Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.     

Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.     

条鳅亚科鱼类一新属新种

本文描述了云南省条鳅亚科鱼类一新属和一新种。根据形态特征并结合区系间的相互关系,探讨了属的分类地位。     

西双版纳热带季节雨林植物种类多样性的一种研究方法

一、方法1.样地的选择样地分别选取热带干性季节雨林的典型代表——以箭毒木(Antiaris toxicaria)、龙果(Pouteria grandifolia)为标志的群落,以千果榄仁(Terminalia myriocarpa)、番龙眼     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.     

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.     

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.     

Quantitative measurements of force and displacement using an optical trap.

We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.