Compromised disease resistance in saponin-deficient plants.

Saponins are glycosylated plant secondary metabolites found in many major food crops [Price, K. R., Johnson, I. T. & Fenwick, G. R. (1987) CRC Crit. Rev. Food Sci. Nutr. 26, 27-133]. Because many saponins have potent antifungal properties and are present in healthy plants in high concentrations, these molecules may act as preformed chemical barriers to fungal attack. The isolation of plant mutants defective in saponin biosynthesis represents a powerful strategy for evaluating the importance of these compounds in plant defense. The oat root saponin avenacin A-1 fluoresces under ultraviolet illumination [Crombie, L., Crombie, W. M. L. & Whiting, D. A. (1986) J. Chem. Soc. Perkins 1, 1917-1922], a property that is extremely rare among saponins. Here we have exploited this fluorescence to isolate saponin-deficient (sad) mutants of a diploid oat species, Avena strigosa. These sad mutants are compromised in their resistance to a variety of fungal pathogens, and a number of lines of evidence suggest that this compromised disease resistance is a direct consequence of saponin deficiency. Because saponins are widespread throughout the plant kingdom, this group of secondary metabolites may have general significance as antimicrobial phytoprotectants.

Heterologous expression of Septoria lycopersici tomatinase in Cladosporium fulvum: effects on compatible and incompatible interactions with tomato seedlings.

The anti-fungal, steroidal, glycoalkaloid saponin, alpha-tomatine, is present in uninfected tomato plants in substantial concentrations, and may contribute to the protection of tomato plants against attack by phytopathogenic fungi. In general, successful fungal pathogens of tomato are more resistant to alpha-tomatine in vitro than fungi that do not infect this plant. For a number of tomato pathogens, this resistance has been associated with the ability to detoxify alpha-tomatine through the action of enzymes known as tomatinases. In contrast, the biotrophic tomato pathogen Cladosporium fulvum is sensitive to alpha-tomatine and is unable to detoxify this saponin. This paper describes the effects of heterologous expression of the cDNA encoding tomatinase from the necrotroph Septoria lycopersici in two different physiological races of C. fulvum. Tomatinase-producing C. fulvum transformants showed increased sporulation on cotyledons of susceptible tomato lines. They also caused more extensive infection of seedlings of resistant tomato lines. Thus, alpha-tomatine may contribute to the ability of tomato to restrict the growth of C. fulvum in both compatible and incompatible interactions.

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces.

Non-transmissible derivatives of the Streptomyces multi-copy plasmid plJ101 were mobilized, by cointegrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coelicolor A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

Transposition of IS117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions.

IS117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of ORF1 of IS117, presumed to encode a transposase, abolished transposition. Deletion or mutation of ORF2 and ORF3, which overlap each other on opposite strands of IS117, caused a c. 20-fold reduction in integration frequency of the circular form of IS117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. ORF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.

The effect of varying tourniquet applications on the flow pattern of lower extremity radionuclide venography.

Radionuclide venography (RNV) is an accepted, reliable, and simple method for detecting thrombi of the deep venous system of the lower extremity. No universal agreement, however, has been established regarding specific techniques for tourniquet applications. In fact, present data reflect a general consensus that tourniquet use and location other than above the ankles has no appreciable or recognizable effect on study outcome. A prospective study was performed on 20 consecutive patients referred for RNV with the clinical impression of deep venous thrombosis (DVT). Each patient was studied initially with tourniquets above the knee and ankle, then with tourniquets above the ankle only, and finally without tourniquets. On the basis of standard criteria for DVT, 8 out of 20 patients were positive for DVT when the study was performed with tourniquets only above the ankle. Four of the eight positive studies became negative, however, when additional tourniquets were placed above the knees (20% false-positive rate). It is concluded that the routine application of additional tourniquets above the knees would eliminate a significant number of false-positive studies and should be part of an established routine protocol.

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.

Transposition of IS117 (the Streptomyces coelicolor A 3 (2) mini-circle) to and from a cloned target site and into secondary chromosomal sites.

IS117, previously known as the 2.6 kb mini-circle, is a transposable element found in Streptomyces coelicolor A 3(2). It integrates predominantly into one preferred site when introduced into the closely related Streptomyces lividans 66, which lacks IS117. This preferred integration site was deleted from the S. lividans chromosome by replacement with an erythromycin resistance gene delivered by a phi C31 phage vector. When IS117 was introduced into the resulting strain it integrated into many other sites, with some indication of site preference. By cloning a 200 bp fragment centred on the preferred integration site onto a low copy number, self-transmissible Streptomyces plasmid derived from SCP2* it was shown that this sequence is sufficient to define the preferred site: IS117 integrates efficiently into this sequence from its preferred site in the host chromosome and at a lower frequency from the plasmid into the preferred site on the S. lividans chromosome.

Development of genetic systems for the mycobacteria.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.

Lysogeny and transformation in mycobacteria: stable expression of foreign genes.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.

Plasmid pIJ699, a multi-copy positive-selection vector for Streptomyces.

A plasmid vector, pIJ699, which provides positive selection for cloned DNA, was constructed using the replication functions of the Streptomyces wide-host-range multi-copy plasmid pIJ101. The selection for inserts is based on the principle that plasmids with long uninterrupted perfect palindromes (inverted repeats) are 'not viable' in bacteria. For cloning, pIJ699 is digested with BglII. This produces two fragments, one of which is the linearized vector, with two arms of the palindrome at its ends, and the other is a 'spacer' which is needed to keep the inverted repeat sequences apart. The vector fragment is separated from the 'spacer' fragment and ligated with the DNA to be cloned. Plasmids with a fragment of cloned DNA, but not the circularized vector, give rise to thiostrepton-resistant transformants in Streptomyces lividans. The inverted repeat sequences contain a strong transcription terminator which reduces transcriptional read-through both in and out of the cloned fragment. This improves the stability of many hybrid plasmids and facilitates the study of the regulation of cloned genes.

Detection of obstructive uropathy using 99mtechnetium diethylenetriaminepentaacetic acid.

A simple, accurate test with no morbidity and effective for the detection of urinary tract obstruction in patients with normal and impaired renal function would be of significant clinical use. We herein evaluate the ability of 99mtechnetium diethylenetriaminepentaacetic acid to detect the presence or absence of obstruction. Of 107 patients who underwent recent studies 31 had confirmation of the presence or absence of obstruction by excretory urography, retrograde pyelography or other definitive procedures. Patients were studied in the posterior projection wtih perfusion images after intravenous injection of 20 millicuries 99mtechnetium diethylenetriaminepentaacetic acid. Static images of the urinary tract were obtained from 1 to 30 minutes after injection with delayed images up to 24 hours. Obstruction were defined as abnormal retention of activity in the collecting system persisting in the delayed images. Diethylentriaminepentaacetic acid identified correctly 13 of 13 patients proved to have obstruction and 17 of 18 without obstruction. One falsely positive result was owing to pyelocaliectasis. These data indicate a sensitivity of 100 per cent, specificity of 94 per cent and accuracy of 97 per cent.

Sodium trimetaphosphate as a bone-imaging agent. I. Animal studies.

When used is conjunction with stannous ion and 99Tc, the nonsequestering, cyclic, trimeric phosphate anion, (P309)3-, introduced in the form of its sodium salt, exhibits admirable properties as a bone-visualizing agent as demonstrated by animal studies. These studies show that this combinatation is easily prepared reproducibly and, compared to the agents described in the recent literature (all based on sequestering phosphates), is at least equivalent for bone visualization while being considerably less toxic.