Transformants of Fusarium graminearum were derived using linearized DNA of plasmids designed to replace the trichodiene synthase gene, a cutinase gene or a xylanase gene with a hygromycin-resistance marker cassette by homologous recombination between 1-kbp segments of flanking DNA. Most transformants did not exhibit the DNA structure expected of integration by classical double recombination. Instead, they contained linearized plasmid joined end-to-end and variably incorporated into the genome. Transformant types included ectopic integrations and integrations at the target site with or without removal of the targeted gene. We have analyzed a large number of transformants using cloning, PCR and DNA sequencing to determine the structures of their integrated DNA, and describe a model to explain their derivations. The data indicate that 1–3 copies of input DNA are first joined end-to-end to produce either linear or circular structures, probably mediated by the non-homologous end-joining (NHEJ) system. The end-joins typically have 1–5 nucleotides in common and are near or within the original cleavage site of the plasmid. Ectopic integrations occur by attaching linear DNA to two ends of genomic DNA via the same joining mechanism. Integration at the target site is consistent with replication around circularized input DNA, beginning and ending within the flanking homologous DNA, resulting in the integration of multiple copies of the entire structure. This results in deletion or duplication of the target site, or leaves one copy at either end of the integrated multimer. Reiterated DNA in the more complex structures is unstable due to homologous recombination, such that conversion to simpler forms is detected.

Background There is concern about the stigma of mental illness, but it is difficult to measure stigma consistently. Aims To develop a standardised instrument to measure the stigma of mental illness. Method We used qualitative data from interviews with mental health service users to develop a pilot scale with 42 items. We recruited 193 service users in order to standardise the scale. Of these, 93 were asked to complete the questionnaire twice, 2 weeks apart, of whom 60 (65%) did so. Items with a test–retest reliability kappa coefficient of 0.4 or greater were retained and subjected to common factor analysis. Results The final 28-item stigma scale has a three-factor structure: the first concerns discrimination, the second disclosure and the third potential positive aspects of mental illness. Stigma scale scores were negatively correlated with global self-esteem. Conclusions This self-report questionnaire, which can be completed in 5–10 min, may help us understand more about the role of stigma of psychiatric illness in research and clinical settings.

Lifeline Australia Inc. provides a free 24-hour telephone counselling and referral service to all Australians. The trained telephone counsellors of the service record information on many of their calls in Lifeline's Client Service Management Information System (CSMIS). This paper presents a descriptive summary of a national CSMIS data set, which was compiled during a 3-month period in 2003. The CSMIS data provided a clear national profile of the callers to the service. The results of this study support the hypothesis that callers are generally seeking social support from the service. The discussion explores the implications of this finding for Lifeline and other generalist counselling and referral services and their capacity to offer suicide intervention to the community.

The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA− strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75–79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O’Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232–237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.

In 1992 a study by Beggan claimed it had confirmed the existence of Nuttin's 'mere ownership effect'. This study examined an alternative mechanism for Beggan's findings in the form of forced compliance cognitive dissonance. Seventy-three participants volunteered for the study (66 females and seven males). Results support the view that cognitive dissonance, accepting ownership of a target that was earlier perceived with negative affectivity, is a sufficient condition to enhance estimates of a target object. Ownership per se was not found to be a sufficient condition to enhance owner's estimates of an owned object. In light of this finding cognitive dissonance is offered as a possible mechanism for explaining the previous reports of a perceived ownership effect. This mechanism may also be applicable to instant endowment and mere ownership phenomena. Copyright (c) 2005 John Wiley & Sons, Ltd.

A telephone survey was used to examine rural residents? (n=102) perceptions and knowledge of a well-established national telephone counselling and referral service - Lifeline. Residents in rural Australia experience generally poorer access and availability to health-related services than their metropolitan counterparts. They may also have problems with confidentiality and stigmatisation in using what services are available in their area. Although this was a noncomparative study, it was reasoned that these barriers to help-seeking in rural areas would mean their population would know and value a service such as Lifeline, which provides equitable and anonymous support and referrals to all Australians. The results showed that the service was known, valued, and supported strongly by the respondents. The findings supported the belief that telephone counselling and referral has an important and unique place in rural health support and referral.

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B(12)-dependent homocysteine methyltransferase for methionine biosynthesis.

4-Oxalocrotonate tautomerase (4-OT) converts 2-oxo-4E-hexenedioate (1) to 2-oxo-3E-hexenedioate (3) through the dienol intermediate, 2-hydroxy-2,4-hexadiene-1,6-dioate (2). Previous studies established that the isomerization of1to3is primarily a suprafacial process. It was also suggested that the 6-carboxylate group of the substrate maintains the regio- and stereochemical fidelity of the reaction by anchoring the substrate at the active site. A subsequent study suggested an additional role for the 6-carboxylate group in the mechanism: the enzyme may utilize the binding energy of the carboxylate group to facilitate catalysis. In order to explore the role of the carboxylate group in the mechanism further, the nonenzymatic rate constants for mono- and dicarboxylated substrates were measured and compared to the rates obtained for the corresponding enzymatic reactions. The results show that the missing carboxylate group has a profound effect on enzymatic catalysis as evidenced by the significant decreases (a 104- and a 105-fold reduction) in the values ofkcat/Kmobserved for the two monocarboxylated substrates. A comparison of the nonenzymatic rate constants indicates that the reducedkcat/Kmvalues cannot be explained on the basis of the chemical reactivities. The stereochemical course of the 4-OT-catalyzed reaction was also determined using 2-hydroxy-2,4Z-heptadiene-1,7-dioate. The stereochemical analysis reveals that the presence of the carboxylate group improves the stereoselectivity of the enzyme-catalyzed ketonization of 2-hydroxy-2,4Z-heptadiene-1,7-dioate to 2-oxo-[3-2H]-4Z-heptene-1,7-dioate in2H2O—a result that is consistent with its previously assigned role. These findings provide further evidence that the substrate's carboxylate group contributes to the mechanism of the enzyme in two ways: it anchors the substrate at the active site and it facilitates catalysis by destabilizing the substrate or by stabilizing the transition state.

Rhizobium (Sinorhizobium) meliloti strains capable of dissimilatory nitrous oxide reduction (Nos+) carry a nosRZDFY gene cluster on a 10.1 kb EcoRI fragment of the nod megaplasmid near the fixGHIS genes. These nos genes are arranged in three complementation groups and the 10.1 kb EcoRI fragment is sufficient to confer Nos activity to R. meliloti strains lacking such activity. An overlapping HindIII fragment containing the nosRZDFY genes but missing a 0-6 kb HindIII-EcoRI downstream segment was found incapable of imparting Nos activity to strains unable to reduce nitrous oxide, suggesting the presence of other nos gene(s) in this region. Tn5 introduced near the HindIII site resulted in mutants with a Nos- phenotype. Complete sequence analysis of nosY showed that it was well-conserved with respect to that of Pseudomonas stutzeri. Two previously unreported genes downstream of nosY in R. meliloti were also revealed. Contiguous with nosY was a sequence showing 63% identity with the ORFL protein of P. stutzeri. It appeared to be in the same operon as nosDFY and was predicted to encode a membrane lipoprotein similar to the putative NosL of P. stutzeri. Unlike the latter protein, however, amino acid sequences typical of metal-binding sites and cysteine residues indicative of the active site of protein disulphide isomerase were absent in the predicted NosL of R. mellioti. The Tn5 mutations resulting in a Nos- phenotype were localized within a 966 nucleotide gene 31 nucleotides downstream of nosDFYL with the same orientation. The new gene, nosX, was determined to be in a separate complementation group. It encoded a periplasmic protein with homology in the C-terminal domain with Rnff of Rhodobacter capsulatus and with a hypothetical Escherichia coli protein, YOJK. It was concluded that there are seven genes constituting the nos cluster in R. meliloti. They are organized in four complementation groups and in the same orientation, spanning a distance of about 9 kb on the nod megaplasmid.

4-Oxalocrotonate tautomerase from Pseudomonas putida mt-2 and 5-(carboxymethyl)-2-hydroxymuconate isomerase from Escherichia coli C catalyze the same reaction on substrates that differ only by a carboxymethyl group. While the structural resemblances between the substrates for these two proteins suggest that the enzymes might be evolutionarily related, the existing literature does not indicate an obvious link. It was found that both enzymes ketonize 2-hydroxymuconate (1)-the substrate for 4-oxalocrotonate tautomerase. In an effort to uncover similarities in the mechanisms, the stereochemical courses of both enzymatic reactions were examined, utilizing 1 in (H2O)-H-2. In each case, the product, 2-oxo-3-(E)-hexenedioate (2), was trapped and processed to [2-H-2(1)]glutaric acid by chemical degradative procedures. The configuration was established by comparing the molar ellipticity of the isolated glutaric acid to that of a sample generated by a stereoselective synthesis. It is concluded that 4-OT and CHMI ketonize 1 stereospecifically to (S)-2-oxo-3-(E)-[5-H-2(1)]hexenedioate. The mechanism and the evolution of 4-OT and CHMI and their respective pathways are discussed in the context of these results.

We have previously shown that human placental estradiol-17β dehydrogenase (EC 1.1.1.62; 17β-EDH) catalyzes the conversion of estradiol-17β to estrone and stereospecifically reduces NAD+ to [4-pro-S]NADH, ([4-B]NADH). Subsequently, this enzyme was found to reduce the ketone function at C-20 of progesterone, and evidence indicates that both activities reside at the same active site. This study was done to further elucidate spatial arrangements of cofactor and the 21-carbon substrate as they bind at the active site. The cofactor, [4B-3H]NADPH, was generated with homogeneous 17β-EDH from term human placenta, utilizing [17α-3H]estradiol-17β and NADP+. The resulting [4B-3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 μM) with a large molar excess of progesterone (150 μM) as substrate in the presence of the enzyme. Following incubation, the steroid reactants and products were extracted, separated by high-performance liquid chromatography and quantitated as to mass and tritium content. Oxidized and reduced cofactor were separated by ion-exchange chromatography and similarly quantitated. In all incubations, equimolar amounts of 20α-hydroxy-4-pregnen-3-one (20α-OHP) and NADP+ were obtained. Radioactivity was stoichiometrically transferred from [4B-3H]NADPH to the steroid product ([3H]20α-OHP). These results further substantiate a single active site for both 17β- and 20α-dehydrogenation enzyme activities. In addition, the enzyme is B-side specific, catalyzing the transfer of the 4B-hydrogen from the dihydronicotinamide moiety of the cofactor, for both C-18 and C-21 steroid substrates. Since the 20α-dehydrogenation by other enzyme sources has always been demonstrated to be an A-side specific reaction, this observation represents an important exception to the Alworth-Bentley rules of enzyme stereospecificity.

The stereospecificity of hydrogen transfer between steroid (17-hydroxypro-gesterone) and both natural cofactors by bovine testicular 20α-hydroxy steroid dehydrogenase (20α-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17β-dehydrogenase (EC 1.1.1.62) utilizing [17α-3H]estradiol-17β and NAD+ or NADP+ respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20α-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20α-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20α-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor.

A collection of symbiotically defective mutants of Rhizobium meliloti JJ1c10 was derived by Tn5 mutagenesis using the suicide vector pGS9. They include two Nod − and about 250 Fix − mutants. The mutants were found to be heterogenous in acetylene reduction activity and in the morphology and ultrastructure of the nodules which they induced. Over 90% were found to contain bona fide Tn5 insertions in a variety of DNA restriction fragments. When Tn5-carrying DNA segments cloned from 24 of the mutants were introduced into the equivalent location in the genome of the wild-type strain by recombination-mediated replacement, only eight produced a symbiotically defective phenotype similar to that of the original mutant. This result indicated that many of the symbiosis mutations were not directly caused by Tn5 insertion. DNA segments apparently containing mutated fix genes but not containing Tn5 were found in eight mutants by identifying cosmids carrying wild-type DNA which complemented their symbiosis defects. Probing of the DNA of these mutants with their complementing cosmids revealed no detectable physical alteration of the homologous DNA. A segment of DNA including the hsn and nifHDK genes was favoured for these non-Tn5 mutations. Three regions of the genome in which Tn5 caused fix mutations were identified. One of these was the known megaplasmid nod-nif region. The other two regions, designated fix-e5 and fix-h21, were found to be chromosomal. Mutants in one of these chromosomal regions fluoresced more intensely on calcofluor plates than the wild type.

ISRm1, an insertion sequence present in Rhizobium meliloti strain 1021, has been identified as the cause of the Nod− phenotypes in two mutants of another strain, JJ1c10. The insertions were found to be at different sites, though only about 100 base pairs apart within the nodC gene. ISRm1 causes no mutations in the nifHDK gene region of strain JJ1c10, as it does at high frequency in strain 1021. In JJ1c10 ISRm1 inserted at high frequency into a region of the genome adjacent to copies of other reiterated DNA segments. The target region was not required for symbiotic nitrogen fixation.

AbstractMica and polystyrene in four ratios were sheared for three different times, each at three speeds, and at two temperatures in the viscoelastic region, in a screw mixer. From precise density measurements on the resulting composites, void volume fractions were calculated and found to be proportional to mica concentration and independent of total screw rotations. Experimental void/mica ratios were represented by a kinetic model; they decreased from a single value common to all conditions to a steady‐state value proportional to the square root of the screw speed. Void formation and elimination occur simultaneously. The goodness of the fit to the data is insensitive to the details of the kinetic model. The characteristic rate constant depends only on temperature, increasing at the same rate as the reciprocal of the low‐frequency dynamic viscosity. Cavitation in regions of diverging flow of the mixer may inhibit void elimination.

Downstream from its colicin and immunity genes (col, imm), Escherichia coli plasmid ColE3-CA38 contains a 0.81-kb DNA segment, the hic region, which is required for high colicin production. Characterization of derived plasmids, carrying the col-imm operon but varying in the hic region, showed that the latter functions in lacuna production, colicin release, cell death, and lysis. The hic gene expression after induction was shown to be dependent on the col gene promoter. The nucleotide sequence of the 0.81-kb region was determined and the hic gene localized to its imm-distal portion following an open reading frame (ORF) with no known function. There are two overlapping ORFs in that portion of the sequence, one of which was identified as the hic gene by its partial homology to lysis gene H of CloDF13. The 3' half of the hic gene is non-essential and contains a terminator-like DNA sequence. Preceding the gene, there are also inverted repeats which may attenuate its transcription.