Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

Cricket Paralysis Virus, a Potential Control Agent for the Olive Fruit Fly, Dacus oleae Gmel

Representatives of several families of insect viruses were tested for growth and pathogenicity in the olive fruit fly, Dacus oleae Gmel. The viruses included nuclear polyhedrosis viruses, an iridovirus, two picornaviruses, and Trichoplusia ni small RNA virus (a member of the Nudaurelia β family), in addition to two naturally occurring viruses of the olive fruit fly. Two viruses, one of the two picornaviruses (cricket paralysis virus [CrPV] and the iridovirus (type 21 from Heliothis armigera), were found to replicate in adult flies. Flies which were fed on a solution containing CrPV for 1 day demonstrated a high mortality with 50% dying within 5 days and nearly 80% dying within 12 days of being fed. The virus was transmissible from infected to noninfected flies by fecal contamination. The CrPV which replicated in the infected flies was demonstrated to be the same as input virus by infection of Drosophila melanogaster cells and examination of the expressed viral proteins, immunoprecipitation of the virus purified from flies, and electrophoretic analysis of the structural proteins.     

Isolation of Different Agrobacterium Biovars from a Natural Oak Savanna and Tallgrass Prairie

Populations of agrobacteria in excess of 10⁵ CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.     

Complementary Methodologies To Identify Specific Agrobacterium Strains

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Influence of Physical Disruption on Growth of Attached Bacteria

Attached bacterial populations cultured without an exogenous carbon source or grown in conjunction with attached diatoms incorporated [³H]thymidine at a rate between four and five times lower than that of replicate bacterial populations which were dispersed before being assayed.     

Evidence for the existence of an expressed minor variant tRNAPhe in yeast

Two expressed brewer's yeast tRNAsPhe, a major and a minor one, have been purified and sequenced. The major tRNAPhe corresponds to the already known tRNAPhe, whereas the minor one differs from the former in the substitution of T6-A67 by C6-G67 base pair in the "acceptor stem". The minor tRNAPhe contaminates all preparations of yeast tRNAPhe except those prepared by polyacrylamide gel electrophoresis.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.