive pattern. PKU is caused by mutations in the gene encoding the phenylalanine hydroxylase (PAH) enzyme. This review cites the most prominent methods for the detection of mutations in the PAH gene. Since the image of PKU transcends »simple« monogenic disease, the known data on non-coding PAH gene variants and their role and PKU modifier genes have been further reviewed. It has been shown that there is a significant correlation between mutant PAH genotypes and PKU phenotypes. However, genotype-phenotype correlation inconsistencies have also been found. This review discusses the possible causes of phenotypic inconsistencies, such as oversight of more than two mutations present in the patient’s PAH genotype, pitfalls of patient phenotypic classification (plasma phenylalanine concentration and phenylalanine tolerance), the inter-allelic complementation (positive and negative) phenomenon. A new therapeutic approach, tetrahydrobiopterin (BH4) supplementation therapy, is an important innovation in the course of PKU patients’ treatment. How - ever, in countries where the BH4-loading test and BH4-supplementation therapy are not available, a genotype-based estimation of responsiveness to the therapy is a valuable approach. It enables BH4-potential benefit estimation, which provides vital information both for the patient and for the population. An optimal molecular diagnostics algorithm, established according to the published mutation frequencies in Serbian PKU patients, has been suggested. In the future, the molecular-genetic algorithm for PKU could be expanded to include a variety of transcriptional regulatory elements located in noncoding PAH gene regions and yet to be discovered modifier genes.

Tetrahydrobiopterin, its Mode of Action on Phenylalanine Hydroxylase, and Importance of Genotypes for Pharmacological Therapy of Phenylketonuria

In about 20%-30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor tetrahydrobiopterin therapy. These patients can be diagnosed by an oral tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with tetrahydrobiopterin were categorized as predictive for tetrahydrobiopterin responsiveness or non-responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.

Effects of compound heterozygosity at the Xpd locus on cancer and ageing in mouse models

XPD is a helicase subunit of transcription factor IIH, an eleven-protein complex involved in a wide range of cellular activities including transcription and nucleotide excision DNA repair (NER). Mutations in NER genes including XPD can lead to a variety of overlapping syndromes with three general categories of symptoms in addition to sun (UV) sensitivity: severe skin cancer predisposition as in xeroderma pigmentosum (XP), segmental progeria as in trichothiodystrophy (TTD) and Cockayne syndrome (CS), and a combination of both as in XP/CS and XP/TTD. Genetic background and compound heterozygosity are two factors potentially complicating straightforward interpretations of genotype–phenotype relationship at the XPD locus. Previously we showed that the presence of two different mutant Xpd alleles in compound heterozygous mice could in principle contribute to disease heterogeneity through biallelic effects, including dominance of one mutant allele over another and interallelic complementation between mutant alleles, in a tissue-specific manner. Here we report on the interaction between different mutant alleles in compound heterozygous mice carrying one XP/CS-associated allele (XpdG602D) and one TTD-associated allele (XpdR722W) relative to homozygous controls in an isogenic background over a range of metabolic and UV-induced DNA damage-related phenotypes. We found complementation of metabolic phenotypes including body weight and insulin sensitivity, but none for any of the measured responses to UV irradiation. Instead, we found dominance of the partially functional TTD allele over the XPCS allele in most aspects of the response to UV irradiation including sunburn and skin cancer in vivo or cellular proliferation and DNA damage foci formation in vitro. These data support to a model of genotype–phenotype relationship at the XPD locus in which interactions between different recessive diseases alleles are a potent source of disease heterogeneity in compound heterozygous patients.

Construction of diploid zygotes by interallelic complementation of ade6 in Schizosaccharomyces japonicus

The construction of diploid cells eases genetic analysis in haploid genetic systems because diploid cells allow for the characterization of essential genes. Here, we report the construction of diploid cells using ade6 point mutants that suppress each other via interallelic complementation in the fission yeast Schizosaccharomyces japonicus var japonicus (Sz. japonicus). We constructed an ade6-domK mutant in addition to the previously described ade6-domE. Phenotypes of both mutants exhibited adenine auxotrophy and red colonies. The mutations complemented the phenotypes in a mutually dependent manner. Diploid zygotes, in which the two mutations were introduced simultaneously into the same cells, were isolated by selecting for adenine independence. Such diploid cells are apparently larger in size than haploid cells, yet have a similar nuclear/cytoplasmic ratio, and thus the nuclear size control that has been reported in Sz. pombe is also present in Sz. japonicus.

Heterotetrameric forms of human phenylalanine hydroxylase: Co-expression of wild-type and mutant forms in a bicistronic system

Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~ 50% tetramer; ~ 10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~ 80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~ 95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH 4 supplementation.

Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling

BackgroundSince the identification of BRI1 (BRASSINOSTEROID-INSENSITIVE1), a brassinosteroids (BRs) receptor, most of the critical roles of BR in plant development have been assessed using various bri1 mutant alleles. The characterization of individual bri1 mutants has shown that both the extracellular and cytoplasmic domains of BRI1 are important to its proper functioning. Particularly, in the extracellular domain, regions near the 70-amino acid island are known to be critical to BR binding. In comparison, the exact function of the leucine rich-repeats (LRR) region located before the 70-amino acid island domain in the extracellular cellular portion of BRI1 has not yet been described, due to a lack of specific mutant alleles.ResultsAmong the mutants showing altered growth patterns compared to wild type, we further characterized cp3, which displayed defective growth and reduced BR sensitivity. We sequenced the genomic DNA spanning BRI1 in the cp3 and found that cp3 has a point mutation in the region encoding the 13th LRR of BRI1, resulting in a change from serine to phenylalanine (S399F). We renamed it bri1-120. We also showed that overexpression of the wild type BRI1 protein rescued the phenotype of bri1-120. Using a GFP-tagged bri1-120 construct, we detected the bri1-120 protein in the plasma membrane, and showed that the phenotypic defects in the rosette leaves of bri1-301, a kinase-inactive weak allele of BRI1, can be restored by the overexpression of the bri1-120 proteins in bri1-301. We also produced bri1-301 mutants that were wild type in appearance by performing a genetic cross between bri1-301 and bri1-120 plants.ConclusionsWe identified a new bri1 allele, bri1-120, whose mutation site has not yet been found or characterized. Our results indicated that the extracellular LRR regions before the 70-amino acid island domain of BRI1 are important for the appropriate cellular functioning of BRI1. Also, we confirmed that a successful interallelic complementation occurs between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.

A neonatal‐onset succinyl‐CoA:3‐ketoacid CoA transferase (SCOT)‐deficient patient with T435N and c.658‐666dupAACGTGATT p.N220_I222dup mutations in the OXCT1 gene

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidotic crises and no apparent symptoms between them. Here, we report a Japanese case of neonatal-onset SCOT deficiency. The male patient presented a severe ketoacidotic crisis, with blood pH of 7.072 and bicarbonate of 5.8 mmol/L at the age of 2 days and was successfully treated with intravenous infusion of glucose and sodium bicarbonate. He was diagnosed as SCOT deficient by enzymatic assay and mutation analysis. At the age of 7 months, he developed a second ketoacidotic crisis, with blood pH of 7.059, bicarbonate of 5.4 mmol/L, and total ketone bodies of 29.1 mmol/L. He experienced two milder ketoacidotic crises at the ages of 1 year and 7 months and 3 years and 7 months. His urinary ketone bodies usually range from negative to 1+ but sometimes show 3+ (ketostix) without any symptoms. Hence, this patient does not show permanent ketonuria, which is characteristic of typical SCOT-deficient patients. He is a compound heterozygote of c.1304C > A (T435N) and c.658-666dupAACGTGATT p.N220_I222dup. mutations in the OXCT1 gene. The T435N mutation was previously reported as one which retained significant residual activity. The latter novel mutation was revealed to retain no residual activity by transient expression analysis. Both T435N and N220_I222 lie close to the SCOT dimerization interface and are not directly connected to the active site in the tertiary structure of a human SCOT dimer. In transient expression analysis, no apparent interallelic complementation or dominant negative effects were observed. Significant residual activity from the T435N mutant allele may prevent the patient from developing permanent ketonuria.

Interallelic complementation for pyrimidine requirement in rudimentary mutants of Drosophila melanogaster.

Complementation at the rudimentary locus in Drosophila melanogaster is reported for the nutritional requirement observed earlier in the rudimentary mutants. It is demonstrated that this complementation parallels that for wing phenotype, and that at the same time it is a more potent and quantitative test for complementation at this locus. The possible molecular basis for the observed complementation is discussed in the light of current knowledge of the genetic regulation of pyrimidine biosynthesis in other organisms.

Genetics of postzygotic reproductive isolation in plants

Postzygotic reproductive isolation, based on negative interactions of genes, is a key aspect of divergent speciation in plants and animals. The effect of the interaction manifests as a drastic reduction in fitness of hybrids of the first and subsequent generations, which is expressed as hybrid lethality, weakness or sterility. Both simple genetic control of genetic incompatibility, which includes interallelic negative complementation or epistatic interactions of a limited number of genes, and more complex control, based on epistatic interactions of many genes, have been described in plants. It is thought that genes for reproductive isolation are nonuniformly distributed over the genome and are related to differential adaptation. The mosaic organization of the genomes in this respect provides restoration of their structural and functional integrity upon interspecies hybridization in natural conditions. Many cultured and wild plant species, in contrast to animals, were found to be polymorphic at genes controlling interspecies incompatibility. This fact facilitates genetic analysis of incompatibility and broadens perspectives in studying the structure, functions, and molecular evolution of the genes controlling postzygotic reproductive isolation, in view of the possible leading role of these genes in adaptive speciation.

Genetic Analysis of chaste, a New Mutation of Drosophila melanogaster Characterized by Extremely Low Female Sexual Receptivity

By screening about 2,000 P-element-insertion lines of Drosophila melanogaster, we isolated a new behavioral mutant line, chaste (chst), the females of which display extraordinarily strong rejection behavior against courting males. The chst mutation is mapped to the muscleblind (mbl) locus at 54B on the right arm of chromosome 2. The reduced sexual receptivity in chst mutant females is reversed to the wild-type level by introducing a transgene, which expresses either the mblB+ or mblC+ isoform, demonstrating that chst is an allele of mbl. Among the P-elements inserted upstream of the mbl gene, those inserted in the same orientation as that of mbl express the chst phenotype, whereas a P-element inserted in the opposite orientation does not. This finding implies that the former P-elements induce the mutant phenotype by a mechanism that is sensitive to the direction of transcription (e.g., transcriptional interference). The mbl alleles, with deletions near the transcription start site and/or in part of the exons, complement the chst mutation in the sexual receptivity phenotype, but not in the lethality phenotype of mbl mutations. Such interallelic complementation of the sexual receptivity phenotype in the mbl locus disappears in the presence of a mutant copy of zeste (z), a gene encoding a protein that mediates transvection. We suggest that the mbl gene function is required for the normal development of neural substrates that regulate female sexual receptivity.