SUB-CHROMATID REARRANGEMENTS IN TRILLIUM ERECTUM. I. ORIGIN AND NATURE OF CONFIGURATIONS INDUCED BY IONIZING RADIATION

Wilson , G. B. (Michigan State U., East Lansing.), A. H. Sparrow , and Virginia Pond . Subchromatid rearrangements in Trillium erectum. I. Origin and nature of configurations induced by ionizing radiation. Amer. Jour. Bot. 46(4): 309–316. Illus. 1959.—Microsporocytes of Trillium erectum were x-irradiated with 25 r at various stages of meiotic prophase and at first metaphase. Analysis of these cells at the following first and second anaphase revealed that post-pachytene irradiation produces 2-side-arm bridges which are indicative of half-chromatid exchanges. The occurrence of these bridges and knowledge of the structure and spatial relationship of chromatid strands in T. erectum have led to certain conclusions regarding the target and the number of strands broken by a single event: (1) the most likely target for primary effects is the 4 associated half-chromatids of a half-bivalent. The results of irradiation experiments suggest that the half-bivalent is effectively as well as structurally quadripartite at stages following pachytene. (2) Consideration of the configurations which would result from breakage and rejoining of 2, 3 or all 4 strands of the half-bivalent indicates that only 2 of the 4 half-chromatids are broken by a single event. Exchanges between 2 half-chromatids of sister chromatids will produce two recognizable types of 2-side-arm bridges: one with a true dicentric half-chromatid and one in which the bridge results merely from an interlocking of coils. Whether a 2-side-arm bridge appears at first or second meiotic anaphase is determined by the position and number of chiasmata between the point of breakage and the kinetochore. No 2-side-arm bridges have been detected at microspore anaphase following meiotic prophase irradiation. The types of configurations which might be expected at microspore metaphase as a result of broken 2-side-arm bridges are noted.     

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.Abbreviations 35S promoter isolated from CaMV - CaMV cauliflower mosaic virus - Adh1 maize gene encoding Alcohol dehydrogenase-1 enzyme - BMS suspension cultures of the Black Mexican Sweet maize variety     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets

Bacillus thuringiensis screening programs based on the official potency bioassay using third-instar larvae and on a neonate bioassay were developed for Heliothis armigera, Earias insulana, and Spondoptera littoralis. In these bioassays, the diets were standardized to be suitable, with minor modifications, for feeding of the three lepidopterans. The bioassay protocol was based on determination of the LC50 of the microbial standard HD-1-S-80 in the insects susceptible to B. thuringiensis var. kurstaki strains. This was followed by preliminary screening of B. thuringiensis strains at the LC50 of the B. thuringiensis standard. The B. thuringiensis strains causing 100% mortality at this LC50 in the larvae were selected for potency determinations. The neonate bioassay was suitable for accurate determinations of potencies also in S. littoralis--a representative of insects weakly susceptible to the HD-1 standard. The role of the official and the neonate bioassays in developing microbial control programs is discussed.     

Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli.

The fec region of the Escherichia coli chromosome determines a citrate-dependent iron(III) transport system. The nucleotide sequence of fec revealed five genes, fecABCDE, which are transcribed from fecA to fecE. The fecA gene encodes a previously described outer membrane receptor protein. The fecB gene product is formed as a precursor protein with a signal peptide of 21 amino acids; the mature form, with a molecular weight of 30,815, was previously found in the periplasm. The fecB genes of E. coli B and E. coli K-12 differed in 3 nucleotides, of which 2 gave rise to conservative amino acid exchanges. The fecC and fecD genes were found to encode very hydrophobic polypeptides with molecular weights of 35,367 and 34,148, respectively, both of which are localized in the cytoplasmic membrane. The fecE product was a rather hydrophilic but cytoplasmic membrane-bound protein of Mr 28,189 and contained regions of extensive homology to ATP-binding proteins. The number, structural characteristics, and locations of the FecBCDE proteins were typical for a periplasmic-binding-protein-dependent transport system. It is proposed that after FecA- and TonB-dependent transport of iron(III) dicitrate across the outer membrane, uptake through the cytoplasmic membrane follows the binding-protein-dependent transport mechanism. FecC and FecD exhibited homologies to each other, to the N- and C-terminal halves of FhuB of the iron(III) hydroxamate transport system, and to BtuC of the vitamin B12 transport system. FecB showed some homology to FhuD, suggesting that the latter may function in the same manner as a binding protein in iron(III) hydroxamate transport. The close homology between the proteins of the two iron transport systems and of the vitamin B12 transport system indicates a common evolution for all three systems.     

Inhibition of lipopolysaccharide O-antigen synthesis by colicin M

Colicin M inhibits peptidoglycan biosynthesis at the level of the bactoprenyl carrier lipid. Since the synthesis of O-antigen also requires bactoprenyl carrier lipid, the effect of colicin M on O-antigen biosynthesis was studied using a colicin-sensitive strain of Salmonella typhimurium. Determination of O-antigen intermediates by two different methods showed that bactoprenyl-dependent O-antigen biosynthesis was inhibited by colicin M. Synthesis of both O-antigen and peptidoglycan was almost immediately inhibited following colicin addition. This was followed some 20 min later by cell lysis. The only known common step between O-antigen and peptidoglycan synthesis is formation of bactoprenyl phosphate by dephosphorylation of bactoprenyl pyrophosphate. Determination of bactoprenyl phosphates showed an accumulation of bactoprenyl pyrophosphate in colicin-treated cultures. It was concluded that dephosphorylation of the bactoprenyl lipid carrier was inhibited by colicin M, and this in turn prevented both O-antigen and peptidoglycan synthesis.