Natural occurrence of Fusarium mycotoxins in field samples from the 1992 Wisconsin corn crop.

Analysis of 98 moldy corn samples collected in Wisconsin between November 1992 and January 1993 for Fusarium toxins by various immunochemical assays revealed overall average mycotoxin concentrations of 305.6, 237.7, and 904.3 ng/g for type A trichothecenes (TCTCs), deoxynivalenol (DON)-related type B TCTCs (total DON), and zearalenone (ZE), respectively. A small portion (5.1%) of the samples was found to be contaminated with high levels ( > 1 microgram/g) of type A TCTCs and total DON during the whole survey. Over 40% of the samples had 100 to 1,000 ng of total DON per g, while 17% of the samples had the same levels of type A TCTCs. The analytical data were consistent with those from mycological examinations for the samples in which various toxic Fusarium spp., including F. sporotrichioides, F. poae, and F. graminearum, were found. The samples received in November 1992 had relatively low concentrations of toxin; the average levels of type A TCTCs and total DON were 9.9 and 79 ng/g, respectively. The toxin concentrations became progressively higher in the samples received in December. The average levels for the type A TCTCs and total DON increased to 920 and 335 ng/g, respectively. However, the levels of ZE were higher in the samples collected earlier. The average levels for samples collected in November and late December were 1,195 and 242 ng/g, respectively. Analysis of selected samples by high-performance liquid chromatography monitoring with an enzyme-linked immunosorbent assay revealed that T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and T-2 tetraol (T-2-4ol) were common in these samples. Statistical analysis revealed a weak correlation between the levels of total type A TCTCs and total DON in the samples (r = 0.18, P = 0.09), but a strong correlation between the levels of ZE and total type B TCTCs (r = 0.75, P < 0.0001) was found. The mycotoxin levels of total type A TCTCs, total DON-related type B TCTCs, and ZE in the cobs (5.2, 3.9, and 21 micrograms/g, respectively) were considerably higher than those in the kernels (1.0, 0.5, and 0.5 microgram/g, respectively). The type A toxin levels increased from a range of 14 to 35 ng/g to a range of 110 to 538 ng/g after the moldy corn samples were held at 5 degrees C for 8 days in the laboratory.     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

Characterization of cis-acting elements in light regulation of the nuclear gene encoding the A subunit of chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.

We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.     

Water potentials for developing cladodes and fruits of a succulent plant, including xylem-versus-phloem implications for water movement

Developing cladodes had lower water potentials and developingfruits had higher water potentials than the underlying cladodesof the widely cultivated prickly pear cactus, Opuntia ficus-indica.The 0.06 MPa lower value in 4-week-old daughter cladodes indicateda typical water potential gradient from the underlying clad-odealong the xylem of –0.2 MPa m^–1; the 0.17 MPa highervalue in 4-week-old fruits, which decreased to 0.07 MPa by 10weeks, implicated the phloem as their supplier of water. Thephloem sap of the underlying cladodes had an osmotic pressureof only 0.90 to 0.98 MPa, so the phloem could supply a relativelydilute solution to the photosynthetically dependent fruits (daughtercladodes of O. ficus-indica are photosynthetically independentat 4 weeks). Although the water potentials were similar foradjacent tissues, the osmotic pressures were lower for the water-storagecompared with the photosynthetic tissue; the osmotic pressureswere higher for xylem sap from fruits, for which xylary flowapparently occurred toward the underlying cladodes, than fordaughter cladodes. The relative capacitance (change in relativewater content divided by change in tissue water potential) wasapproximately 0.71 MPa^–1 for the water-storage tissueand the photosynthetic tissue of both daughter cladodes andfruits at 4 weeks of age. When these organs approached maturityat 10 weeks, the relative capacitance increased about 40% fortheir water-storage tissue, but decreased 30% for their photosynthetictissue. As the plant water content decreases during drought,about twice as much water will thus be lost per unit volumeof the water-storage tissue compared with the photosynthetictissue of maturing fruits and cladodes. Key words: Opuntia ficus-indica, phloem, relative water content, water capacitance, water potential     

Pitaya (stenocereus spp., Cactaceae): An ancient and modern fruit crop of Mexico

Pitayasfrom various species were an important edible fruit in semiarid lands of tropical and subtropical Mexico in ancient times. Recently, farmers have been cultivating plants selected from the wild, such as Stenocereus queretaroensis in the Sayula Basin of Jalisco. These cacti can flower and produce fruit before the onset of the summer rainy period. Their fruits have an attractively colored pulp (often dark red) with digestible seeds and without the nasty glochids found on cactus pears. The sugar content is 10 to 11%. The shelf life is only a few days, as the fruits tend to dehisce longitudinally. Pitayas bring a competitive price in local markets, resulting in a substantial financial return with relatively low inputs of water, fertilizer, and pesticides.     

Analysis of peptides from known proteins: Clusterization in sequence space

A combinatorial sequence space (CSS) model was introduced to represent sequences as a set of overlapping k-tuples of some fixed length which correspond to points in the CSS. The aim was to analyze clusterization of protein sequences in the CSS and to test various hypotheses about the possible evolutionary basis of this clusterization. The authors developed an easy-to-use technique which can reveal and analyze such a clusterization in a multidimensional CSS. Application of the technique led to an unexpectedly high clusterization of points in the CSS corresponding to k-tuples from known proteins. The clusterization could not be inferred from nonuniform amino acid frequencies or be explained by the influence of homologous data. None of the tested possible evolutionary and structural factors could explain the clusterization observed either. It looked as if certain protein sequence variations occurred and were fixed in the early course of evolution. Subsequent evolution (predominantly neutral) allowed only a limited number of changes and permitted new variants which led to preservation of certain k-tuples during the course of evolution. This was consistent with the theory of exon shuffling and protein block structure evolution. Possible applications of sequence space features found were also discussed.Correspondence to: H.A. Lim     

Inheritance and expression of a transgene insert in an aneuploid tobacco line

A T-DNA locus comprising nptII, uidA and nos genes — all under the control of the nos promoter (this locus was designated K because it encodes resistance to Kanamycin) - was found to be inherited erratically in a transgenic tobacco line. This anomalous behavior was partially explained following a karyotype analysis of plants representing several generations: these plants were aneuploids, presumably for the K-containing chromosome. During four generations of sexual propagation, transgenic plants that were either trisomic or tetrasomic for the K-containing chromosome (i.e. 2n=49 or 2n=50, respectively) were obtained. The trisomic plants (2n=48+1) were virtually indistinguishable phenotypically from normal euploids (2n=4x=48), whereas the tetrasomic plants (2n=48+2) were smaller, had somewhat misshapen leaves and exhibited reduced fertility. Although the amount of NPTH protein in different trisomic (K--, KK-, KKK) and tetrasomic (KK--, KKK-) plants was generally consistent with a K dosage effect, the genetic behavior of each trisomic — with respect to segregation of Kan^R and marker gene activity in progeny — was unique and not completely explicable by invoking aneuploidy. Specifically, unexpected gains or losses of K could occur, suggesting the formation of double reductional gametes and/or frequent gene conversion at this locus. The susceptibility of K locus marker genes to trans-inactivation in the trisomic and tetrasomic lines was tested by crossing in partially homologous silencing loci. In all transgenotypes tested, the three K marker genes were sensitive to trans-silencing, which was accompanied by methylation in all copies of the nos promoter. In addition to this directed inactivation/methylation, the K locus could also undergo infrequent, spontaneous partial methylation, which produced stable epialleles. In most plants, however, the multiple copies of the nos promoter at this locus remained unmethylated and active through four generations in all transgenotypes examined. The significance of these results for irregular inheritance patterns, aneuploid syndromes and homology-dependent gene silencing is discussed.     

Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β-1,4-exoceilobiohydrolase analogous to Trichoderma reesei CBH II

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.