Isolation and characterization of variants ofRhizobium leguminosarum bvphaseoli

Summary Seven spontaneous variants ofR. leguminosarum byphaseoli were isolated from three stock cultures by their diversity in colony morphology. Five variants had small opaque or translucent colonies and two variants, large gummy colonies. There were marked differences between strains, but not between variants of the same strain, in the utilization of carbon sources. The large gummy colony variants were more tolerant to streptomycin and novobiocin and less effective or ineffective in N₂ fixation than the variants of small non-gummy colonies; they were also of higher competitive ability than the other variants from the same strain. At least for one strain, competitiveness seemed less subject to variation.

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Resumen A partir de cultivos de referencia, y debido a la diversidad morfológico de sus colonias, se aislaron siete variantes espontaneas deR. leguminosarum bvphaseoli. Cinco de las variantes presentaron colonias pequeñas, opacas o traslucidas. Las colonias de las otras dos variantes eran grandes y de aspecto gomoso. En la utilización de fuentes de carbono se observaron diferencias marcadas entre distintas cepas pero no entre variantes de la misma cepa. Las colonias grandes y de aspecto gomoso eran más tolerantes a la estreptomicina y a la novobiocina, menos eficaces que las variantes de colonias pequeñas en cuanto a la fijación de N₂, y mostraron además una mayor habilidad competitiva frente a las otras variantes de la misma cepa. Al menos en una de las cepas la competitividad estaba menos sujeta a variaciones.

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Résumé Sept variants spontanés deR. leguminosarum bvphaseoli ont été isolés à partir de trois cultures stock sur la base de la diversité de la morphologie de leurs colonies. Cinq variants se présentaient en colonies petites, opaques à translucides, trandis que deux variants se présentaient en grandes colonies visqueuses. Il y avait des différences marquées entre souches mais pas entre variants de la même souche, quant à l'utilisation de sources carbonées. Les variants se présentant en grandes colonies visqueuses étaient plus tolérants à la streptomycine et à la novobiocine et moins efficaces voire inefficaces pour la fixation de l'N₂, que les variants des petites colonies non visqueuses; elles présentaient aussi une capacité de compétition plus élevée que les autres variants de la même souche. Au moins pour une souche, la capacité de compétition semblait moins sujette à variation.

     

Metabolic activity interferometer: description and calibration of an interferometric method to measure growth of mycobacteria

An interferometer that measures the refractive index changes due to bacterial metabolism is described. The apparatus permits simultaneous and real time measurement of bacterial growth in several samples of slowly growing mycobacteria. The error sources are discussed and the sensitivity of the apparatus is tested. For the species Mycobacterium bovis BCG and M. smegmatis, a relation between refractive index change and bacterial concentration is determined experimentally and the time constant of bacterial growth is measured.

<Emphasis Type="Italic">Carapichea lucida</Emphasis> (<Emphasis Type="Italic">Rubiaceae: Psychotrieae</Emphasis>), a new species from Eastern Bahia,Brazil

A new species of Carapichea was discovered in the Atlantic Forest of Bahia, Brazil. Carapichea lucida J. G. Jardim &; Zappi is described, illustrated, and its phenology, habitat and conservation status are discussed.     

Xanthine phosphoribosyltransferase from Leishmania donovani. Molecular cloning, biochemical characterization, and genetic analysis

Xanthine phosphoribosyltransferase (XPRT) from Leishmania donovani is a unique enzyme that lacks a mammalian counterpart and is, therefore, a potential target for antiparasitic therapy. To investigate the enzyme at the molecular and biochemical level, a cDNA encoding the L. donovani XPRT was isolated by functional complementation of a purine auxotroph of Escherichia coli that also harbors deficiencies in the prokaryotic phosphoribosyltransferase (PRT) activities. The cDNA was then used to isolate the XPRT genomic clone. XPRT encodes a 241-amino acid protein exhibiting approximately 33% amino acid identity with the L. donovani hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and significant homology with other HGPRT family members. Southern blot analysis revealed that XPRT was a single copy gene that co-localized with HGPRT within a 4.3-kilobase pair (kb) EcoRI fragment, implying that the two genes arose as a result of an ancestral duplication event. Sequencing of this EcoRI fragment confirmed that HGPRT and XPRT were organized in a head-to-tail arrangement separated by an approximately 2.2-kb intergenic region. Both the 3.2-kb XPRT mRNA and XPRT enzyme were significantly up-regulated in Deltahgprt and Deltahgprt/Deltaaprt L. donovani mutants. Genetic obliteration of the XPRT locus by targeted gene replacement indicated that XPRT was not an essential gene under most conditions and that the Deltaxprt null strain was competent of salvaging all purines except xanthine. XPRT was overexpressed in E. coli and the recombinant protein purified to homogeneity. Kinetic analysis revealed that the XPRT preferentially phosphoribosylated xanthine but could also recognize hypoxanthine and guanine. K(m) values of 7.1, 448.0, and >100 microM and k(cat) values of 3.5, 2.6, and approximately 0.003 s(-1) were calculated for xanthine, hypoxanthine, and guanine, respectively. The XPRT gene and XPRT protein provide the requisite molecular and biochemical reagents for subsequent studies to validate XPRT as a potential therapeutic target.     

Observing bacterial activity interferometrically

It is shown that bacterial activity, even of slowly growing species, can be detected by precise interferometric measurements of refractive index changes of the culture medium. The bacteria-containing sample is kept in an isothermal block together with a reference liquid without bacteria. The biological activity is obtained from the difference of the index changes of these samples. Experiments were performed with Bacilo Calmette-Guérin. The order of magnitude of the observed total refractive index change was compatible with theoretical estimates based on the amount of available oxygen. An unexpected positive index change during the lag phase was observed, which might permit fast diagnostics in medical applications. This technique may provide cheap and quick tests of bacterial susceptibility with respect to antibiotics.     

Double-strand breaks on artificial chromosomes in yeast

Yeast artificial chromosomes composed primarily of bacteriophage λ DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII. On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB). This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes. The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert. Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII. Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes. Received: 15 May 1998; in revised form: 26 September 1999 / Accepted: 18 November 1999     

High germinal instability of the (CTG)n at the SCA8 locus of both expanded and normal alleles

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of late-onset, neurodegenerative disorders for which 10 loci have been mapped (SCA1, SCA2, SCA4-SCA8, SCA10, MJD, and DRPLA). The mutant proteins have shown an expanded polyglutamine tract in SCA1, SCA2, MJD/SCA3, SCA6, SCA7, and DRPLA; a glycine-to-arginine substitution was found in SCA6 as well. Recently, an untranslated (CTG)n expansion on chromosome 13q was described as being the cause of SCA8. We have now (1) assessed the repeat size in a group of patients with ataxia and a large number of controls, (2) examined the intergenerational transmission of the repeat, and (3) estimated the instability of repeat size in the sperm of one patient and two healthy controls. Normal SCA8 chromosomes showed an apparently trimodal distribution, with classes of small (15-21 CTGs), intermediate (22-37 CTGs), and large (40-91 CTGs) alleles; large alleles accounted for only0.7% of all normal-size alleles. No expanded alleles (>/=100 CTGs) were found in controls. Expansion of the CTG tract was found in five families with ataxia; expanded alleles (all paternally transmitted) were characterized mostly by repeat-size contraction. There was a high germinal instability of both expanded and normal alleles: in one patient, the expanded allele (152 CTGs) had mostly contraction in size (often into the normal range); in the sperm of two normal controls, contractions were also more frequent, but occasional expansions into the upper limit of the normal size range were also seen. In conclusion, our results show (1) no overlapping between control (15-91) and pathogenic (100-152) alleles and (2) a high instability in spermatogenesis (both for expanded and normal alleles), suggesting a high mutational rate at the SCA8 locus.     

A step towards non-invasive characterization of the human frontal eye fields of individual subjects

Background

Identifying eye movement related areas in the frontal lobe has a long history, with microstimulation in monkeys producing the most clear-cut results. For humans, however, there is still no consensus about the location and the extent of the frontal eye field (FEF). There is also no simple non-invasive method for unambiguously defining the FEF in individual subjects, a prerequisite for clinical applications. Here we explore the use of magnetoencephalography (MEG) for the non-invasive identification and characterization of FEF activity in an individual subject.

Methods

We mapped human brain activity before, during and after saccades by applying tomographic analysis to MEG data. Statistical parametric maps and circular statistics produced plausible FEF loci, but no unambiguous definition for individual subjects. Here we first computed the spectral decomposition and correlation with electrooculogram (EOG) of the tomographic brain activations. For each of these two measures statistical comparisons were made between different saccades.

Results

In this paper, we first review the frontal cortex activations identified in earlier animal and human studies and place the putative human FEFs in a well-defined anatomical framework. This framework is then used as reference for describing the results of new Fourier analysis of the tomographic solutions comparing active saccade tasks and their controls. The most consistent change in the dorsal frontal cortex was at the putative left FEF, for both saccades to the left and right. The asymmetric result is consistent with the 1-way callosal traffic theory. We also showed that the new correlation analysis had its most consistent change in the contralateral putative FEF. This result was obtained for EOG latencies before saccade onset with delays of a few hundreds of milliseconds (FEF activity leading the EOG) and only for visual cues signaling the execution of a saccade in a previously defined saccade direction.

Conclusions

The FEF definition derived from microstimulation describes only one of the areas in the dorsal lateral frontal lobe that act together to plan, prepare and execute a saccade. The definition and characterization of these areas in an individual subject can be obtained from non-invasive MEG measurements.