Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis

A mosquitocidal toxin gene, cloned from Bacillus thuringiensis subsp. israelensis, was introduced into mutant crystal-negative B. thuringiensis subsp. israelensis cells. Partial toxicity to mosquitos was restored. The 58-kilodalton cloned gene product is a minor protein component of B. thuringiensis subsp. israelensis crystals and is structurally related to a major, 135-kilodalton crystal toxin.     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'

The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the `empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl<sub>2</sub>, or by pretreatments up to 20 minutes with p-chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H<sub>2</sub>O<sub>2</sub>, by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The `steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates.     

Inhalation anesthesia in adult cattle

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.     

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.     

Identification of the dye gene product, mutational loss of which alters envelope protein composition and also affects sex factor F expression in Escherichia coli K-12

Summary The product of the dye gene of Escherichia coli, mapping at 99–100 min, is required for expression of the sex factor F, and also appears to be involved in the regulation of envelope proteins. Mutation of dye thus results in loss of expression of the F-factor (Fex^–, i.e. male sterility, and dye sensitivity (Dye^s). We have isolated a plasmid, pRB38, in which a 6 kb SalI fragment carrying the dye ⁺ gene was cloned into the plasmid pACYC184. This 6 kb SalI fragment also carries two nearby markers, chlG, involved in the synthesis of the molybdenum cofactor, and phoM, required for constitutive expression of alkaline phosphatase.Some of the polypeptides synthesised by pRB38 were identified using the maxi-cell procedure. The product of the dye gene was found to be a polypeptide of M_r=29,000. Thus derivatives of pRB38 in which the transposon was inserted into dye, resulting in a Dye^S Fex^– phenotype when these plasmids were in a dye strain, failed to produce this polypeptide and in some cases produced a truncated product. Such insertions also resulted in a Chl^r and Pho^– phenotype when the plasmid was in a (dye-chlG-phoM) phoR strain, although complementation tests suggested that the phoM ⁺ and chlG ⁺ genes were still intact. Insertions of into the promoter distal end of dye did not result in a Dye^S Fex^– phenotype, although a truncated Dye protein was synthesised, and a Chl^r Pho^– phenotype was produced.It has been suggested (Gaffney et al. 1983) that the dye (=sfrA) gene product is necessary for F-factor expression because it is required for translocation of the F-factor TraJ protein to the outer membrane. Our results suggest that the Dye protein is also required for expression of the molybdenum cofactor and of alkaline phosphatase, and could perhaps be involved in the translocation of these proteins to the membrane.     

The disappearance of isocitrate lyase from the green alga Chlorella fusca studied by immunoprecipitation

When acetate-adapted cultures of Chlorella fusca were transferred to nitrogen-free medium containing glucose, isocitrate lyase activity was lost over a period of about 25 h. Using a combination of in vivo isotope labelling and immunoprecipitation with anti-isocitrate lyase IgG it was shown that: 1. The onset of loss of enzyme activity preceeded the complete cessation of enzyme synthesis. 2. Disappearance of isocitrate lyase activity was accompanied by loss of enzyme protein, without accumulation of antigenic protein distinguishable from the normal subunit polypeptide of the enzyme, as judged by SDS gel electrophoresis of immunoprecipitated samples from supernatant cell-free extracts. 3. SDS gel electrophoresis of immunoprecipitated isocitrate lyase revealed the presence of antigenic protein bands of M_r about twice that of the normal subunit polypeptide, but the appearance of these apparent dimer forms did not obviously correlate with enzyme degradation. 4. Isoelectric focusing of immunoprecipitated isocitrate lyase showed that the enzyme became progressively more oxidised during the period of its degradation in vivo. 5. By titrating crude broken cell suspensions with anti-isocitrate lyase antibody, preliminary evidence was obtained for transfer of the enzyme from the soluble fraction to an insoluble form as part of the process of disappearance.