Isolation, characterization and expression of the gene that encodes D-arabinitol dehydrogenase in Candida tropicalis.

The gene (ARD) that encodes NAD-dependent D-arabinitol dehydrogenase (ArDH) in the pathogenic fungus Candida tropicalis (Ct) was cloned by transforming Escherichia coli (Ec) BW31M (araCc) with a plasmid library of Ct genomic DNA and selecting for D-arabinitol-utilizing (D-arab+) clones. Plasmid DNA from a D-arab+ clone retransformed fresh Ec BW31M cells to D-arab+; these cells produced both ArDH catalytic activity and a 31-kDa protein recognized by antibodies to native Ct ArDH. The plasmid contained an 846-bp open reading frame (ORF) that encoded a deduced protein of 282 amino acids (aa) (30,748 Da). Four partial aa sequences from Ct ArDH were present in the deduced aa sequence, thus verifying that Ct ARD had been cloned. Ct ArDH was 95% identical to ArDH from Candida albicans (Ca), 85% identical to a xylitol dehydrogenase (XDH) from Pichia stipitis (Ps) and 20-25% identical to many other short-chain dehydrogenases. Ct ArDH, Ca ArDH and Ps XDH were typical short-chain dehydrogenases except that they lacked an N-terminal Gly that is conserved in other members of this family. Thus, these enzymes may represent a subclass of closely-related fungal pentitol dehydrogenases. Large amounts of recombinant ArDH (re-ArDH) were produced in Ec and purified by dye ligand affinity chromatography. The physical and catalytic properties of re-ArDH were similar to those of native Ct ArDH, and re-ArDH and native ArDH performed similarly in an automated enzymatic assay for D-arabinitol in human serum.

Serum D-arabinitol measured by automated quantitative enzymatic assay for detection and therapeutic monitoring of experimental disseminated candidiasis: correlation with tissue concentrations of Candida albicans.

In order to further understand serum D-arabinitol (DA) as a marker for the diagnosis of disseminated candidiasis and for monitoring response to antifungal therapy, we studied the serum levels of this Candida carbohydrate metabolite by rapid automated enzymatic assay in rabbits with experimental disseminated candidiasis. The enzymatic reaction steps were performed on a standard automated clinical chemistry analyser. As a correction for renal impairment, data were expressed as serum D-arabinitol/creatinine ratio (DA/Cr). Serum creatinine concentrations were determined from the same sample with the same instrument, thereby allowing rapid determination of the DA/Cr within one laboratory. The DA/Cr was determined in 321 samples from 132 rabbits. The mean serum DA/Cr in 31 normal non-infected rabbits was 1.51 +/- 0.2 microM mg-1 dl-1. Among 84 rabbits with disseminated candidiasis and pre-terminal samples, there was a direct correlation between DA/Cr and tissue concentration of Candida albicans (r = 0.80; P < 0.001). A threshold of elevated DA/Cr (> or = 3.0 microM mg-1 dl-1) was evident in rabbits with a tissue concentration of C. albicans > or = 3 x 10(4) colony forming units (CFU) g-1. Elevated DA/Cr was detected in 48 (89%) of 54 rabbits at a C. albicans tissue concentration of > or = 3 x 10(4) CFU g-1 vs. one (3%) of 30 rabbits with < 3 x 10(4) CFU g-1 (P < 0.0001). Among all 101 rabbits with disseminated candidiasis, an elevated DA/Cr was detected at any point during infection in 60 (92%) of 65 rabbits having a C. albicans tissue concentration > or = 3 x 10(4) CFU g-1 vs. 13 (36%) of 36 rabbits with < 3 x 10(4) CFU g-1 (P < 0.0001). The relationship between the tissue response to antifungal therapy and change in DA/Cr was then further analysed. Ten (91%) of 11 rabbits with a tissue-proven response to antifungal therapy (defined as > or = 10(2)-fold reduction of CFU g-1 in comparison to untreated controls) had a > 50% reduction in elevated DA/Cr levels. By comparison, 10 (83%) of 12 treated rabbits with no response to therapy had persistently elevated DA/Cr levels (P < 0.001). These findings provide an experimental basis for understanding the patterns of expression of serum DA in disseminated candidiasis and further indicate that serial DA/Cr measurements may be useful for diagnosis and therapeutic monitoring of disseminated candidiasis.

An automated enzymatic method for measurement of D-arabinitol, a metabolite of pathogenic Candida species.

An automated enzymatic method was developed for the measurement of D-arabinitol in human serum. The assay is based on a novel, highly specific D-arabinitol dehydrogenase from Candida tropicalis. This enzyme catalyzes the oxidation of D-arabinitol to D-ribulose and the concomitant reduction of NAD+ to NADH. The NADH produced is used in a second reaction to reduce p-iodonitrotetrazolium violet (INT) to INT-formazan, which is measured spectrophotometrically. The entire reaction sequence can be performed automatically on a COBAS MIRA-S clinical chemistry analyzer (Roche Diagnostic Systems, Inc., Montclair, N.J.). Replicate analyses of human sera supplemented with D-arabinitol over a concentration range of 0 to 40 microM demonstrated that the pentitol could be measured with an accuracy of +/- 7% and a precision (standard deviation) of +/- 0.4 microM. Serum D-arabinitol measurements correlated with those determined by gas chromatography (r = 0.94). The enzymatic method is unaffected by L-arabinitol, D-mannitol, or other polyols commonly found in human serum. Any of 17 therapeutic drugs potentially present in serum did not significantly influence assay performance. Data illustrating the application of the assay in patients for possible diagnosis of invasive candidiasis and the monitoring of therapeutic intervention are presented. The automated assay described here was developed to facilitate the investigation of D-arabinitol as a serum marker for invasive Candida infections.

Identification, purification, and characterization of a D-arabinitol-specific dehydrogenase from Candida tropicalis.

A novel D-arabinitol (DA) dehydrogenase was identified and purified more than 300-fold from Candida tropicalis. The enzyme is specific for DA and catalyzes the NAD(+)-dependent oxidation at carbon 4 to yield D-ribulose. Purification was accomplished by a combination of protamine sulfate and ammonium sulfate precipitation and dye ligand chromatography on a reactive yellow 86 column. The apparent Km of the enzyme for DA ([NAD+] = 2.2 mM) is 39.8 mM. The apparent Km for NAD+ ([DA] = 384 mM) is 0.12 mM. The pH-optimum for the enzymatic oxidation of DA is approximately 10. Cofactor stereospecificity studies demonstrate that the enzyme catalyzes transfer of the 4(S) hydrogen of NADH with D-ribulose as substrate. The polyol substrate specificity of the present DA dehydrogenase makes the enzyme potentially useful for the development of a simple and specific method for the measurement of DA, a metabolite of pathogenic Candida spp. which has been described as a marker for disseminated candidiasis.

Sustained ethylene production in Agrobacterium-transformed carrot disks caused by expression of the T-DNA tms gene products.

Agrobacterium-infected carrot disks continually produced elevated levels of ethylene. Ethylene production was mediated by the elevated levels of auxin synthesized in transformed tissues.

Viroid replication: equilibrium association constant and comparative activity measurements for the viroid-polymerase interaction.

The binding and replication of purified potato spindle tuber viroid (PSTV) by DNA-dependent RNA polymerase II from wheat germ was studied in analytical ultracentrifugation experiments and in vitro transcription assays. The equilibrium association constant for the viroid-polymerase interaction is 1.9 X 10(7) M-1. Both ultraviolet and fluorescent monitoring during the sedimentation experiments showed two distinguishable viroid-polymerase complexes. These are interpreted as resulting from a 1:1 and 2:1 enzyme-to-viroid binding stoichiometry. A265/A280 ratios across the sedimenting boundaries, the sedimentation velocity of the complexes, as well as electron microscopic data support this interpretation. The role of viroid secondary structure in enzyme binding and polymerization is discussed in the light of these results and compared with binding and polymerization data for virusoid RNA, single- and double-stranded RNA, and double-stranded DNA.

Physical characterization of a kinetoplast DNA fragment with unusual properties.

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to phi X174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix (Marini, J.C., Levene, S.D., Crothers, D.M., and Englund, P.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7664-7668). In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 degrees C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w]. Thermal denaturation reveals a triphasic melting curve.

Dynamics and interactions of viroids.

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. III. High resolution thermal denaturation and circular dichroism studies.

High resolution thermal denaturation and circular dichroism studies were performed on a series of six recombinant DNA restriction fragments. The fragments varied in size from 132 to 193 bp and contained Escherichia coli wild type and UV5 lactose promoters both with and without homopolymer insertions of poly(dA).poly(dT) and poly(dG).poly(dC). A differential thermal destabilization of the wild type promoter region, as compared to the UV5 promoter, was observed when dA70.dT70 was inserted into the -60 region or both DNAs. This effect may depend, in part, on the differences in the base composition between adjoining cooperative units in the fragments. The relatively larger effect of the AT sequence on the wild type promoter region may be correlated with the increased levels of in vitro transcription activity described in the preceding paper (Klein, R. D., and Wells, R. D. (1982) J. Biol. Chem. 257, 12962-12969). Stretches of homopolymeric GC base pairs stabilized the wild type and UV5 promoter regions by over 2.5 degrees C. CD studies could not detect conformational differences between DNAs containing the wild type or UV5 promoter. The presence of homopolymers had a marked effect on the CD spectra of the fragments.

Computer programs to assist in high resolution thermal denaturation and circular dichroism studies on nucleic acids.

Computer programs are described that direct the collection, processing, and graphical display of numerical data obtained from high resolution thermal denaturation (1-3) and circular dichroism (4) studies. Besides these specific applications, the programs may also be useful, either directly or as programming models, in other types of spectrophotometric studies employing computers, programming languages, or instruments similar to those described here (see Materials and Methods).

Circular dichroism spectra of twelve short DNA restriction fragments of known sequence: a comparison of measured and calculated spectra.

The CD spectra of twelve DNA restriction fragments ranging in size from 12 to 360 base pairs are reported. Since the sequences of these fragments are known, it is possible to calculate their CD spectra from a set of nearest neighbor contributions derived from a combination of synthetic polydeoxyribonucleotides. While the calculations lead to good agreement in the negative band at approximately 245 nm, they generally reproduce the positive band at approximately 270 nm only poorly. The experimentally observed positive band consists of two peaks centered around 270 and 285 nm. The comparison of calculated and measured spectra reveals that end effects lead to increased disagreement for fragments smaller than approximately 40 base pairs. The disagreement between calculated and measured spectra can be partially attributed to the fraction of next nearest neighbors in the DNAs, which are also in the spectral components. Thus, the sequence specific CD contributions in the long wavelength region of the spectra extend at least to next nearest neighbor nucleotides and may extend beyond.

High resolution experimental and theoretical thermal denaturation studies on small overlapping restriction fragments containing the Escherichia coli lactose genetic control region.

The distribution of thermal stability in the Escherichia coli lac control region is evaluated from the melting behavior of 5 short (80-219 base pairs (bp)) sequenced DNA restriction fragments containing various parts of this sequence. The thermal denaturation of these fragments was measured at 3 salt concentrations. The previous notion that the melting curves for small fragments are sharp and asymmetric in 0.01 M Na+ and broadened and less asymmetric at 0.105 and 0.505 M Na+ is confirmed and the possible explanations are discussed. The existence of two thermodynamic boundaries in this region is also confirmed. The exact location of the boundary upstream of the cyclic AMP receptor protein (CAP) binding site is accurately determined from melting experiments at 260 and 282 nm. The secondary boundary located between the promoter and operator sequence is apparent at the two higher salt concentrations and begins to disappear at the lower salt concentration. The physical interpretation of the melting experiments is compared to the results of theoretical predictions derived from the known sequence of the fragments.

Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments.

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.

High resolution thermal denaturation analyses of small sequenced DNA restriction fragments containing Escherichia coli lactose genetic control loci.

Differential melting curves are reported for four DNA restriction fragments (789, 301, 203, and 95 base pairs in length) spanning the lactose control region. All but the smallest melt with two or more subtransitions. Maps are proposed which identify the positions of regions of different thermal stability in the sequences. The sizes of regions comprising subtransitions range from 60 to 200 base pairs. An analysis is made of the cooperativity exhibited between regions in the sequence. The effect on the shape of the differential melting curves of Na+ between 10 mM and 0.5 M as well as that of Mg2+ and glycerol has been determined. An 81-bp-long sequence of unusual thermal stability occurs at the lactose promoter. Its TM change, resulting from the above change in salt concentration, is out of step by 1.5 degree C with the neighboring DNA sequence. The potential biological significance of this behavior is discussed.