The timing of phenotypic suppression of an aggregation defect by an aggregation-stimulating factor from Polyspboadylium violaceum

Abstract. A class of aggregation deficient mutants of Polysphondylium violaceum ( aggA ) had previously been shown to aggregate in the presence of an excreted, dialyzable product (D factor) prepared from wild type amoebae. We have characterized further the development of the aggA mutants in liquid culture. In the absence of an external source of D factor, aggA mutants never become aggregation competent. D factor must be added to the mutants in order for them to be able to aggregate when removed from liquid culture and plated on a surface. The ability of D factor to stimulate the development of aggregation competence can be illustrated with both the aggA mutant and wild type amoebae. D factor is only required by the aggA mutants at a late stage in development of aggregation competence and does not have to be present continuously during incubation. Wild type amoebae provided with additional D factor become aggregation competent earlier than amoebae incubated without additional D factor. These data suggest that the amoebae develop most of the biochemical functions necessary in order to aggregate and that D factor is necessary to trigger aggregation. One of these biochemical functions, development of aggregation-specific adhesion sites, has been shown to occur in the aggA mutant in the absence of D factor.     

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylium violaceum

An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group aggA. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. after partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the aggA mutants. The three major components (DfA, DfB, and DfC) show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.     

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylirrm violaceurn

Abstract. An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group agg A. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. After partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the agg A mutants. The three major components (Df_A, Df_B, and Df_C), show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.     

Dictyostelium mutants lacking DIF,a putative morphogen

DIF is an endogenous extracellular signal that may control differentiation of D. discoideum cells. It is a dialyzable, lipid-like factor that induces stalk cell formation among isolated amebae incubated in vitro with cAMP. To examine the consequences of DIF deprivation, we have isolated several mutant strains that are impaired in DIF accumulation, and whose inability to make stalk cells in vitro and during normal development on agar can be corrected by the addition of exogenous DIF. Little DIF is made by the mutants, and morphological development on agar stops after the cells have aggregated, but before a slug forms. In these DIF-deprived conditions, prespore cells can differentiate, but prestalk cells cannot.     

Mutants of Group D1Salmonella Carrying the Somatic Antigen of Group A Organisms: Isolation and Serological Characterization

O antigen mutants were obtained from Salmonella durban, a group D(1) organism, by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Serological studies demonstrated that the mutants lost the O-9 antigen factor of the parent organism but acquired the O-2 factor specific to group A Salmonella. Lipopolysaccharides of the mutant strains contained paratose which determines the specificity of O-2 factor. Tyvelose, present in the wild-type lipopolysaccharide, was not found in the mutants. H antigens and other biological characteristics of the mutant strains were the same as those of the wild-type organism. The present finding implies that group A Salmonella species might be derived from group D(1) organisms.     

Isolation of conjugation-constitutive mutants of colicin factor Ib

Summary Colicin factor ColIb-P9 is known to act as a sex factor in E. coli or Salmonella. Although ColIb-P9 confers mating ability on its host bacteria, this ability appears to be repressed since only a small proportion of cells in a culture of a colicinogenic strain are able to pair with, and transmit the factor to recipient bacteria. We have isolated mutants of ColIb-P9 which confer constitutive donor ability on their host. De-repression in these mutants is probably due to failure to produce repressor, rather than to insensitivity to repressor. As the colicin production by the mutants is still repressed, colicin synthesis and conjugation ability are subject to independent systems of regulation.     

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.     

Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.     

Induction of normal levels of genetic transformation in a class of endonuclease-defective mutants of Pneumococci.

Pneumococcal mutants were isolated that showed no zones of DNA hydrolysis around the bacterial colonies, even after prolonged incubation on the surface of agar plates containing DNA and methylgreen. Lysates of such mutants contained only very low levels of endonuclease activity; the mutants were also defective in genetic transformation even though they could still bind DNA to their surface. However, the same mutants could be made to undergo normal, high frequency genetic transformation by treatment with the activator protein, under appropriate conditions. The same treatment caused no detectable increase in the endonuclease level. Poor transformability of these mutants seems to be related to an inhibitory factor(s) released into the growth medium. Activation in the presence of this factor(s) leads to an abnormal (non-productive) DNA adsorption-both in mutant and in wild type pneumococci.     

The regulation of cellular slime mold development: a factor causing development of Polysphondylium violaceum aggregation-defective mutants.

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.     

Genetic and biochemical studies of phosphomannose isomerase deficient mutants ofSaccharomyces cerevisiae

Summary Three mannose-negative mutants ofSaccharomyces cerevisiae have been isolated. These mutants showed growth inhibition when mannose was added to a growth medium containing glycerol or fructose. Crosses between wild type mutants showed segregation of 2⁺/2⁻. Crosses between the mutants themselves showed that they were closely linked. Two mutants (XM3 and D2) showed characteristics of allelic structural alteration of phosphomannoseisomerase. Mutant D4 had a deficiency of phosphomannoseisomerase activity, but with a normal thermostability. Revertants from D4 had a normal thermostability.     

Mutants of Pichia guilliermondii yeasts with multiple sensitivity to antibiotics and antimetabolites. I. The selection and properties of the mutants

Riboflavin deficient mutant Pichia guilliermondii MS1 which requires approximately 1000-fold lower concentration of exogenous vitamin B2 for growth when compared with a non-adapted riboflavin deficient mutants of this species was isolated by means of of UV-irradiation. The growth of the mutant was strongly inhibited by actinomycin D and L-canavanine. The revertant MS8 and MS14 which synthesized riboflavin were selected from the strain MS1. These revertants posses a multiple sensitivity to actinomycin D, rifamycin, euflavine, mitomycin C, antimycin A, 8-azaadenine, 8-azaguanine, L-canavanine and 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine. The ability to utilized glycerol and ethanol as a sole carbon source for growth was impaired in these mutants. The mutants which can utilize glycerol were isolated from the strain MS14. Such mutants were resistant to actonomycin D. Mutation (s) which determines a multiple sensitivity and inability to utilized glycerol was recessive.     

Parasexual genetic analysis of aggregation-deficient mutants of Dictyostelium discoideum.

One hundred and thirty-nine independent, nitrosoguanidine-induced mutants blocked early in development were isolated in two haploid strains of D. discoideum. Forty of these developmental mutants were completely aggregation-deficient on bacterial lawns (Class I mutants) and these mutants were selected for parasexual genetic analysis. By fusing the Class I mutants with developmentally-competent strains the developmental mutations in 39 of these mutants were shown to be recessive; the remaining mutation appeared to be partially dominant. Complementation analysis of the developmental mutations in the Class I strains identified 5 complementation groups. Statistical analysis of the complementation data suggests that there are approximately 40 genes in this organism which will completely block aggregation when mutated and perhaps as many as 150 genes involved in some aspect of the aggregation process. Linkage analysis of 18 Class I developmental mutations revealed that 10 of these mutations map in linkage group II at a minimum of 5 loci.     

Murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein

Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L-R654H-H716D-E1035D) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.     

Promotion of Citrate Transport of the Transport-negative Mutants of Aerobacter cloacae

The “citrate transport-enhancing factor” obtained from Aerobacter cloacae did stimulate uptake of radioactive citrate by Escherichia coli, having an intrinsic barrier against citrate permeation. In order to prove function of the factor in the cells of Aerobacter, citrate transport-negative mutants of A. cloacae were isolated. These mutants were found to be lacking in the factor. Addition of the factor to these mutants resulted in stimulation of uptake of citrate. These results evidenced that the factor played an essential role in the citrate transport system of A. cloacae.     

D-核糖生产菌的选育

将枯草芽胞杆菌通过紫外线诱变得到了莽草酸缺陷突变株,在２８株突变株中有１０株积累Ｄ－核糖。这些菌株均属戊糖磷酸途径的非氧化支路缺失突变株。对这些菌株的产核糖能力进行了验证、培养基中芳香族氨基酸的浓度影响Ｄ－核糖的积累     

Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene.

Five hundred thirty-five lacY mutants were isolated from an Escherichia coli strain carrying the lactose operon on an F' factor, either without mutagenesis or after mutagenesis with 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine. Crosses against 48 independently isolated deletions ending in the lacY gene divided the gene into 36 deletion groups. Suppressibility studies with 7 nonsense suppressor strains classified 276 mutants as nonsense mutants and 78 as missense (or nonsuppressible) mutants. One hundred seventy-nine mutants were "leaky" and could not be so allocated, and two were found to have small internal deletions. Nonsense mutants could in many cases be subdivided even within deletion groups on the basis of their suppressibility pattern, giving a total of 70 groups of nonsense mutants. Studies of these mutants allow the following conclusions: lactose and melibiose most probably do not have separate binding sites on the permease; the lacY region most likely consists of one cistron, and so both active transport and facilitated diffusion are functions of one protein; and finally, there is probably no small defined region of the permease responsible for energy coupling of transport. Furthermore, the strains and the analysis form the basis for a future functional study of the permease by biochemical techniques.     

Identification of genes involved in growth autonomy of hematopoietic cells by analysis of factor-independent mutants

The factor-dependent myeloid precursor cell line D35 mutates spontaneously at a frequency greater than 2.4 x 10(-7) to growth factor autonomy. This frequency could be increased at least 20-fold by retrovirus insertional mutagenesis. The isolation and characterization of factor-independent mutants allowed the identification of genes involved in growth autonomy. Mutants could be subdivided into two sets: those that secreted a stimulating factor (10/11) and those that did not (1/11). In one case, the factor released was distinct from previously characterized growth factors. In most mutants (6/9), the activation of a growth factor gene was associated with rearrangement that could be attributed to the insertion of a transposable-like element either 5' or 3' of the factor coding region in all cases examined, excluding oncogene involvement. All factor-independent mutants were tumorigenic, consistent with the hypothesis that growth-factor independence initiated by aberrant growth factor gene activation is an important and early step in tumorigenesis.