The genetic fine structure of the complex locus aro3 involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe.

Summary The complex locus aro3 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis. By comparing the complementation map and the meiotic recombination map, the aro3 locus could be subdivided into the five adjacent subregions A, B, C, D and E. Out of 115 aro3 alleles, 26 nonsense alleles and 30 missense alleles could be identified by the criteria of nonsense suppressor sensitivity and leakiness, respectively. Most alleles with a pleiotropic complementation pattern are of the nonsense type. We conclude from the polarity of the complementation patterns characterising the nonsense alleles that the translation direction proceeds from subregion A to subregion E. Antipolar effects in complementation are more frequent than in the analogous system of the arom gene cluster of Neurospora crassa.This work formed part of a Ph.D. thesis submitted to the University of Bern     

Genetic and Cytogenetic Analysis of the 43a-E Region Containing the Segment Polarity Gene Costa and the Cellular Polarity Genes Prickle and Spiny-Legs in Drosophila Melanogaster

A cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster is presented. Within this interval 27 complementation groups have been identified by extensive F(2) screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described.     

Nonsense-missense suppression in yeast

Summary A dominant suppressor has been discovered which suppresses polarity type mutations, noncomplementing ones and those belonging to short nonpolar complementation groups at the ad ₂ locus of S. cerevisiae. The suppressor translates two types of nonsense previously established at ad ₂. These data together with theoretical consideration of relationships between nonsense-translating anticodons and different codons make it possible to suggest a mechanism for the observed suppression. According to this mechanism two nonsense types of ad ₂ are identified as amber and ochre codons.     

Bacteriophage Mu DNA replication is stimulated by non-essential early functions

Summary The replication of a spontaneous Kil^– mutant of bacteriophage Mu has been investigated. The Kil^– mutation (Mucts62-13/4), which was introduced into a defective prophage, is pleiotrophic, leading to the loss of also the Gam, Cim and Sot functions. The mutation is caused by an insertion with the characteristics of IS1, located just outside the B gene.Mucts62-13/4 phages form extremely small plaques on wildtype indicator strains. The replication of the insertion mutant as compared to Mucts62 is strongly reduced. Normal replication could be restored by relieving the polarity of the insertion or by complementation with defective prophages which express all early functions. Apparently, early genes other than A and B are involved in normal Mu DNA replication.     

An amber map of Salmonella phage P22

Summary Forty five amber mutants of Salmonella phage P22 were assigned to 12 complementation groups. Two amber mutants could not be classified into any one particular group.Ten of the complementation groups were mapped by two-factor crosses. The map is circular and about 65 units long.The remaining two complementation groups each consist of a single member which form very small plaques. Attempts to map these two mutants by two-factor crosses were unsuccessful.     

The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa

Cellular polarity is a fundamental property of every cell. Due to their extremely fast growth rate (>/=1 microm/s) and their highly elongated form, filamentous fungi represent a prime example of polarized growth and are an attractive model for the analysis of fundamental mechanisms underlying cellular polarity. To identify the critical components that contribute to polarized growth, we developed a large-scale genetic screen for the isolation of conditional mutants defective in this process in the model fungus Neurospora crassa. Phenotypic analysis and complementation tests of ca. 950 mutants identified more than 100 complementation groups that define 21 distinct morphological classes. The phenotypes include polarity defects over the whole hypha, more specific defects localized to hyphal tips or subapical regions, and defects in branch formation and growth directionality. To begin converting this mutant collection into meaningful biological information, we identified the defective genes in 45 mutants covering all phenotypic classes. These genes encode novel proteins as well as proteins which 1) regulate the actin or microtubule cytoskeleton, 2) are kinases or components of signal transduction pathways, 3) are part of the secretory pathway, or 4) have functions in cell wall formation or membrane biosynthesis. These findings highlight the dynamic nature of a fungal hypha and establish a molecular model for studies of hyphal growth and polarity.     

Structural and functional analysis of the scute locus in Drosophila melanogaster

Summary The functional expression of 12 scute alleles in homozygotes and compounds of Drosophila melanogaster at 14°, 22°, 30°C is analysed. Based on the data obtained, linear maps for bristles and mutations are built. The basic features of the maps, clustering and polarity, are invariable with respect to temperature, scute gene dosage and cross direction. In addition local dominance of the norm over bristle reduction was produced by the scute mutation; different types of complementation reactions were established for each bristle. The gene scute is treated as an operon-like system, composed of 3–4 cistrons with each controlling the formation of bristles on a particular region of the fly's body. This model argues well with the structure of maps constructed and implies a post-translational level of initial events of bristle-formation process.This paper is based on the report presented at XIV International Congress of Genetics (Moscow, August 1978)     

Retroviral gene transfer for the assignment of Fanconi anemia (FA) patients to a FA complementation group

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure, cancer susceptibility, and a variety of developmental defects. The disease is clinically heterogeneous; eight different complementation groups (FA A–H) and, thus, genetic loci have been discovered. Two genes, FAA and FAC, have been cloned. Disease-associated mutations have been detected and rapid mutation screening makes possible the assignment of patients without resorting to time-consuming cell fusion and complementation analysis. Amplification of specific cDNAs from RNA followed by direct or indirect sequence analysis is a standard method for mutation detection. During the course of such examinations of the FAC gene, we have noted that frequently only one of the expressed alleles is successfully amplified. This can lead to false assignment of patients to a complementation group. As we report here, such cases can be rapidly clarified by retroviral gene transfer and complementation analysis. Received: 30 July 1997 / Accepted: 13 October 1997     

Amber mutants of Salmonella-phage P22 in genes engaged in the establishment of lysogeny

Summary Phage mutants were isolated with amber mutations in genes necessary for establishment of lysogeny. These mutants form turbid plaques on su ⁺ strain 527R1 and clear plaques of different types on LT2. According to complementation tests, fourteen mutants fall in the c ₂ gene, four in the c ₃ gene but no amber mutants were found belonging to the c ₁ gene. Pulse labelling experiments to follow DNA synthesis after phage infection were done with the mutants classified by complementation tests. Furthermore the labelling experiments demonstrated that the nonleaky c ₃ amber mutants displayed the same DNA synthesis pattern as c ₁ missense mutants. Since these c ₃ amber mutants complement missense c ₁ mutants it is concluded that the c ₃ and c ₁ genes must act together for the first transient repression of DNA synthesis, i.e., seven minutes after infection. It is suggested that clear plaque forming c ₁ amber mutants cannot be isolated because of polarity leading to defectivity of lysogenic as well as of lytic functions.The majority of the experiments presented are a part of the dissertation of H. D. Dopatka at the University of Göttingen.     

Selective activation by the guanine nucleotide exchange factor Don1 is a main determinant of Cdc42 signalling specificity in Ustilago maydis

The highly conserved GTP-binding proteins Cdc42 and Rac1 regulate cytokinesis, establishment of cell polarity and vesicular trafficking. In the dimorphic fungus Ustilago maydis , Rac1 is required for cell polarity and budding, while Cdc42 is essential for cell separation during cytokinesis. The same cell separation defect is also observed in mutants that lack Don1, a guanine nucleotide exchange factor (GEF) of the Dbl family. We have generated a series of chimeric GTP-binding proteins consisting of different portions of Cdc42 and Rac1. In vivo complementation analysis revealed that a short region encompassing amino acids 41–56 determines signalling specificity. Remarkably, substitution of a single amino acid at position 56 within this specificity domain is sufficient to confer Cdc42 function to Rac1 in vivo . Expression of Rac1^W56F in Δ cdc42 mutant cells resulted in complementation of the cell separation defect. In vitro GDP/GTP exchange assays demonstrated that the Dbl family GEF Don1 is highly specific for Cdc42 and cannot activate Rac1. However, if Rac1^W56F is used as a substrate, Don1 is able to stimulate GDP/GTP exchange. Together these data indicate that activation by the GEF Don1 is an important determinant of Cdc42-specific signalling in vivo .     

Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels

Background: The frizzled (fz) gene of Drosophila encodes the founding member of the large family of receptors for the Wnt family of signaling molecules. It was originally studied in the adult epidermis, where it plays a key role in the generation of tissue polarity. Mutations in components of the fz signal transduction pathway disrupt tissue polarity; on the wing, hairs normally point distally but their polarity is altered by these mutations.Results: We devised a method to induce a gradient of fz expression with the highest levels near the distal wing tip. The result was a large area of proximally pointing hairs in this region. This reversal of polarity was seen when fz expression was induced just before the start of hair morphogenesis when polarity is established, suggesting that the gradient of Fz protein acted fairly directly to reverse hair polarity. A similar induction of the dishevelled (dsh) gene, which acts cell autonomously and functions downstream of fz in the generation of tissue polarity, resulted in a distinct tissue polarity phenotype, but no reversal of polarity; this argues that fz signaling was required for polarity reversal. Furthermore, the finding that functional dsh was required for the reversal of polarity argues that the reversal requires normal fz signal transduction.Conclusions: The data suggest that cells sense the level of Fz protein on neighboring cells and use this information in order to polarize themselves. A polarizing signal is transmitted from cells with higher Fz levels to cells with lower levels. Our observations enable us to propose a general mechanism to explain how Wnts polarize target cells.     

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.     

Complementation system for Helicobacter pylori

Previously Langford et al. (2006) developed the pIR203C04 complementation system for Helicobacter pylori, which can be used to complement and restore phenotypic effects in H. pylori mutant, and furthermore they used the complementation system in vivo experiments to animals without altering the ability of strain SSI to colonize mice. In their previous study, the pIR203C04 was able to transform 26695, SSI, J99, and 43504 H. pylori strains by an electroporation method. However, in the present study using a natural transformation the pIR203C04 transformed only 26695 H. pylori but not SSI, J99, 7.13, and G27 H. pylori strains. Since the useful complementation system has a limitation of narrow selection among H. pylori strains, we redesigned the complementation system for the improvement. The same intergenic chromosomal site between hp0203 and hp0204 was utilized for the new complementation system because the insertion at the intergenic site didn’t show any polar effects and disruption of other H. pylori genes. The genome sequence analysis showed that the intergenic regions among H. pylori strains may have too low homology to each others to do a homologous recombination. Thus, in addition to the short intergenic region, the fragments of the new complementation system included 3′ conserved parts of hp0203 and hp0204 coding regions. Between the fragments there are a chloramphenicol acetyltransferase cassette and multicloning sites, resulting in pKJMSH. DNA fragment of the interest can be cloned into the multicloning sites of pKJMSH and the fragment can be integrated at the intergenic region of H. pylori chromosome by the homologous recombination. Indeed, by the natural transformation, pKJMSH was able to transform all five H. pylori strains of 26695, SSI, J99, 7.13, and G27, which are common for the investigation of molecular pathogenesis. Thus, the new pKJMSH complementation system is applicable to most H. pylori wild-type stains.     

Molecular basis for genetic complementation in propionyl CoA carboxylase deficiency

The extent of genetic complementation in polyethylene glycol-induced heterokaryons of propionyl CoA carboxylase deficient fibroblast lines was determined by comparing enzyme activity changes over time in pairwise fusions of the three major complementation groups, bio, pcc A and pcc C, with the activity changes in similarly mixed but unfused cultures. Maximum complementation between bio and pcc. A or pcc C lines was attained within 24 h after fusion and was not inhibited by cycloheximide. In contrast, the complementation between pcc A and pcc C lines only attained 50% of the maximum restored carboxylase activity by 24–36 h and the increase was 93% inhibited by cycloheximide. Maximum restoration of activity was not achieved until 72–96 h after fusion. Removal of cycloheximide at 24 h permitted complementation to take place. Our studies suggest that intergenic complementation between the bio and pcc A or pcc C lines is due to the contribution within the heterokaryon of normal enzymes from each of the respective lines, resulting in almost immediate, protein synthesis-independent, partial restoration of carboxylase activity. Complementation between pcc A and pcc C lines also appears to be intergenic but probably results from the de novo synthesis of normal subunits and protomers which assemble into normal or stabilized propionyl CoA carboxylase molecules with restored enzyme activity.     

Organization of the histidine-3 region of Neurospora

Summary The histidine-3 region of Neurospora specifies the structure of three enzymes of the histidine biosynthetic pathway viz., PR-AMP 1,6-cyclohydrolase, PR-ATP pyrophosphohydrolase, and histidinol dehydrogenase. Point mutations in this region may either affect individual enzyme activities or all of the three activities simultaneously. Attempts have been made to fractionate the three enzymes in order to understand the organization of the his-3 region. Gel-filtration on Sephadex G-200 revealed a single peak of activity for pyrophosphohydrolase and dehydrogenase. The two enzymes also emerged as one peak on chromatography through a DEAE-cellulose column eluted with linear salt gradient. Sedimentation characteristics of cyclohydrolase, pyrophosphohydrolase, and dehydrogenase were identical on sucrose density-gradient centrifugation. The molecular weight of this aggregate was estimated to be about 140,000. Other enzymes of histidine biosynthesis could be readily separated by these methods. Furthermore, mutational events occurring at several points across the genetic map seem to influence the three activities simultaneously. These results suggest that the polypeptides coded by the his-3 region are organized into a functional aggregate or multi-enzyme complex. Analysis of existing four-point cross data indicates that recombination within his-3 region is polarized. This polarity, according to the model proposed by Bernstein (1964), is towards the centromere and proceeds in a direction opposite to translation as deduced from complementation.     

Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region

Summary Mutants with Tn5 insertions in the vir region of the Agrobacterium tumefaciens TiC58 plasmid are unable to form crown-gall tumors. Complementation tests of these vir region mutants were carried out by constructing merodiploids in a recombination-deficient strain. Each merodiploid possessed a mutant TiC58 plasmid and a recombinant plasmid containing either the homologous wild-type DNA region or the homologous region containing a second Tn5 insertion. The analysis identified six complementation groups. Mutations in one of these complementation groups were not complemented in trans and represent a cis-dominant locus. The mutation in one complementation group showed variation in host range.     

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

Internal reinitiation of translation in polar mutants of the trpB gene of Salmonella typhimurium.

Summary The trpB gene of S. typhimurium codes for the bifunctional component II subunit of the AS-PRT complex which catalyzes the first two steps of tryptophan biosynthesis. It has previously been shown that the amino-terminal 40% of the component II molecule possesses the catalytic sites determining glutamine amidotransferase (GAT) activity, demonstrable indirectly by complementation with component I, the product of trpA, in the synthesis of anthranilic acid from chorismic acid and glutamine (AS activity), while its carboxy-terminal 60% possesses the catalytic sites determining anthranilate-PRPP phosphoribosyl transferase (PRT) activity, demonstrable by direct enzymatic assay. Here we further demonstrate the functional independence of the two regions of the component II subunit by providing evidence for the existence of monofunctional (GAT^-, PRT⁺) carboxy-terminal restart fragments of component II in certain chain terminating trpB mutants. Nonsense and frameshift mutants of the operator-proximal portion (region 1) of trpB have been found to grow well in media supplemented with anthranilic acid, implying the presence of PRT activity in the cell. Analysis of extracts of these strains has demonstrated the presence of low, but variable levels of PRT activity, but no GAT activity. Correlation of the map location of these mutations with the intensity of their polar effects on the expression of operator-distal genes suggests the existence of at least two gradients or units of polarity within region 1. Furthermore, in double mutant polarity tests, multiplicatiove polar effects were found in certain region 1 trpB-trpB double mutants strains. Taken together, these results lead us to conclude that at least two sites for reinitiation of translation exist within region 1 of trpB which can be activated by the presence of a nearby chain terminating codon. Such reinitation leads to the synthesis of labile carboxy-terminal restart fragments of component II which possess PRT function, but lack GAT function.