Joined suppression of rough phenotype and of red colour ofade2-1 mutants inSaccharomyces cerevisiae

A reciprocal suppression of rough phenotype was found in rough mutants of two prototrophic strains ofSaccharomyces cerevisiae on medium containing 1 % glucose. Suppression affects at least three independent rough loci and is strongly influenced by the genetic background. It is accompanied by suppression of red pigmentation inade2-1 mutants, and this too responds similarly to the genetic background. There is evidence for at least two independent loci that modify the action of the suppressor of both rough and red. The suppressor segregates regularly and is linked to one of the tested rough loci. It is not a super-suppressor and probably is not involved in the regulatory mechanism of purine biosynthesis either. An involvement of glucose metabolism in the suppression can be assumed from the lifting of the suppression of rough phenotype on lactate medium.     

Antisuppressors in yeast

Summary 1. Twenty-three mutations, mapping at eight different loci, were shown to prevent detectable suppression of ade2-1 and can1-100 by the ochre suppressor, ocSUPQ2. 2. Some reduce the efficiency of suppression of his5-2 and lys1-1 also. 3. Mutants from each of the eight loci prevented the lethal phenotype of ocSUPQ2 in [psi+] strains. They are, therefore, antisuppressor mutations. 4. One of the loci, asu3, is centromere-linked; two of the loci, asu2 and asu6, are linked to each other.     

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.     

An extragenic suppressor ofprp24-1 defines genetic interaction betweenPRP24 andPRP21 gene products ofSaccharomyces cerevisiae

The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.     

Cloning of the Agrobacterium tumefaciens C58 trpE gene by complementation in Escherichia coli

Summary The trpE gene of Agrobacterium tumefaciens C58 was cloned from a gene library by complementation in Escherichia coli. It was shown to be unlinked to trpD gene in this organism. It was also shown that the nontumorigenic phenotype of tryptophan auxotrophs of A. tumefaciens could be complemented by addition of exogenous tryptophan. The role of bacterially synthesised tryptophan in the process of tumour formation is discussed.Abbreviations Ap ampicillin - Cm chloramphenicol - Gent gentamycin - Km kanamycin - dATP deoxyadenosine 5-triphosphate - IAA indole acetic acid - NB nutrient broth - MinAB minimal Agrobacterium medium     

Reversion of a streptomycin-dependent strain of Escherichia coli

Summary A streptomycin dependent, spectinomycin resistant mutant ofEscherichia coli was used to select spontaneous phenotypic revertants to non-dependence on streptomycin. The ribosomes from one such revertant, which is inhibited by both streptomycin and spectinomycin, were analyzedin vitro. The altered protein responsible for the suppression of the streptomycin dependent phenotype was identified; this protein is 30S-10. The genetic locus for this mutation is a newly identified locus and it has been positioned close to thestr locus. The identification of the altered component responsible for the suppression of the spectinomycin resistant phenotype may be the same as that for the streptomycin dependent phenotype, but this is unproven.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity.

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-111 mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-111-bearing strain is the consequence of a single mutation. The rho-111 mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-111 mutation did not alter in vivo expression of either the tryptophan or histidine operons.     

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant

Summary We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485–510 kb) whereas sipC and sipD both map to 82 min (3850–3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.     

Rye-derived powdery mildew resistance gene <Emphasis Type="Italic">Pm8</Emphasis> in wheat is suppressed by the <Emphasis Type="Italic">Pm3</Emphasis> locus

Genetic suppression of disease resistance is occasionally observed in hexaploid wheat or in its interspecific crosses. The phenotypic effects of genes moved to wheat from relatives with lower ploidy are often smaller than in the original sources, suggesting the presence of modifiers or partial inhibitors in wheat, especially dilution effects caused by possible variation at orthologous loci. However, there is little current understanding of the underlying genetics of suppression. The discovery of suppression in some wheat genotypes of the cereal rye chromosome 1RS-derived gene Pm8 for powdery mildew resistance offered an opportunity for analysis. A single gene for suppression was identified at or near the closely linked storage protein genes Gli-A1 and Glu-A3, which are also closely associated with the Pm3 locus on chromosome 1AS. The Pm3 locus is a complex of expressed alleles and pseudogenes embedded among Glu-A3 repeats. In the current report, we explain why earlier work indicated that the mildew suppressor was closely associated with specific Gli-A1 and Glu-A3 alleles, and predict that suppression of Pm8 involves translated gene products from the Pm3 locus.     

Regulation of tryptophan metabolism in Coprinus cinereus: Isolation and characterisation of mutants resistant to 5-fluoroindole

171 mutations conferring resistance to the indole analogue 5-fluoroindole (5 FI) were isolated in the filamentous basidiomycete fungus Coprinus cinereus. 5 FI is thought to be toxic because it is converted intracellularly to 5-fluorotryptophan (5 FT) which feedback inhibits the first enzyme of the tryptophan biosynthetic pathway, anthranilate synthase. Mutations were assigned to five loci, iar-1-iar-5 on the basis of functional analyses and mapping experiments. iar-5 mutations mapped in the anthranilate synthase structural gene and gave rise to an enzyme feedback resistant to tryptophan and its analogue. Mutants at other loci had regulatory changes. iar-1 and iar-3 mutants had elevated levels of two pathway enzymes measured (anthranilate synthase and tryptophan synthase) and were cross resistant to analogues of other aromatic amino acids suggesting that the entire aromatic pathway was derepressed. iar-3 mutants were unable to degrade metabolically derived typtophan to anthranilic acid unlike iar-1 mutants which excreted high levels of anthranilic acid. iar-2 mutants appeared to have a constitutive degradative pathway. iar-4 mutants had a blocked degradative pathway and unusual levels of tryptophan pathway enzymes.Abbreviations 5 FI 5-fluoroindole - 5 FT 5-fluorotryptophan - pFP para-fluorophenylalanine - mFT meta-fluoro-tyrosine     

The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID

The centromere-specific histone H3 variant CENP-A plays a crucial role in kinetochore specification and assembly. We chose a genetic approach to identify interactors of the Drosophila CENP-A homolog CID. Overexpression of cid in the proliferating eye imaginal disk results in a rough eye phenotype, which is dependent on the ability of the overexpressed protein to localize to the kinetochore. A screen for modifiers of the rough eye phenotype identified mutations in the Drosophila condensin subunit gene Cap-G as interactors. Yeast two-hybrid experiments also reveal an interaction between CID and Cap-G. While chromosome condensation in Cap-G mutant embryos appears largely unaffected, massive defects in sister chromatid segregation occur during mitosis. Taken together, our results suggest a link between the chromatin condensation machinery and kinetochore structure.Electronic Supplementary Material Supplementary material is available for this article at     

Chemical and Photochemical Modification of Colicin E1 and Gramicidin A in Bilayer Lipid Membranes

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.Abbreviations: AlP_cS₃, almininum trisulfophthalocyanine; BLM, bilayer lipid membrane; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidyl-glycerol; DPhPG, diphytanoylphos-phatidylglycerol; DPhPg, diphytanoylphosphatidylcholine; gA, gramicidin A; NBS, N-bromosuccinimideThis revised version was published online in August 2005 with a corrected cover date.     

Genomic dissection of pod shattering in common bean: mutations at non‐orthologous loci at the basis of convergent phenotypic evolution under domestication of leguminous species

The complete or partial loss of shattering ability occurred independently during the domestication of several crops. Therefore, the study of this trait can provide an understanding of the link between phenotypic and molecular convergent evolution. The genetic dissection of ‘pod shattering’ in Phaseolus vulgaris is achieved here using a population of introgression lines and next‐generation sequencing techniques. The ‘occurrence’ of the indehiscent phenotype (indehiscent versus dehiscent) depends on a major locus on chromosome 5. Furthermore, at least two additional genes are associated with the ‘level’ of shattering (number of shattering pods per plant: low versus high) and the ‘mode’ of shattering (non‐twisting versus twisting pods), with all of these loci contributing to the phenotype by epistatic interactions. Comparative mapping indicates that the major gene identified on common bean chromosome 5 corresponds to one of the four quantitative trait loci for pod shattering in Vigna unguiculata. None of the loci identified comprised genes that are homologs of the known shattering genes in Glycine max. Therefore, although convergent domestication can be determined by mutations at orthologous loci, this was only partially true for P. vulgaris and V. unguiculata, which are two phylogenetically closely related crop species, and this was not the case for the more distant P. vulgaris and G. max. Conversely, comparative mapping suggests that the convergent evolution of the indehiscent phenotype arose through mutations in different genes from the same underlying gene networks that are involved in secondary cell‐wall biosynthesis and lignin deposition patterning at the pod level.     

Two Arabidopsis methylation-deficiency mutations confer only partial effects on a methylated endogenous gene family

In Arabidopsis a SWI2/SNF2 chromatin remodeling factor-related protein DDM1 and a cytosine methyltransferase MET1 are required for maintenance of genomic cytosine methylation. Mutations in either gene cause global demethylation. In this work we have assessed the effects of these mutations on the PAI tryptophan biosynthetic gene family, which consists of four densely methylated genes arranged as a tail-to-tail inverted repeat plus two unlinked singlet genes. The methylation mutations caused only partial demethylation of the PAI loci: ddm1 had a strong effect on the singlet genes but a weaker effect on the inverted repeat, whereas met1 had a stronger effect on the inverted repeat than on the singlet genes. The double ddm1 met1 mutant also displayed partial demethylation of the PAI genes, with a pattern similar to the ddm1 single mutant. To determine the relationship between partial methylation and expression for the singlet PAI2 gene we constructed a novel reporter strain of Arabidopsis in which PAI2 silencing could be monitored by a blue fluorescent plant phenotype diagnostic of tryptophan pathway defects. This reporter strain revealed that intermediate levels of methylation correlate with intermediate suppression of the fluorescent phenotype.     

A cytological analysis of the Scutoid mutation of Drosophila melanogaster,and its induced revertants

The Scutoid mutation of Drosophila melanogaster is associated with two small transpositions within region 35 of the polytene chromosome arm 2L. A number of induced revertants of Sco have been recovered and the majority of these are chromosome aberrations. The localisation of at least one breakpoint of these aberrant revertants to the proximal breakpoint of the proximal transposition of Sco, a breakpoint that juxtaposes two loci (noc and sna), normally separated by several loci is evidence that the Scutoid phenotype results from a fusion of noc and sna.     

SCM2, a tryptophan permease in Saccharomyces cerevisiae,is important for cell growth

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm2Δ1-Δ4) cause slow growth, whereas one disrupted allele (scm2Δ5) is lethal. Cells with both the scm2Δ1 and trp1-Δ101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm2Δ1 or trp1-Δ101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.

     

The effect of the plating medium on the recovery of nonsense suppressors in Saccharomyces cerevisiae

The recovery of nonsense suppressors in a strain of Saccharomyces cerevisiae carrying five ochre mutations, tr, hi, ly, ar, and ad, is affected by the plating medium. The highest frequency is observed on tryptophanless medium, while the lowest is observed on adenineless medium. Experiments showed that exogenous histidine inhibits suppressor expression and that exogenous adenine relieves this inhibition. In histidine-independent strains, mutation expression requires adenine. A model, based on the role of RNA in supersuppression and on the biosynthetic pathways of histidine and adenine, is proposed to account for the observed data. It cannot, however, account for the high frequency of suppressors on tryptophanless medium. The tentative conclusion is drawn either that mis-reading of the tryptophan nonsense codon by mutated tRNA is facilitated by the neighboring bases or that the type of acceptable amino acid is less rigorously limited in the mutated site of the tryptophan locus than in those of the other suppressible loci.This work was in part supported by the Calouste Gulbenkian Foundation and by the Medical Research Council (Grant No. G969/24/B).     

The Effects of Calcium and the Ionophore A23I87 on Modulation, Nitrogen Fixation and Growth of Soybeans

Tryptophan synthase in Phycomyces blakesleeanus. Part II: Activity of tryptophan synthase in Phycomyces blakesleeanus depending on the light and the content of zinc ions in the culture medium Five-day-old cultures of Phycomyces blakesleeanus show notice-able differences in the phenotype, depending on the culture conditions (permanent light, permanent dark, zinc deficiency, zinc sufficiency) and related to the distribution of tryptophan synthase activity between mycelium and sporangiophores. Permanent light and the presence of zinc ions in the medium during culturing have an antagonistic influence on the tryptophan synthase. The activity of the enzyme is being reduced in the sporangiophores and increased in the mycelium by the influence of light, while zinc ions in the culture medium increase the activity in the sporangiophores at simultaneous reduction in the mycelium. The importance of tryptophan synthase and tryptophan for the development of the fungus in relation to the metabolism of indole acetic acid is discussed.     

Genetics of barley hooded suppression

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.