Chinese hamster cells mutant at the hypoxanthine-guanine phosphoribosyltransferase (GPRT) locus. V. Complementation analysis of ts mutants

Four temperature-sensitive HPRT clones were used for hybridological analysis, which led to increase in complementation rate about 5 times. The probability of complementation, in respect of the HPRT locus proved to be rather high: 14 of 45 hybridization-tested mutants had complementation ability (including 3 ts mutants). Analysis of the complementation rate among mutants revealed clear-cut dependence on the selection conditions: clones grown in a medium with 8-azaguanine showed most frequent complementation. The use of mutants with a new phenotype in hybridization analysis revealed four additional complementation groups, three of which are made of temperature-sensitive clones. Biochemical analysis revealed the presence of hybrid forms of the HPRT enzyme in all hybrids tested. This confirms the intragenic character of complementation. At present, the functional map of the HPRT locus is represented by 9 groups, including a group of mutants with no complementation ability.     

Female Sterile Mutations on the Second Chromosome of Drosophila Melanogaster. I. Maternal Effect Mutations

In mutagenesis screens for recessive female sterile mutations on the second chromosome of Drosophila melanogaster 529 chromosomes were isolated which allow the homozygous females to survive, but cause them to be sterile. In 136 of these lines, mutant females produce morphologically normal eggs which cannot support normal embryonic development. These "maternal-effect" mutations fall into 67 complementation groups which define 23 multiply hit and 44 singly hit loci. In eggs from 14 complementation groups development is blocked before the formation of a syncytial blastoderm. In eggs from 12 complementation groups development is abnormal before cellularization, 17 complementation groups cause abnormal cellularization, 12 complementation groups cause changes in cellular morphology in early gastrulation stages, and 12 complementation groups seem to affect later embryonic development.     

Products of complementation between temperature-sensitive mutants of simian virus 40.

Temperature-sensitive mutants of the D complementation group of simian virus 40 exhibit delayed complementation. Analysis of the thermal stability, kinetic profiles in temperature shift experiments, and progeny of complementation have led to the hypothesis that delayed complementation is not true complementation, but the result of a very low level of leakiness, followed by phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants at the restrictive temperature.     

Kinetics of Mitochondrial Complementation

Mitochondrial complementation (enhancement of oxidative activity of mitochondrial occured when mitochondria of some inbrveds of eron were mixed in vitro. It was found that mixtures composed of various proportions of mitochondria of inbreds exhibited complementation. Extracts of mitochondria of one inbred did not promote complementation when they were added to infact mitochondria of inbreds exhihited complententation. Extracts of mitochondria of on einbred did not promote complementation when they were added to infact mitochondria of the second inbred. Serial dillutions of mixtures resulted in a rapid reduction of mitochondrial oxidation (QO₂ N) suggesting that intimale association between mitochondria was minimized by the dilution and that this decreased complementation. Similar dilutions of mitochondria from inbreds did not decrease their specific activity. Kinetics of mitochondrial oxidation were measured polarographically and it was found that complementation was measurable immediately (1 min) following the mixing. This suggested that physical contact between mitochondria was necessary for complementation.     

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selection protocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules. They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocols designed, in many cases, to isolate different classes of receptor mutants. The identification of most membrane mutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to several lectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty may subsequently be determined by complementation analysis with selected members of 10 recessive, glycosylation-defective complementation groups defined by this laboratory. In an attempt to identify new complementation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratories have been investigated for their lectin resistance and complementation properties. Only one new complementation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3, or 8. Although no evidence for intragenic complementation was observed, indirect evidence for different mutations within some genes was obtained. Seven of the independent isolates fell into complementation group 1, reflecting the high probability of isolating the Lec1 phenotype from Chinese hamster ovary populations. The results emphasize the importance of performing a genetic analysis before biochemical characterization of putative new membrane mutants.     

Novel segregation patterns of infecting-mutant genotypes in plate complementation tests among amber mutants of bacteriophage BF23

Amber mutants of bacteriophage BF23 were classified into two functional groups, types I and II, by the yields of the infecting-mutant genotypes in plate complementation tests. Type I mutants produced their genotypes at levels more than 20% of the total progeny phages, and type II mutants did so at levels of less than 5%. Comparison of the results of plate complementation tests with those of extract complementation tests revealed that all the type I mutants were defective in the tail formation, while most type II mutants were defective in the formation of either mature heads (type IIa) or both mature heads and tails (type IIb). Since in extract complementation tests the activated phages are always of genotypes corresponding to mutations defective in only the tail formation, the plate complementation test is comparable with the extract complementation test when judged on the basis of the yield of the mutant genotypes. Of 29 complementation groups, 8 type I, 14 type IIa, and 5 type IIb mutants were identified. Previously, amber mutations of BF23 were mapped on four genetic segments. These segments were ordered in one linkage map by crosses between deletion and amber mutants.     

Peroxisomal disorders: complementation analysis using beta-oxidation of very long chain fatty acids

Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as well as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demonstrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

Genetic classification of riboflavin-dependent mutants of Pichia guilliermondii yeasts]

114 riboflavinless mutants were selected from the genetic line of Pichia guilliermondii yeast. By means of accumulation test the mutants were divided into five biochemical groups. In genetic experiments seven complementation classes were found among 106 mutants. The strains of the I biochemical group, accumulating no specific products, corresponded to complementation class rib1; II group, accumulating 2,4,5-triaminopyrimidine - to the class rib2; III group, accumulating 2,6-dihydroxy-4-ribitylaminopyrimidine - to the class rib3; the mutants of the IV group, accumulating 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine, were divided into three complementation classes rib4, rib5 and rib6; the mutants of the V group, acculumating 6,7-dimethyl-8-ribityllumazine, corresponded to the class rib7. Two mutants of the IV biochemical group within complementation classes rib4 and rib5 were detected could not grow in the medium with diacetyl without riboflavin. Intragenic complementation was found within classes rib6 and rib7. No linkage between mutations of different complementation classes was detected.     

Allelic Complementation in the First Gene for Histidine Biosynthesis in SACCHAROMYCES CEREVISIAE. II. Complementation Mapping of Mutants and a Subunit Model of the Enzyme

Sixty-two alleles of the histidine-1 (his1) gene were tested for complementation. The 44 complementing mutants fell into 31 complementation groups which were used to construct a complex complementation map with 18 complementation units. Cluster analysis of the complementation map by either visual inspection or the computer method of Gillie and Peto (1969) shows two very definite clusters.The molecular weight estimate of the his1 enzyme, phosphoribosyl adenosine triphosphate: pyrophosphate phosphoribosyltransferase, is 1.8 . 10(5) by sucrose density gradient analysis and 2.4 . 10(5) by Sephadex gel chromatography. Correlating the length of the his1 gene to the molecular weight of the enzyme indicates that this enzyme is composed of 6 subunits, as is the analogous enzyme in Salmonella typhimurium.A model of the subunit and tertiary and quaternary structure of the enzyme has been developed from consideration of the genetic and complementation data, the distribution of the various mutant types within the gene, and the biochemical properties of the enzyme encoded by the his1 gene.     

Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.     

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes.     

Genetic Fine Structure of the Y Chromosome of DROSOPHILA HYDEI

A genetic map of the Y chromosome of Drosophila hydei has been constructed from deletion/complementation experiments, with the aid of male sterile mutants of the Y chromosome. A central conclusion of our experiments is that not more than a single complementation group can be detected in each of the lampbrush loop forming sites. Additional complementation groups, functionally independent of lampbrush loops, reside between these loci. Six complementation groups have been defined by several methods of mapping. An additional ten complementation groups are indicated, but their exact definition requires further investigation. The "synthetic sterility" of mutations in these ten loci contributes to the difficulty in unequivocally establishing their individual boundaries. Mapping problems also arise from the instability of certain mutants.     

Molecular Mapping of a Gene Cluster Flanking the Drosophila Dopa Decarboxylase Gene

Nine lethal complementation groups flanking the Drosophila Dopa decarboxylase (Ddc) gene, have been localized within 100 kb of cloned chromosomal DNA. Six of these complementation groups are within 23 kb of DNA, and all ten complementation groups, including Ddc, lie within 78-82 kb of DNA. The potential significance of this unusually high gene density is discussed.     

Isolation and characterization of temperature-sensitive mutants of Sendai virus

Sixteen temperature-sensitive mutants of Sendai virus were isolated from mutagenized stocks (10 mutants, designated numerically) and persistently infected cultures (6 mutants, designated alphabetically). Based on complementation tests, virion-associated activities, thermal inactivation, and viral RNA and hemadsorbing antigen synthesis as well as virion production in chick lung embryo cells at nonpermissive temperature, these mutants were divided into seven groups as follows. i) HANA group mutants (ts-5, -9, -10, -201), defective in hemagglutinin-neuraminidase protein, complementation group I. ii) F group mutants (ts-18, -108), defective in hemolytic and cell-fusing activity, complementation group II. iii) Ts-43, defective in RNA polymerase activity, complementation group III. iv) Ts-23, defective in RNA polymerase activity, interfered with the other mutants in complementation tests. v) Ts-25, defective in the incorporation of hemagglutinin-neuraminidase protein into the virion at the stage of virus assembly. vi) Ts-110, belongs to F group mutants on one hand, but is considered to carry another undetermined defect. vii) C group (carrier culture-borne group) mutants (ts-a, -b, -c, -d, -e, -f), defective lesion not yet determined and belong to neither complementation group I nor II. Assignment of mutants in groups iv), v), vi), and vii) to complementation groups could not be achieved.     

Interallelic complementation and recombination at therudimentary wing locus inDrosophila melanogaster

A study of 22 independentrudimentary wing mutants confirmed the occurence of complementation among half of these mutants. Recombination studies show that a minimum of three loci occur paralleling the number of loci deduced from complementation. The data are discussed in relationship to the more extensive studies of complementation inNeurospora.     

Assembly of the mitochondrial membrane system. Genetic complementation of mit- mutations in mitochondrial DNA of Saccharomyces cerevisiae.

A method has been devised to test intergenic complementation of mutations in the mitochondrial DNA of Saccharomyces cerevisiae. The test is based on the observation that diploids issued from pairwise crosses of certain mit- mutants with deficiencies in cytochrome oxidase, or coenzyme QH2-cytochrome c reductase, acquire high levels of respiratory activity shortly after zygote formation. Under our experimental conditions neither biochemical complementation, interallelic complementation, nor recombination has been found to contribute to any significant extent toward the respiration measured in the diploids at early times. The test has been used to study the number of complementation groups represented by a large number of mit- mutants. Results of pairwise crosses of mutants in the oxi 1, oxi 2, oxi 3, cob 1, and cob 2 loci indicate that complementation occurs between the oxi and cob loci between different oxi loci but not between the two cob loci. The five loci have, therefore, been assigned to four different complementation groups.     

Complementation and Epistasis in Viral Coinfection Dynamics

Coinfection in RNA virus populations results in two important phenomena, complementation and recombination. Of the two, complementation has a strong effect on selection against deleterious mutations, as has been confirmed in earlier studies. As complementation delays the purging of less-fit mutations, coinfection may be detrimental to the evolution of a virus population. Here we employ both deterministic modeling and stochastic simulation to explore the mechanisms underlying the interactions between complementation and other evolutionary factors, namely, mutation, selection, and epistasis. We find that strong complementation reduces slightly the overall fitness of a virus population but substantially enhances its diversity and robustness, especially when interacting with selection and epistasis.     

Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens

The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.     

Dietary complementation by wild birds: Considerations for field studies

Free-living birds must satisfy fluctuating nutrient requirements in diverse and varying environments. Ingesting nutritionally complementary foods may be the most effective means by which wild birds match nutrient ingestion and nutrient needs. Dietary complementation may occur fortuitously when foods chosen in response to non-nutritive factors (e.g. competition, predation risk, food colour), or on the basis of energy density, also fulfill specific nutrient needs (passive dietary complementation). In some environments, especially during productive phases (e.g. reproduction), free-living birds may rely on nutrient appetites to ensure their choice of foods satisfies their nutrient needs (active dietary complementation). Meeting nutrient needs through dietary complementation can be facilitated, complicated, or impeded by any of several environmental or organism determinants of food choice. Nutrient appetites, exogenous food stores, and endogenous nutrient stores are three organismal determinants that are probably the most important in facilitating dietary complementation.