Mutations of Anacystis nidulans that affect cell division

Summary Following nitrosoguanidine treatment of the blue-green alga Anacystis nidulans strain 6311, mutants which grow predominantly as short or long filaments are commonly produced. Cytological study shows that the filaments are multinucleate, coenocytic structures. Such mutations are therefore best interpreted as ones that impair cell division in a normally unicellular organism.     

siRNA-mediated gene silencing of MexB from the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa

To gain insights into the effect of MexB gene under the short interfering RNA (siRNA), we synthesized 21 bp siRNA duplexes against the MexB gene. RT-PCR was performed to determine whether the siRNA inhibited the expression of MexB mRNA. Changes in antibiotic susceptibility in response to siRNA were measured by the E-test method. The efficacy of siRNAs was determined in a murine model of chronic P. aeruginosa lung infection. MexB-siRNAs inhibited both mRNA expression and the activity of P. aeruginosain vitro. In vivo, siRNA was effective in reducing the bacterial load in the model of chronic lung infection and the P. aeruginosa-induced pathological changes. MexB-siRNA treatment enhanced the production of inflammatory cytokines in the early infection stage (P ＜ 0.05). Our results suggest that targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. [BMB Reports 2014; 47(4): 203-208]     

Mutations that affect the regulation of phs in Salmonella typhimurium.

The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs-lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations cis-acting and that phs is not subject to autoregulation.     

Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.     

Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions.

The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989). A chimeric influenza virus A/WSN/33 NA containing the influenza B virus cytoplasmic tail rescued influenza A virus infectivity. The transfectant virus had less NA incorporated into virions than A/WSN/33, indicating that the cytoplasmic tail of influenza virus NA plays a role in incorporation of NA into virions. However, these results also suggest that the influenza A virus and influenza B virus cytoplasmic tail sequences share common features that lead to the production of infectious virus. Transfectant virus was obtained with all cytoplasmic tail mutants generated by site-directed mutagenesis of the influenza A virus tail, except for the mutant resulting from substitution of the conserved proline residue, presumably because of its contribution to the secondary structure of the tail. No virus was rescued when the cytoplasmic tail was deleted, indicating that the cytoplasmic tail is essential for production of the virus. The virulence of the transfectant viruses in mice was directly proportional to the amount of NA incorporated. The importance of the NA cytoplasmic tail in virus assembly and virulence has implications for use in developing antiviral strategies.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

Mutations in the par genes of Caenorhabditis elegans affect cytoplasmic reorganization during the first cell cycle

A dramatic reorganization of cytoplasm occurs during the first cell cycle in embryos of the nematode, Caenorhabditis elegans. We present here the results of a quantitative study of some of the events during this reorganization in wild-type embryos and in par mutant embryos. The par mutations define a set of genes required for cytoplasmic localization in early embryos. We show that par mutations lead to defects in several events of the reorganization. Mutations in all four of the par genes we studied lead to defects in pseudocleavage and asymmetric redistribution of cortical microfilaments. In addition, some of the par mutations affect streaming of cytoplasm, migration of the pronuclei, and asymmetric shortening of the embryo. We propose that the major function of the par genes might be to orchestrate this initial reorganization of cytoplasm.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.     

Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly.

The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33. Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33. Neither strain showed major defects in ribosome assembly. However, when the mutations were transferred to other strains of E. coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated. In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced. The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.     

Residues that affect human Argonaute2 concentration in cytoplasmic processing bodies

Sequence-specific gene silencing triggered by double-stranded RNA is a fundamental gene regulatory mechanism present in almost all eukaryotes. Argonaute2 (Ago2) is the central protein component of RNA-induced silencing complex (RISC), and resides in cytoplasmic processing bodies (P-bodies). In the present study, we demonstrated one human mutant Ago2 protein containing 6 point mutations (G32W, F128L, R196Q, P458S, T741A, S752G) failed to accumulate in P-bodies. Analysis of the different Ago2 revertants indicates the S752 as a key amino acid for P-body localization of Ago2. The S752 is evolutionary conserved in the Piwi domain of Ago2 homologs from worms, insects, plants and mammals. We further showed the single point mutation S752G interfering the interaction between Ago2 and Dcp1a, a key component of P-bodies.     

Mutations that overcome plasmid-mediated relaxation affect (p)ppGpp

Summary A recombinant plasmid, pMY3, was constructed in this laboratory to express the amber suppressor allele, Su⁺7, of the tRNA^Trp gene from E. coli (Yarus, 1979a). This plasmid also relaxes control of the synthesis of all stable RNA species in its host cell after amino acid deprivation. Guanosine penta and tetra-phosphate (MSII and MSI) concentrations are reduced to about one-half the levels achieved by starving the host cells carrying the cloning vehicle (pMB9) alone.We now show that the relaxation conferred on cells carrying pMY3 can be overcome by at least three different missense mutations at the chromosomal spoT locus. In these stringent, plasmid-carrying strains, the ppGpp levels attained during starvation are equivalent to or higher than that of the host cell carrying the vehicle alone.In vitro mutagenesis of the relaxing plasmid with EMS, followed by transformation and screening for plasmid-bearing stringent cells, yielded four stringent revertants of the relaxing locus. Cells carrying these mutants plasmids all have normal stringent responses to amino acid starvation, and again, elevate (p)ppGpp levels equal to or greater than 80% LS286 (pMB9) levels.Despite pMY3s modest effect on its host's MSI levels during the steady state of starvation, an obvious correlation exists between the concentration of that nucleotide and the host's ability to respond stringently. We therefore believe that the plasmid intervenes in MS metabolism. Measurements of the in vivo rates of decay of MSI and MSII after reversal of isoleucine starvation show that pMY3 has no effect on those reactions. The most likely mechanism of plasmid action is therefore inhibition of MS synthesis.Nonstandard Abbreviations MSI ppGpp - MSII pppGpp - EMS ethyl methane sulfonate - TCA trichloroacetic acid     

Mutations that affect production of branched RNA-linked msDNA in Myxococcus xanthus.

A deletion mutation of the gene (msd-msr) for the branched RNA-linked msDNA of Myxococcus xanthus was constructed by replacing the chromosomal 0.7-kilobase (kb) SmaI-XhoI fragment encompassing msd-msr with a 1.4-kb fragment carrying a gene for kanamycin resistance. It was found that this deletion strain (delta msSX) could not produce msDNA, although it still contained another species of msDNA, mrDNA (msDNA, reduced size). No apparent differences between delta msSX and the wild-type strain were observed in terms of cell growth, morphogenesis, fruiting-body formation, or motility. Both a deletion mutation at the region 100 base pairs upstream of msd and an insertion mutation at a site 500 base pairs upstream of msd showed a significant reduction of msDNA production, indicating that there is a cis- or trans-acting positive element in this region. When the 3.5-kb BamHI fragment carrying msd-msr from Stigmatella aurantiaca was inserted into the M. xanthus chromosome, the S. aurantiaca msDNA was found to be produced in M. xanthus.     

Linkage relationships of mutations endowing Streptococcus pyogenes with resistance to antibiotics that affect the ribosome

Summary Several mutations conferring resistance to streptomycin, kanamycin, spectinomycin, erythromycin, and lincomycin on the group A streptococcal strain 56188 have been mapped by two- and three-point crosses using transduction with bacteriophage A25. The markers are located in two linkage regions too distant to be cotransduced. One harbors the streptomycin and kanamycin loci which are transduced jointly at 78% and the other bears loci for spectinomycin (spc), erythromycin (eryA), and lincomycin (linA) resistance, in this order. spc and linA are cotransduced at a frequency of about 27%. Analysis of three-point crosses involving spc-4, eryA300, and linA12 according to the Wu model for random general transduction shows consistency of the theoretical predictions with the experimental data and indicates that the intervals of the above sequence are about 22% and 6% of the average length of the transduced piece.     

Mutations that affect vacuole biogenesis inhibit proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae

In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.     

Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln⁰, a flightin null mutant, 2) Mhc¹³, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc⁶, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln⁰ and Mhc¹³ but not in Mhc⁶. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln⁰ and Mhc¹³ but not in Mhc⁶. Tension generation under isometric conditions was not impaired in fln⁰. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles. flight muscles; muscle mutants; myosin     

Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptotene and bouquet stages

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.