The SalI (SalGI) restriction-modification system of Streptomyces albus G

The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively. These enzymes are responsible for restriction and modification of bacteriophages. Some phages carry genes that interfere with SalI-specific modification. The sal genes have been cloned in a Streptomyces host-vector system. Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx. 1 kb into the sal genes. Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA.     

Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI.

The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G.     

Temperature-Sensitive Modification and Restriction Phenotypes of an Escherichia coli dnaD Mutant

A mutant of Escherichia coli temperature-sensitive for deoxyribonucleic acid synthesis, dnaD, was found to have temperature-sensitive modification and restriction phenotypes. In contrast to the original observation by Carl (1970), the mutant could support the growth of λ phage at 41 C. However, the λ phages thus produced were able to form plaques with normal plating efficiency only on E. coli C, a restriction-less strain, but not on E. coli K. Since the λ phages produced in the mutant at 30 C could form plaques equally well on both E. coli strains, it was concluded that the dnaD mutant has a temperature-sensitive modification phenotype. Furthermore, since the dnaD mutant allowed some growth of unmodified λ·C phages at 41 C but less at 30 C, the mutant is also temperature sensitive in restriction. The relationship, if any, between temperature-sensitive deoxyribonucleic acid synthesis and temperature-sensitive modification-restriction in the dnaD mutant is not known. Similar experiments were done with three dnaC mutants and one dnaA mutant. Two dnaC mutants were found to have altered restriction phenotypes at 41 C, but none of the mutants were defective in modification.     

Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.     

Altered restriction endonuclease cleavage pattern of simian virus 40 DNA.

Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101--1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaIII site at position 0.21 and a defective HinIII site at 0.655 map units, and (iii) tsC219 carries a new HinIII site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.     

Complementation between temperature-sensitive and deletion mutants of reovirus.

A very low level of complementation has been found in conventional crosses between various classes of temperature-sensitive (ts) mutants of reovirus. A more definitive test for complementation was devised through a plaque assay on cell monolayers mixedly infected with defective reovirions lacking the L1 segment and prototype ts mutants from one or other of the known classes of reovirus mutants. An increase in the number of plaques on the mixedly infected plates over that on control plates infected with defective virions or ts mutants alone indicated that the ts mutant had been complemented by the defective virus. Class A, B, D, F, and G mutants were complemented at 39 C by the defective viruses, whereas class C and E mutants were not. In tests to determine whether complementation was reciprocal it was found that the defective virions were complemented by a class G mutant but not by the class C mutant. This and previous work (D.A. Spandidos and A. F. Graham, 1975) has therefore shown that of the seven known classes of ts mutants the class C mutant is the only one that neither complements nor is complemented by the defective virions. For this reason the class C ts mutation has been assigned to the L1 segment of the viral genome.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.     

A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state.

A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids.     

Mutant of Vesicular Stomatitis Virus Which Allows Deoxyribonucleic Acid Synthesis and Division in Cells Synthesizing Viral Ribonucleic Acid

Some temperature-sensitive mutants of vesicular stomatitis virus were tested for their ability to block the initiation of deoxyribonucleic acid (DNA) synthesis and division in serum-stimulated hamster embryo fibroblasts at the nonpermissive temperature. Although the parental strain blocked these processes, one particular mutant allowed essentially normal DNA synthesis and division. By autoradiography, it was shown that individual cells infected with this mutant could synthesize viral ribonucleic acid and at the same time initiate DNA synthesis and divide. Cells infected with such conditional defective mutants appear to be suitable for studies on the effects of persistent viral infections on molecular and cellular functions in proliferating cell populations.     

Restriction-deficient mutants of Staphylococcus aureus.

A series of restriction-deficient mutants was isolated from non-lysogenic strains of Staphylococcus aureus belonging to phage groups I and II. Some mutants were sensitive to all phages tested. With one possible exception, all the mutants were unaffected in their modification systems. The breakdown of DNA of phages, restricted in the parental strains, was reduced in both the mutants that were tested. The restriction in propagating strain 3A could be transduced to its restriction-deficient mutant. The transduction efficiency increased after ultraviolet irradiation of the transducing phage suggesting that the gene for restriction is present on the bacterial chromosome.     

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.     

Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA (tmRNA)

An Escherichia coli strain with a deletion in the ssrA gene that encodes 10Sa RNA (tmRNA) was used to screen for temperature-sensitive (ts) mutants whose ts phenotypes were suppressible by introduction of the wild-type ssrA gene. Mutants in four different genes were isolated. Ts mutants of this type were also obtained in a screen for mutations in thyA, the structural gene for thymidylate synthase. The ThyA activity in crude extracts prepared from the ts mutants was temperature-sensitive. The presence of the ssrA gene caused an increase in the total amount of the temperature-sensitive enzyme expressed, rather than suppressing the ts activity of the enzyme itself. SsrA-DD, a mutant form of 10Sa RNA, suppressed the ts phenotype of a thyA mutant, suggesting that degradation of a tagged peptide was not required for suppression of the ts phenotype. Considering the fact that ssrA-suppressible mutants could be isolated as temperature-sensitive mutants with mutations in different genes, it seems evident that trans-translation can occur on mRNA that is not lacking its stop codon.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.     

Temperature-sensitive developmental mutants of Caenorhabditis elegans

About 200 temperature-sensitive mutants of the nematode Caenorhabditis elegans have been isolated. At restrictive temperature, the mutants are blocked in the reproductive life cycle. They have been placed into six broad categories based on their defective phenotypes. The six categories are: (1) mutants blocked in embryogenesis; (2) mutants defective in gonadogenesis; (3) mutants defective in spermatogenesis; (4) mutants that accumulate at an intermediate growth stage; (5) mutants that produce sterile adult progeny; (6) mutants that have a temperature-sensitive morphological defect that interrupts the reproductive life cycle. The critical times of temperature sensitivity have been measured using temperature-shift experiments. Most of the gonadogenesis and spermatogenesis mutants are temperature sensitive during the period of cellular differentiation rather than proliferation. The temperature responses of the gonadogenesis and zygote-defective mutants indicate a common association between functions in gonadogenesis and early embryogenesis. Many of the mutants placed in different categories share other temperature-sensitive phenotypes upon close examination. This implies that many of the functions required for development are general metabolic reactions under increased demand during differentiation and embryogenesis.     

Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis.

A new mutant of Salmonella typhimurium was isolated which possesses a temperature-sensitive defect in the synthesis of 3-deoxy-D-manno-octulosonic acid. The defect in 3-deoxy-D-manno-octulosonic acid synthesis is due to a temperature-sensitive 3-deoxy-D-manno-octulosonate-8-phosphate synthetase, and the mutant accumulates an incomplete lipid A under nonpermissive conditions. Evidence is presented which indicates that the incomplete lipid A molecule is most likely identical in structure to the lipid A precursor synthesized by previously characterized mutants conditionally defective in 3-deoxy-D-manno-octulosonic acid synthesis. However, unlike related mutants which undergo growth stasis under nonpermissive conditions, the accumulation of lipid A precursor in the new mutant results in cell death at elevated temperatures.     

Isolation and Characterization of Temperature-Sensitive Respiratory Mutants of NEUROSPORA CRASSA

Filtration-enrichment and inositol-less death methods of mutant isolation, coupled with a screen for cyanide-insensitive respiration, proved to be highly efficient methods for isolating temperature-sensitive (ts) nuclear Neurospora mutants having defective respiration. Eighteen different ts respiratory mutants have been isolated. Most of them are pleiotropic and defective in one or more of the following phenotypes: cytochrome aa3, b, and c (individual or multiple defects); oligomycin inhibition of ATPase activity; respiration and its inhibition by KCN and salicyl hydroxamic acid; and growth rates in liquid and solid media at 25 degrees and 38 degrees. Among these mutants are the first cytochrome c mutant of Neurospora and an extranuclear ts ATPase mutant. An added bonus was the fact that over half of the mutants were affected either in ribosome assembly or in protein synthesis in the mitochondrion. We have yet to find any mutants completely lacking activities associated with the respiratory chain. However, the wide spectrum of mutants isolated here, along with those currently available, constitutes a considerable resource for investigating respiration in obligate aerobes.     

TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.