Linker-assisted immunoassay and liquid chromatography/mass spectrometry for the analysis of glyphosate.

A novel, sensitive, linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was compared to on-line solid-phase extraction (SPE) with high-performance liquid chromatography/mass spectrometry (HPLC/MS) for the analysis of glyphosate in surface water and groundwater samples. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitotic attachment of glyphosate to horseradish peroxidase hapten. Thus, L'ELISA recognized the derivatized glyphosate more effectively (detection limit of 0.1 microg/L) and with increased sensitivity (10-100 times) over conventional ELISA and showed the potential for other applications. The precision and accuracy of L'ELISA then was compared with on-line SPE/HPLC/MS, which detected glyphosate and its degradate derivatized with 9-fluorenylmethyl chloroformate using negative-ion electrospray (detection limit 0.1 microg/ L, relative standard deviation +/- 15%). Derivatization efficiency and matrix effects were minimized by adding an isotope-labeled glyphosate (2-13C15N). The accuracy of L'EUSA gave a false positive rate of 18% between 0.1 and 1.0 microg/L and a false positive rate of only 1% above 1.0 microg/L The relative standard deviation was +/- 20%. The correlation of L'ELISA and HPLC/MS for 66 surface water and groundwater samples was 0.97 with a slope of 1.28, with many detections of glyphosate and its degradate in surface water but not in groundwater.

Germ cell specific promoter drives ectopic transgene expression during embryogenesis.

In this study, we used the male germ cell-specific phosphoglycerate kinase 2 (Pgk2) promoter to generate Pgk2Cre transgenic mice to allow investigation of genes critically involved in meiosis. The Pgk2 promoter had been used previously to target transgene expression to spermatocytes and spermatids in several laboratories including ours. In several Cre targeting experiments using other promoters, ectopic Cre expression had been observed, but the timing and extent of this expression was not analyzed. We demonstrate that in adult mice the Pgk2Cre transgene is expressed specifically in spermatocytes and spermatids, as expected. However, in offspring from matings of Pgk2Cre mice and an H19loxP indicator strain, we discovered that recombination events had occurred in several, but not all, tissues to varying extents. The lacZ-loxP transgenic indicator strain was next used to uncover ectopic Cre expression even in single cells, which indicated that the Pgk2Cre transgene is expressed between days 11 and 15 during embryogenesis in several tissues and organs. Using an RT PCR assay we were unable to detect endogenous Pgk2 mRNA during embryogenesis or in adult tissues other than testis. In conclusion, the Pgk2 promoter is a valid choice for targeting gene expression to meiotic male germ cells, since transient ectopic expression is unlikely to have a discernable effect in most studies, but it may be inappropriate for utilization with Cre recombinase.

Kinesin light-chain KLC3 expression in testis is restricted to spermatids.

Kinesins are tetrameric motor molecules, consisting of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs) that are involved in transport of cargo along microtubules. The function of the light chain may be in cargo binding and regulation of kinesin activity. In the mouse, two KLC genes, KLC1 and KLC2, had been identified. KLC1 plays a role in neuronal transport, and KLC2 appears to be more widely expressed. We report the cloning from a testicular cDNA expression library of a mammalian light chain, KLC3. The KLC3 gene is located in close proximity to the ERCC2 gene. KLC3 can be classified as a genuine light chain: it interacts in vitro with the KHC, the interaction is mediated by a conserved heptad repeat sequence, and it associates in vitro with microtubules. In mouse and rat testis, KLC3 protein expression is restricted to round and elongating spermatids, and KLC3 is present in sperm tails. In contrast, KLC1 and KLC2 can only be detected before meiosis in testis. Interestingly, the expression profiles of the three known KHCs and KLC3 differ significantly: Kif5a and Kif5b are not expressed after meiosis, and Kif5c is expressed at an extremely low level in spermatids but is not detectable in sperm tails. Our characterization of the KLC3 gene suggests that it carries out a unique and specialized role in spermatids.

The large high mobility group proteins of rainbow trout are localized predominantly in the nucleus and nucleoli of a cultured trout cell line.

On the basis of immunofluorescence and biochemical studies, it has been suggested that the concentration of nonhistone chromosomal protein high mobility group 1 may be higher in the cytoplasm than in the nucleus of mammalian cells (Bustin, M., and Neihart, N. K. (1979) Cell 16, 181-189). In view of the possible implications of this finding, we have examined the in situ location of trout proteins HMG-T1 and HMG-T2, which are analogous to the mammalian proteins HMG-1 and HMG-2. Antibodies prepared against purified HMG-T2 were shown to react only with HMG-T1 and HMG-T2, but not with any other chromosomal proteins from trout. This has been established using a modified immunoautoradiographic techique involving CNBr-activated paper transfers of proteins separated on regular sodium dodecyl sulfate-polyacrylamide gels. Using the indirect immunofluorescence technique to examine the subcellular location of HMG-T (T1 and T2) proteins in a cultured cell line of rainbow trout, we find that these proteins are located primarily in the nucleus of these cells. The fluorescence in the nucleolar regions is even more intense than in the nonnucleolar regions. The cytoplasmic regions show only a weak fluorescence which may be due to low levels of HMG-T proteins in the cytoplasm, since preincubation of anti-HMG-T2 with purified HMG-T2 abolishes the nuclear as well as cytoplasmic fluorescence.

Organization of the rosy locus in Drosophila melanogaster: further evidence in support of a cis-acting control element adjacent to the xanthine dehydrogenase structural element.

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.

Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T.

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of (14)CO(2) by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of (14)C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO(2) fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO(2) provides a bioassaay for race T toxin which is both reliable and rapid.