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.     

Theste13 + gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

A mitochondrial ribosomal RNA mutation and its nuclear suppressors

Summary We have isolated cold-resistant revertants from a mitochondrial cold-sensitive mutation, cs909, localized in the 21S ribosomal RNA gene.Two types of revertants have been isolated: (1) strong revertants which were shown to be due to a single, nuclear, dominant suppressor; (2) weak revertants which are all due to the presence of a single, nuclear, recessive suppressor.The recessive suppressor, when separated from the mitochondrial mutation, itself confers a cold-sensitive phenotype, that is, there is a mutual suppression between the mitochondrial cold-sensitive mutation and the nuclear cold-sensitive mutation. The suppressor by itself produces modified ribosomes and therefore probably codes for an element of the mitochondrial ribosome such as a ribosomal protein.     

Mutation frequency decline following chemical mutagenesis of Salmonella typhimurium

The occurrence is reported of a mutation frequency decline process (MFD) following treatment of Salmonella typhimurium strain trpC₃ with two chemical mutagens which give rise predominantly to suppressor revertants. With the carcinogen 4-nitroquinoline-N-oxide (4NQO) the results are analogous to those obtained for UV-mutagenesis. In the case of methoxynamine, the process is due to specific excision of premutational lesions, since lethality is low and lethal lesions are non-excisable. Mutants are described which cannot perform MFD of lesions induced by one or both of the chemical mutagens, indicating that the loss of revertants is in each case due to a bacteial repair system rather than to spontaneous degradation of the induced lesion. The mutants, however, were isolated because of an altered response to UV mutagenesis, viz., their ability to express UV-induced mutants in the absence of amino acids to stimulate active post-irradiation protein synthesis. In all other respects tested, their response to UV is identical with that of the parent strain. The hypothesis is discussed that the total absence of UV-induced revertants of the strain S. typhimurium trpC₃ when active protein synthesis is inhibited is due to two processes, first, rapid MFD due to the specific excision of pyrimidine dimers (the predominant UV-lesion) and secondly, the slow excision of other premutational damage which may be other photoproducts or secondary distortions caused by close juxtaposition of several pyrimidine dimers.     

Genetic analysis of mutants of Aspergillus nidulans blocked at an early stage of sporulation.

Three mutants of Aspergillus nidulans, selected to have a block at an early stage of conidiation (asexual sporulation), exhibit similar pleiotropic phenotypes. Each of these mutants, termed preinduction mutants, also are blocked in sexual sporulation and secrete a set of phenolic metabolites at level much higher than wild type or mutants blocked at later stages of conidiation. Backcrosses of these mutants to wild type showed that the three phenotypes always cosegregated. Diploids containing the mutant alleles in all pairwise combinations were normal for all phenotypes, showing that the three mutations are nonallelic. This conclusion was confirmed by the finding that the mutations map at three unlinked or distantly linked loci. Ten revertants of the two least leaky preinduction mutants, selected for ability to conidiate, were found in each case to arise by a second-site suppressor mutation. All of the revertants still showed accumulation of some of the phenolic metabolites but differed from each other in certain components. Three of the revertants retained the block in sexual sporulation. In these cases the suppressor has thus uncoupled the block in asexual sporulation from the block in sexual sporulation. These results are understandable in terms of a model in which preinduction mutations and their suppressors affect steps in a single metabolic pathway whose intermediates include an effector specific for asexual sporulation and a second effector specific for sexual sporulation.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein

We previously described a dominant negative secY ^-d 1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY ^-d 1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity?to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY^-d1. When the secY ^-d 1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY ^-d 1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY^-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex.     

Mutations affecting the regulation of γ-aminobutyrate utilization inEscherichia coli K-12

Four genes,gabCPDT, are involved in the utilization of γ-aminobutyrate (GABA) byEscherichia coli K-12. Thegab gene cluster maps nearrecA andsrl, at 57.5 min.gabP, gabD andgabT specify the synthesis of GABA transport carrier, succinic semialdehyde dehydrogenase (SSDH), and glutamate-succinic semialdehyde transaminase (GSST), respectively;gabC controls the synthesis of all three proteins. GABA-nonutilizing mutants carrying deletions insrl extended into thegab cluster have been isolated. The mutants completely lost the capacity for GABA transport, while preserving full activity of GSST and SSDH, suggesting thatgabC is not a promoter-operator locus or a gene coding for an activator protein. A mutation ingabD (M-16) that abolished SSDH activity had the following additional properties: It exerted a bipolar effect on the neighboring genes, greatly reducing the activities of GSST and SSDH; the polar effect ongabP but not ongabT was fully suppressed by the knownrho mutation suA78; at least three classes of GABA-utilizing revertants of M-16 were obtained: (i) revertants with allgab activities restored to the parental levels; (ii) revertants with SSDH activity still missing, but with the other activities fully repaired; (iii) revertants with no SSDH activity, with GSST partly recovered, but with transport fully repaired. It is suggested that thegab cluster is transcribed bidirectionally from a promoter in thegabD region and that the mutation in strain M-16 may be due to DNA insertion in that region.     

An unusual suicidal interaction inEscherichia coli involving nucleoid protein H-NS

A conditional-lethal mutation (rpoB364) mapping to the gene that encodes the β-subunit of RNA polymerase was obtained inEscherichia coli. This mutation caused cell filamentation at the restrictive growth temperature and partial derepression of the osmotically regulatedproU operon at the permissive growth temperature. Even under the latter condition, transformants of therpoB364 mutant strain carrying the plasmid vector pACYC184, but not those carrying otherpolA-dependent multicopy plasmids such as pACYC177 or pBR322, were killed in early stationary phase; one class of suppressor mutants isolated as survivors within these transformant colonies were further derepressed forproU-lac expression, and the mutation in each of several independent clones of this class was mapped tohns, the gene that encodes the protein H-NS of theE. coli nucleoid. Thehns mutations did not suppress the conditional-lethal growth phenotype of therpoB364 mutant itself. On the other hand, intracellular overproduction of guanosine 3’, 5’-bispyrophosphate (ppGpp) in therpoB364 strain alleviated both the growth inhibition at the restrictive temperature and the pACYC184-mediated stationary-phase lethality. Upon subcloning into pUC19 or into pACYC177, a 105-bpXbal-HindIII fragment from pACYC184 was shown to be sufficient to confer therpoB364 hns ⁺-dependent lethal phenotype. We suggest that the level in stationary-phase cultures of a gene product(s) that interacts with the pACYC184 DNA fragment is altered in therpoB364 hns+derivative (compared to that inrpoB+ orrpoB364 hns strains) and that this results in cell suicide.     

A rapid and simple method for the determination of base substitution and frameshift specificity of mutagens

A rapid method for the determination of mutagenic specificity has been developed which makes use of the ochre mutation (TAA) in the his-4 gene of Escherichiacoli. Reversion to His⁺ may occur by suppressor mutation (Type I) or by mutation within the his-4 gene (Type II). The Type I mutations may be further subdivided with respect to the type of suppressor mutation by their ability to suppress nonsense mutants of bacteriophage T4, thus allowing the identification of the responsible base substitution (Kato et al., 1980). The system has the ability to identify mutagens which produce A:T → G:C transitions since only Type II mutants can arise through this base substitution; and in fact, the system confirms the A:T → G:C specificity of the mutagen, N⁴-hydroxycytidine (Janion and Glickman, 1980) since only Type II mutants were induced by treatment with this base analogue.When this system was further tested with several additional mutagens, the results indicate that ethyl methanesulphonate, methyl nitrosourea and ethyl nitrosourea produce primarily Type I revertants which were primarily G:C → A:T transitions. UV-light, γ-rays, 4NQO and methyl methanesulphonate produced all types of base substitutions. The tester strain was further improved by introducing a series of sequenced trp^? frameshift mutations, thus allowing the simultaneous monitoring of frameshift and base-substitution mutations.     

Genetic analysis of ryanodine receptor function in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis> based on<Emphasis Type="Italic"> unc-68</Emphasis> revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca²⁺-induced Ca²⁺ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca²⁺ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.Communicated by C. P. Hollenberg     

Dominant Suppressors of a Muscle Mutant Define an Essential Gene of CAENORHABDITIS ELEGANS

The sup-11 I locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.     

A slow-growing, ribosomal mutant of Salmonella typhimurium

Summary The mechanisms of S. typhimurium reversion from histidine dependence (his ^–) to histidine independence (his ⁺) were studied. Genetic and phenotypic characteristics of revertants induced by nitrosoguanidine were analyzed. Among them a class of slow-growing revertants was selected. It is found that all of these slow-growing revertants carry the original UGA nonsense mutation within the histidine operon. They are streptomycin sensitive and no specific suppressor(s) for UGA nonsense codon are demonstrable. The suppression takes place in the absence of conventional nonsense UGA suppressor(s). It is seemingly due to a ribosomal mutation which in turn is likely to produce ambiguity in the process of translation and which suppresses the UGA nonsense codon. The rate of both in vivo and in vitro protein synthesis is significantly reduced. The fact that streptomycin, at sublethal doses, reduced the growth rate of these mutants, probably because of the simultaneous burden of two ambiguity factors, suggests that the mutants described may be regarded as a kind of ram (ribosomal ambiguity) mutants with a his ^– sup ^–genotype. Their capacity to translate poly-U is reduced and in that respect they differ from ram mutants of Escherichia coli.     

New Late Gene, dar, Involved in DNA Replication of Bacteriophage T4 I. Isolation, Characterization, and Genetic Location

Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.     

Isolation and characterization of Escherichia coli birA intragenic suppressors

Summary The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions. Twenty-five heatresistant revertants of three temperature-sensitive birA alleles (birA 85, bir A 104 and bir A 879) were isolated and categorized into five growth and six repression classes. The revertants appear to increase biotin activation by raising the specific activity of BirA and/or, increasing the number of enzyme molecules. The 19 bir A 85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations. The bir A 85 revertants included a novel class of bio superrepressor mutations. Repressor titration experiments suggested that many of the bir A 85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids. The majority of the bir A 104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the bir A 104 mutation has been substituted by the wild-type or a chemically similar amino acid.     

Isolation and characterization of cold-sensitive mutations at the benA, beta-tubulin, locus of Aspergillus nidulans

Summary We have isolated large numbers of conditionally lethal -tubulin mutations to provide raw material for analyzing the structure and function of tubulin and of microtubules. We have isolated such mutations as intragenic suppressors of benA33, a heat-sensitive (hs^-) -tubulin mutation of Aspergillus nidulans. Among over 2,600 revertants isolated, 126 were cold-sensitive (cs^-). In 41 of 78 cs^- revertants analyzed, cold sensitivity and reversion from hs^- to hs⁺ were due to mutations linked to benA33. In three cases reversion was due to mutations closely linked to benA33 but cold sensitivity was due to a coincidental mutation unlinked to benA33. In the remaining 34 cases reversion was due to mutations unlinked to benA33. Thirty-three of the revertants in which cold sensitivity and reversion were linked to benA33 were sufficiently cold-sensitive to allow us to select for rare recombinants between benA33 and putative suppressors in a revertant x wild-type (wt) cross. We found only one recombinant among 1,000 or more viable progeny from crosses of each of these revertants with a wt strain. Reversion is thus due to a back mutation or very closely linked suppressor in each case. We have analyzed 17 of these 33 revertants with greater precision and have found that, in each case, reversion is due to a suppressor mutation that maps to the right of benA33. The recombination frequencies between benA33 and the suppressors are very low (less than 1.2×10^-4) in all cases. Five of these 33 revertants have been examined microscopically and in each of them nuclear division and nuclear migration are inhibited at a restrictive temperature. We conclude that at least some and perhaps all of these revertants carry intragenic suppressors of benA33 that, in combination with benA33, cause cold sensitivity by inhibiting the functioning of microtubules at low temperatures. Of the 17 suppressors mapped, 11 map to two clusters. These clusters are likely to define regions particularly important to the functioning of the -tubulin molecule.