Temperate Coliphage HK022: Clear Plaque Mutants and Preliminary Vegetative Map

Wild type phage HK022 was mutagenized by N-methyl-N′-nitro-N-nitrosoguanidine to induce clear plaque mutants. A total of 225 clear plaque mutants were isolated and 198 of these were assignable to one or the other of the two complementation groups of the corresponding cistrons which have been designated as cI and cII, respectively. Approximately 25% of the c mutants were found to be temperature-sensitive (cts); producing turbid plaques at 32 C and clear plaques at 38 C and above. From complementation tests involving cI and cII mutants, bacteria lysogenic for cII prophage were frequently obtained. Double lysogens harboring a cI and a cII prophage were infrequently found and single lysogens harboring only a cI prophage have not been recovered. Bacterial lysogens harboring a prophage carrying a cts mutation in the cI cistron were readily obtainable. However, such lysogens show a lethal phenotype at 40 C and above, although they appear to be fully viable at 32 C. It is shown that by incubation of lysogens harboring a cts mutant of the cI cistron at 42 C, it is possible to isolate cryptic lysogens which are non-immune but harbor at least one of the phage sus⁺ alleles. Genetic data involving cI, cII, and two complementing sus mutants of essential genes are presented. From these data the following vegetative map is deduced: sus4–cII-cI-sus3.     

Complementation experiments between conditional lethal mutants of bacteriophage ?X174

Summary Complementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage X174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.     

Phenotypic Characterization of Adenovirus Type 12 Temperature-Sensitive Mutants in Productive Infection and Transformation

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxyl-amine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for “initiation” of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutated in H12ts505 is required to maintain at least some aspects of the transformed state.     

Isolation and complementation analysis of temperature-sensitive mutants of the PS8 bacteriophage ofAgrobacterium tumefaciens

The isolation, method of complementation analysis and classification of temperature-sensitive (ts) mutants of phage PS8 (Agrobacterium tumefaciens) are described.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37° C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation. Received: 30 June 1997 / Accepted: 23 October 1997     

Genes involved in the regulation of the neutral phosphatase in Chlamydomonas reinhardi

Summary In Chlamydomonas reinhardi, mutations in either of two unlinked genes (PD₂ and PD₃) abolish the activity of the derepressible neutral phosphatase. The question arose whether these genes (or one of them) specify the structure of the enzyme or whether they have a regulatory function.Three mutants producing an active phosphatase at 25°C but not at 35°C were isolated and investigated. One of these mutants (PD ₁₁ ^ts ) was allelic with PD₂, another one (PD ₁₂ ^ts ) was linked to PD₃ and the third one (PD ₁₃ ^ts ) was linked to PD₂.PD ₁₁ ^ts and PD ₁₃ ^ts affected the formation of the neutral phosphatase only whereas PD ₁₂ ^ts interfered with the formation of both neutral and alkaline phosphatases at 35°C. The neutral phosphatase produced by the three mutants at low temperature was not more thermosensitive in vitro than the wild enzyme. Moreover, quite similar K_m values were found in WT, PD ₁₁ ^ts and PD ₁₂ ^ts using naphthyl phosphate as a substrate.On the other hand, revertants of PD ₂ ^- and PD ₃ ^- were isolated: their neutral phosphatases could not be distinguished from the wild enzyme on the basis of their thermosensitivities and K_m values for naphthyl phosphate.These results are consistent with the idea that PD₂ and PD₃ are regulatory genes. Other possible regulatory genes were revealed through PD ₁₂ ^ts and PD ₁₃ ^ts mutations.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique     

Ribosome structure, maturation of ribosomal RNA and drug sensitivity in temperature-sensitive mutants of Saccharomyces cerevisiae

Summary Selected strains of Saccharomyces cerevisiae were mutagenised with nitrosoguanidine and temperature-sensitive mutants isolated. These mutants were screened by twodimensional gel-electrophoresis for the presence of ribosomal proteins with altered mobility relative to parental preparations. Electrophoretic changes were detected in three mutants designated ts205, ts212 and ts417, with the alterations apparently the same in the three cases. All three mutants were more sensitive than were their parents to the antibiotics G418, hygromycin B and MDMP. Mutant ts212 has an abnormal distribution of native ribosomal subunits and appears to be defective in its assembly of the smaller subparticle.     

Genetic studies on Rhizobiophage 16-3

Summary A number of temperature-sensitive (ts) and lysis-deficient mutants were isolated from a bacteriophage of Rhizobium meliloti. The ts mutants were grouped by complementation. Functions were classified in relation to the eclipse and latent period. A genetic map of about 40 units was derived from crosses. The genes on the chromosome of the phage are arranged in the order of their presumed functions during the phage growth. They are located on the chromosome in sequence: immunity-early-late-lysis genes.Abbreviations Ant altered antigene - C immunity gene - h host range - K velocity constant of antiphage serum - L lysis gene - m.o.i. multiplicity of infection - NTG N-methyl-N-nitro-Nnitrosoguanidine - P.F.U. plaque forming unit - Rh.41 Rhizobium meliloti strain 41 - ti thermo-inducible - ts temperature-sensitive     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

Enhanced Mutability Associated with a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.     

The isolation and characterization of bacterial strains (Tab32) that restrict bacteriophage T4 gene 32 mutants

Summary Gene 32 of bacteriophage T4 codes for a single-stranded DNA binding protein. We have isolated mutants of Escherichia coli (called Tab32) that specifically restrict the growth of gene 32 missense mutants and allow normal growth of T4⁺. During infections of Tab32 with 32^tsL171, large amounts of DNA are synthesized and late proteins are made, but very few progeny phage are produced. At least two bacterial mutations are necessary for the restrictive phenotype; these mutations have been mapped to about min 41 and min 64.     

Lethal(1)aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tum^l and tu(1)Sz^ts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from “autoimmune responses” or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.     

Mutant of the glutamine transfer RNA gene as UGA suppressor in Escherichia coli

Summary A UGA suppressor derived from a glutamine tRNA gene of Escherichia coli K 12 was isolated and characterized. Phages carrying the suppressor su⁺2_UGA could be obtained only from a hybrid transducing phage, h ⁸⁰ cI ₈₅₇psu ⁺2_oc, but not from the original transducing phage cI ₈₅₇psu ⁺2_oc. By DNA sequence analysis, it was found that the su ⁺2 UGA suppressor obtained has two mutations; one is in the anticodon (TTATCA), as expected, and the other (CT) is at the 7th position from the 3 end of tRNA ₂ ^Gln . The significance of these mutations and the lethal effect on phage of the increased amounts of UGA suppressor tRNAs are discussed.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

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.     

Antimutators of mitochondrial and nuclear DNA in Saccharomyces cerevisiae. Relationship with gamma-ray sensitivity

Summary In Saccharomyces cerevisiae ten antimutator mutants have been isolated. The spontaneous occurrence of mitochondrial mutants resistant to erythromycin, oligomycin, and diuron is decreased 2-60-fold in these strains. The rate of forward and reverse spontaneous mutations of the nuclear genome is also reduced. The meiotic progenies arising from the crosses of seven mutants (LB₁, LB₂, LB₄, LB₅, LB₆, LB₇, LB₁₀) with an isogenic parental strain exhibit 2:2 segregations and therefore are the result of mutations in a single nuclear gene. The six mutants LB₁, LB₂, LB₄, LB₆, LB₇, LB₁₀ are semidominant and determine six complementation groups. The mutant LB₅ is dominant and therefore cannot be assigned to any complementation group. The mutants. LB₁, LB₄ and LB₁₀ are gamma-ray sensitive and, by tetrad analysis, it has been shown that gamma-ray sensitivity and spontaneous antimutability are the result of a single nuclear gene mutation. The other three mutants LB₃, LB₈ and LB₉ exhibit complex tetrad segregations typical of cytoplasmic inheritance and do not complement each other. However, although the mutations are semidominant, it has not been possible to detect any antimutator cytoductant among some 500 cytoductants carrying the kar1-1 nucleus. These results suggest that either several nuclear genes are involved in the expression of the antimutator phenotype or that the antimutator gene is located on nonchromosomal elements of the nucleus. The present study leads to the conclusion that a large number of nuclear genes are able to control simultaneously the spontaneous mutation rate of nuclear and mitochondrial genes. Since out of the ten antimutator mutants, three are also deficient in the repair of gamma-ray damage, it is also concluded that spontaneous and gamma-ray-induced lesions of DNA can be repaired by the same error-free process.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998     

Behavioral Mutants of DROSOPHILA MELANOGASTER. I. Isolation and Mapping of Mutations Which Decrease Flight Ability

A flight test box was developed and used in the isolation and initial characterization of Drosophila melanogaster mutants defective in flight behavior. Forty-eight mutants were isolated from F₁ progeny of ethyl methanesulfonate-treated males. Genetic mapping and complementation tests show that the mutations reside at thirty-four different sites on the X chromosome. Different mutants show different degrees of flight ability compared to controls. Forty-six mutations are recessive, while two appear to be semi-dominant with respect to flight behavior. In addition to flight defects, five mutants have visible defects, five behave as temperature-sensitive lethals and three exhibit abnormal electro-retinograms. Alleles of each of the previously known behavioral mutations, Hyperkinetic, ether à go-go and Shaker were found. Preliminary studies also suggest that the flight behavioral phenotype of mutations at seven sites is affected by the temperature at which the flies develop.