Interspecific Complementation Research Articles

Adaptive evolution among cytoplasmic piRNA proteins leads to decreased genomic auto-immunity.

In metazoan germlines, the piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). Expression of genomic TEs is proposed to initiate self regulation by increasing the production of repressive piRNAs, thereby "adapting" piRNA-mediated control to the most active TE families. Surprisingly, however, piRNA pathway proteins, which execute piRNA biogenesis and enforce silencing of targeted sequences, evolve rapidly and adaptively in animals. If TE silencing is ensured through piRNA biogenesis, what necessitates changes in piRNA pathway proteins? Here we used interspecific complementation to test for functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. In contrast to piRNA-mediated transcriptional regulators examined in previous studies, these three proteins have cytoplasmic functions in piRNA maturation and post-transcriptional silencing. Across all three proteins we observed interspecific divergence in the regulation of only a handful of TE families, which were more robustly silenced by the heterospecific piRNA pathway protein. This unexpected result suggests that unlike transcriptional regulators, positive selection has not acted on cytoplasmic piRNA effector proteins to enhance their function in TE repression. Rather, TEs may evolve to "escape" silencing by host proteins. We further discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, as well as modest reductions in the efficiency of piRNA biogenesis, suggesting that promiscuous binding of D. simulans Aub and Armi proteins to host transcripts reduces their participation in piRNA production. Avoidance of genomic auto-immunity may therefore be a critical target of selection. Our observations suggest that piRNA effector proteins are subject to an evolutionary trade-off between defending the host genome from the harmful effect of TEs while also minimizing collateral damage to host genes.

Estimation of Evapotranspiration of a Jujube/Cotton Intercropping System in an Arid Area Based on the Dual Crop Coefficient Method

An accurate estimation of crop evapotranspiration (ETc) in intercropping is critical for a theoretical basis for formulating an intercropping irrigation system of fruit trees and improving the soil moisture condition of orchards in arid regions of Southern Xinjiang. Herein, observational data such as soil moisture and plant physiological indicators were measured in jujube/cotton intercropping in the Aksu region from 2015 to 2017. The evapotranspiration of single-crop jujube and cotton was estimated using the modified dual crop coefficient method. Then, based on the proportion of intercropping crops, the soil water stress coefficient was introduced to estimate the evapotranspiration of the jujube/cotton intercropping. The results show that the model has good applicability to simulate single-crop jujube and cotton, and jujube/cotton intercropping. However, compared with single cropping, the accuracy of simulated daily evapotranspiration was decreased. In addition, adjusting the cotton irrigation amount caused the simulation accuracy to further decrease. From the perspective of interspecies complementarity and competition, cotton dominated the intercropping system and had better tolerance to external environmental changes than jujube trees. Intercropping had negative effects on jujube trees in general, and very obvious positive effects on cotton. Based on the 3-year crop yield, 5-year-old jujube trees are recommended for intercropping. During this time, the yield of cotton under the effect of interspecific complementation increased by 26.47%, and the yield of intercropping jujube was similar to that of single crop. As the jujube tree age increases, the effect of increasing cotton production gradually diminishes. The jujube trees also had a significant reduction in yield due to interspecific competition. Our research supports the dual crop coefficient method as appropriate to estimate crop ETc in intercropping and may be further used to improve irrigation scheduling for jujube/cotton intercropping.

GEMMA CUP-ASSOCIATED MYB1, an Ortholog of Axillary Meristem Regulators, Is Essential in Vegetative Reproduction in Marchantiapolymorpha.

A variety of plants in diverse taxa can reproduce asexually via vegetative propagation, in which clonal propagules with a new meristem(s) are generated directly from vegetative organs. A basal land plant, Marchantia polymorpha, develops clonal propagules, gemmae, on the gametophyte thallus from the basal epidermis of a specialized receptacle, the gemma cup. Here we report an R2R3-MYB transcription factor, designated GEMMA CUP-ASSOCIATED MYB1 (GCAM1), which is an essential regulator of gemma cup development in M.polymorpha. Targeted disruption of GCAM1 conferred a complete loss of gemma cup formation and gemma generation. Ectopic overexpression of GCAM1 resulted in formation of cell clumps, suggesting a function of GCAM1 in suppression of cell differentiation. Although gemma cups are a characteristic gametophyte organ for vegetative reproduction in a taxonomically restricted group of liverwort species, phylogenetic and interspecific complementation analyses support the orthologous relationship of GCAM1 to regulatory factors of axillary meristem formation, e.g., Arabidopsis REGULATOR OF AXILLARY MERISTEMS and tomato Blind, in angiosperm sporophytes. The present findings in M.polymorpha suggest an ancient acquisition of a transcriptional regulator for production of asexual propagules in the gametophyte and the use of the regulatory factor for diverse developmental programs, including axillary meristem formation, during land plant evolution.

Effect of Straw Returning and Reduced Tillage on Interspecific Competition and Complementation in Wheat/Maize Intercropping System

Effect of Straw Returning and Reduced Tillage on Interspecific Competition and Complementation in Wheat/Maize Intercropping System

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

The spectrum of Trp− mutants isolated as 5‐fluoroanthranilate‐resistant clones in Saccharomyces bayanus, S. mikatae and S. paradoxus

5-Fluoroanthranilic acid (FAA)-resistant mutants were selected in homothallic diploids of three Saccharomyces species, taking care to isolate mutants of independent origin. Mutations were assigned to complementation groups by interspecific complementation with S. cerevisiae tester strains. In all three species, trp3, trp4 and trp5 mutants were recovered. trp1 mutants were also recovered if the selection was imposed on a haploid strain. Thus, FAA selection may be more generally applicable than was previously described.

Evolutionary diversification of pigment pattern inDaniofishes:differentialfmsdependence and stripe loss inD. albolineatus

The developmental bases for species differences in adult phenotypes remain largely unknown. An emerging system for studying such variation is the adult pigment pattern expressed by Danio fishes. These patterns result from several classes of pigment cells including black melanophores and yellow xanthophores, which differentiate during metamorphosis from latent stem cells of presumptive neural crest origin. In the zebrafish D. rerio, alternating light and dark horizontal stripes develop, in part, owing to interactions between melanophores and cells of the xanthophore lineage that depend on the fms receptor tyrosine kinase; zebrafish fms mutants lack xanthophores and have disrupted melanophore stripes. By contrast, the closely related species D. albolineatus exhibits a uniform pattern of melanophores, and previous interspecific complementation tests identified fms as a potential contributor to this difference between species. Here, we survey additional species and demonstrate marked variation in the fms-dependence of hybrid pigment patterns, suggesting interspecific variation in the fms pathway or fms requirements during pigment pattern formation. We next examine the cellular bases for the evolutionary loss of stripes in D. albolineatus and test the simplest model to explain this transformation, a loss of fms activity in D. albolineatus relative to D. rerio. Within D. albolineatus, we demonstrate increased rates of melanophore death and decreased melanophore migration, different from wild-type D. rerio but similar to fms mutant D. rerio. Yet, we also find persistent fms expression in D. albolineatus and enhanced xanthophore development compared with wild-type D. rerio, and in stark contrast to fms mutant D. rerio. These findings exclude the simplest model in which stripe loss in D. albolineatus results from a loss of fms-dependent xanthophores and their interactions with melanophores. Rather, our results suggest an alternative model in which evolutionary changes in pigment cell interactions themselves have contributed to stripe loss, and we test this model by manipulating melanophore numbers in interspecific hybrids. Together, these data suggest evolutionary changes in the fms pathway or fms requirements, and identify changes in cellular interactions as a likely mechanism of evolutionary change in Danio pigment patterns.

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages inDaniofishes

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.

Pigment patterns: fish in stripes and spots

The analysis of zebrafish pigment pattern development suggests a model for the genes and cell populations required to make an adult pigment pattern and provides a useful starting point for identifying the mechanisms underlying pigment pattern evolution across species.A recent approach has used interspecific hybrids. When zebrafish are crossed to other danios (Figure 1Figure 1), the resulting pigment patterns typically resemble that of zebrafish more than the other species, suggesting that pigment pattern genes in other species often are recessive to those of zebrafish. This has allowed for interspecific complementation tests to determine whether genes identified as zebrafish pigment pattern mutants might contribute to differences between taxa. For example, hybrids between wild-type zebrafish and D. albolineatus form stripes like zebrafish, whereas hybrids between fms mutant zebrafish and D. albolineatus lack stripes, like D. albolineatus, identifying fms as a candidate gene for stripe elaboration in zebrafish, or stripe loss in D. albolineatus.Danio pigment patterns offer an outstanding opportunity for integrative studies that link genetic and cellular mechanisms to behavior, ecology, and natural selection in the wild. In the future, identification of additional genes and cell behaviors required for pigment pattern formation in zebrafish will provide valuable resources and testable hypotheses for dissecting the evolution of neural crest derivatives in danios and vertebrates more generally.

Chickpea facilitates phosphorus uptake by intercropped wheat from an organic phosphorus source

Pot experiments were conducted to investigate interspecific complementation in utilization of phytate and FePO4 by plants in the wheat (Triticum aestivum L.)/chickpea (Cicer arietinum L.) intercropping under sterile and non-sterile conditions. The pots were separated into two compartments by either a solid root barrier to eliminate root contact and solute movement, by a nylon mesh (30 μM) to prevent root contact but permit solute exchange, or not separated between the compartments. Wheat plants were grown in one compartment and chickpea in the other. Two P sources were tested at 60 mg P kg−1 soil (sodium phytate or FePO4). Under non-sterile conditions, the biomass of wheat was significantly greater when the roots were intermingled with chickpea than when the roots were separated from chickpea roots by a solid root barrier or nylon mesh. When phytate–P was applied, P concentrations in wheat (2.9 g kg−1 in shoots and 1.4 g kg−1 in roots) without root barrier between the two species were higher than those in the treatments with nylon mesh or with the solid root barrier separation (1.9 g kg−1 in shoots and 1.0 g kg−1 in roots). In contrast, P concentrations in wheat supplied with FePO4 were similar between the root separation treatments. There was no significant difference in P uptake by chickpea between the P sources or between the root separation treatments, except that P uptake was greater in the phytate treatment with the root barrier. Total P uptake from phytate was increased by 25% without root separation compared to the root separation treatments. Under sterile conditions and supply of phytate–P, the biomass of wheat was doubled when the roots were intermingled with chickpea and increased by a third with the nylon mesh separation compared to that with the solid root barrier. Biomass production in wheat at various treatments correlated with P concentration in shoot. Biomass production and P concentration in chickpea were unaffected by root separation. Total P uptake by plants was 68% greater with root intermingling and 37% greater with nylon mesh separation than that with the solid root barrier. The results suggest that chickpea roots facilitate P utilization from the organic P by wheat.

Zebrafish hybrids suggest genetic mechanisms for pigment pattern diversification in Danio.

Pigment patterns of Danio fishes are a tractable system for assessing the developmental genetic bases for the evolution of adult form in vertebrates. These pigment patterns include multiple horizontal melanophore stripes in the zebrafish D. rerio, a complete absence of stripes in D. albolineatus, a few broad stripes in D. kerri, and a combination of stripes and spots in D. nigrofasciatus. Here we assess the genetics of pigment pattern development and evolution using interspecific hybrids. We first reconstruct the phylogenetic relationships of these species by analyzing mitochondrial 12S and 16S rDNA sequences. We find a clade comprising several small species of danio, and within this clade a sister taxon relationship between D. rerio and D. nigrofasciatus. We also find that the large bodied D. dangila is more closely related to the clade of small danios than other large bodied species. As a first step in evaluating the genetics of pigment pattern diversification in the group, we then examine the phenotypes of interspecific hybrids. Adult pigment patterns of hybrids between D. rerio and other danios are in many respects more similar to D. rerio than the heterospecific danio, demonstrating that alleles of pigment pattern genes in other species typically are recessive to D. rerio alleles. Furthermore, hybrids between two additional striped species (D. kerri, D. nigrofasciatus) and D. albolineatus suggest that striped patterns are dominant or semi-dominant over an absence of stripes. Together, these analyses support a model in which pigment pattern differences between D. rerio and other species result from gain-of-function alleles in D. rerio, or loss-of-function alleles in other danios. Finally, because several D. rerio pigment pattern mutants resemble heterospecific danios, we use interspecific complementation tests to assess potential roles for these loci in pigment pattern diversification. Crosses between other danios and most D. rerio pigment pattern mutants develop stripes, similar to control hybrids with wild-type D. rerio. These complementation phenotypes allow us to exclude most of these loci as having major effect roles in generating pigment pattern differences between species. In contrast, hybrids between fms mutant D. rerio and D. albolineatus fail to develop stripes, similar to D. albolineatus. This non-complementation phenotype identifies changes in fms, or the pathway in which it acts, as candidates for contributing to the evolutionary loss of stripes in D. albolineatus.

Petunia Ap2-like genes and their role in flower and seed development.

We have isolated three Apetala2 (Ap2)-like genes from petunia and studied their expression patterns by in situ hybridization. PhAp2A has a high sequence similarity to the A function gene Ap2 from Arabidopsis and a similar expression pattern during flower development, suggesting that they are cognate orthologs. PhAp2B and PhAp2C encode for AP2-like proteins that belong to a different subgroup of the AP2 family of transcription factors and exhibit divergent, nearly complementary expression patterns during flower development compared with PhAp2A. In contrast, all three PhAp2 genes are strongly expressed in endosperm. The phenotype of the petunia A-type mutant blind cannot be attributed to mutations in the petunia Ap2 homologs identified in this study, and reverse genetics strategies applied to identify phap2a mutants indicate that PhAp2A might not be essential for normal perianth development in petunia. Nevertheless, we show that PhAp2A is capable of restoring the homeotic transformations observed in flowers and seed of the ap2-1 mutant of Arabidopsis. Although the interspecific complementation proves that PhAp2A encodes a genuine Ap2 ortholog from petunia, additional factors may be involved in the control of perianth identity in this species.

Divergence of larval morphology between Drosophila sechellia and its sibling species caused by cis-regulatory evolution of ovo/shaven-baby.

We report an extreme morphological difference between Drosophila sechellia and related species of the pattern of hairs on first-instar larvae. On the dorsum of most species, the posterior region of the anterior compartment of most segments is covered by a carpet of fine hairs. In D. sechellia, these hairs have been lost and replaced with naked cuticle. Genetic mapping experiments and interspecific complementation tests indicate that this difference is caused, in its entirety, by evolution at the ovo/shaven-baby locus. The pattern of expression of the ovo/shaven-baby transcript is correlated with this morphological change. The altered dorsal cuticle pattern is probably caused by evolution of the cis-regulatory region of ovo/shaven-baby in the D. sechellia lineage.

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.

Interspecific complementation between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation.

Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10.

Cloning Mammalian Genes Using cDNA Expression Libraries in Saccharomyces cerevisiae

Cloning genes by complementation is a powerful method for gene isolation in prokaryotic and smaller eukaryotic organisms. This technique has not been used extensively in larger eukaryotes because of the difficulty of introducing large numbers of clones into these organisms and the general lack of mutations in the systems in which complementation is feasible (e.g., mammalian tissue culture). Recent advances in transformation methods and cDNA expression vector technology have made possible the isolation of genes from potentially any organism by complementation in yeast. Many genes from larger eukaryotes have already been isolated using interspecific complementation of yeast mutations, including important cell cycle regulators such as CDK2 and cyclins C, D, and E. The recent development of artificial selections in yeast, such as the one-hybrid and two-hybrid systems, has broadened the menu of genes that can be isolated by expression beyond strict homologs of yeast genes. The ability to construct and utilize cDNA expression libraries in yeast is an emerging technology that holds great promise for future gene isolation. This article discusses general considerations for cloning by complementation in yeast using heterologous cDNA expression libraries. Strategies for complementation of ts and null mutations in essential genes are presented together with transformation and selection protocols.

The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase

Interspecific complementation of a. Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI- ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.

Molecular cloning and characterization of the recA gene of Rhizobium phaseoli and construction of recA mutants

The Rhizobium phaseoli recA gene has been cloned by interspecific complementation of the Fec phenotype of bacteriophage lambda. The cloned gene restored the recombination proficiency and conferred resistance to DNA-damaging agents (methyl methanesulfonate and nitrofurantoin) to an Escherichia coli recA mutant. The direction of transcription and the localization of the recA gene were determined by mutagenesis with phage MudIIPR13 and heterologous hybridization with an E. coli recA probe. An R. phaseoli recA::Spcr mutation was introduced in two R. phaseoli strains by homogenotization. The R. phaseoli recA mutants were more sensitive to DNA-damaging agents and exhibited a 100-fold reduction in recombination frequency as compared with their parental strains. A deletion of the symbiotic plasmid abolishing nodulation was found at high frequency (10(-2)) in R. phaseoli CNF42. This event was recA dependent. In R. phaseoli CFN285, two events of symbiotic instability were found at high frequency (10(-3]: one was a deletion in the symbiotic plasmid, and the other was the loss of whole symbiotic plasmid. In the CFN285 recA::Spcr mutant, only the loss of the symbiotic plasmid was observed.

A previously unrecognized glutamine synthetase expressed in Klebsiella pneumoniae from the glnT locus of Rhizobium leguminosarum

Using glnT DNA of Rhizobium meliloti as a hybridization probe we identified a R. leguminosarum biovar phaseoli (R. l. phaseoli) locus (glnT) expressing a glutamine synthetase activity in Klebsiella pneumoniae. A 2.2 kb DNA fragment from R. l. phaseoli was cloned to give plasmid pMW5a, which shows interspecific complementation of a K. pneumoniae glnA mutant. The cloned sequence did not show cross-hybridization to glnA or glnII, the genes coding for two glutamine synthetase isozymes of Rhizobium spp. While in previous reports on glnT of R. meliloti and Agrobacterium tumefaciens no glutamine synthetase activity was detected, we do find activity with the glnT locus of R. l. phaseoli. The glutamine synthetase (GSIII) activity expressed in a K. pneumoniae glnA strain from pMW5a shows a ratio of biosynthetic to transferase activity 10(3)-fold higher than that observed for GSI or GSII. GSIII is similar in molecular weight and heat stability to GSI.

Intragenic interspecific complementation of glucose 6-phosphate dehydrogenase in human-hamster cell hybrids

A new variant of human glucose 6-phosphate dehydrogenase (G6PD), provisionally designated G6PD Harilaou, was observed in a Greek boy affected by severe hemolytic anemia. G6PD Harilaou was associated with very severe deficiency of enzyme activity, which measured about 1% of normal in the patient's fibroblasts. By fusion of Harilaou fibroblasts with a similarly enzyme-deficient mutant CHO cell line, we have isolated a hybrid cell line that has a G6PD activity 5-10 times higher than either of the parental cells. By electrophoretic analysis we show that most of this activity is associated with a hybrid dimeric G6PD molecule consisting of one hamster and one human subunit. We suggest that this heterologous quasi-interallelic complementation is effected by a catalytically abnormal hamster subunit stabilizing a catalytically abnormal and unstable Harilaou subunit. This approach may be useful for the study of dimer formation and stability in human G6PD.