Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5 region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.     

Direct evidence for the cell surface location of a protease-inhibitor complex on intact leukaemia cells

The interaction of a protease with two fluorescent inhibitors has been studied using intact fixed leukaemia cells as the source of the membrane bound enzyme. Fresh rat leukaemia cells were disrupted and the cytosol collected; this extract was known to contain a protein inhibitor of guanidinobenzoatase (GB) associated with leukaemia cells. All the cytosolic proteins were derivatised with Texas red acid chloride. Leukaemia cells with latent GB failed to bind the Texas red inhibitor protein but did so after activation of GB. Competition experiments with 9-amino acridine (a fluorescent marker for the active site of GB) demonstrated that the Texas red-inhibitor protein could only bind to intact leukaemia cells when the active centre of GB was not already occupied by 9-amino acridine. This competition between these two fluorescent inhibitors demonstrated their specificity for GB. The use of intact leukaemia cells and the high molecular weight of the inhibitor protein precludes the possibility of any interaction between GB and inhibitor within the cells. It is concluded that GB and the GB-inhibitor complex of latent GB are located on the external surface of intact leukaemia cells.     

Molecular evidence for Acanthocephala as a subtaxon of Rotifera

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters. Correspondence to: J.R. Garey     

Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer.

For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.     

Misting and nitrogen fertilization of shoots of a saltmarsh grass: effects upon fungal decay of leaf blades

We conducted a 12-week field manipulation experiment in which we raised the nitrogen availability (ammonium sulfate fertilization to roots) and/or water potential (freshwater misting) of decaying leaf blades of a saltmarsh grass (smooth cordgrass, Spartina alterniflora) in triplicate 11-m² plots, and compared the manipulated plots to unmanipulated, control plots. The ascomycetous fungi that dominate cordgrass leaf decomposition processes under natural conditions exhibited a boosting (>2-fold) of living standing crop (ergosterol content) by misting at the 1 st week after tagging of senescent leaves, but afterwards, living-fungal standing crop on misted blades was equivalent to that on control blades, confirming prior evidence that Spartina fungi are well adapted to natural, irregular wetting. Misting also caused 2-fold sharper temporal declines than control in instantaneous rates of fungal production (ergosterol synthesis), 5-fold declines in density of sexual reproductive structures that were not shown by controls, and 2-fold higher rates of loss of plant organic mass. Extra nitrogen gave a long-term boost to living-fungal standing crop (about 2-fold at 12 weeks), which was also reflected in rates of fungal production at 4 weeks, suggesting that saltmarsh fungal production is nitrogen-limited. Although bacterial and green-microalgal crops were boosted by manipulations of nitrogen and/or water, their maximal crops remained 0.3 or 2% (bacteria or green microalgae, respectively) of contemporaneous living-fungal crop. The fungal carbon-productivity values obtained, in conjunction with rates of loss of plant carbon, hinted that fungal yield can be high (>50%), and that it is boosted by high availability of nitrogen. We speculate that one partial cause of high fungal yield could be subsidy of fungal growth by dissolved organic carbon from outside decomposing leaves.     

The Mouse Glutathione PeroxidaseGpx2Gene Maps to Chromosome 12; Its PseudogeneGpx2-psMaps to Chromosome 7

TheGPX2gene codes for GSHPx-GI, a glutathione peroxidase whose mRNA is readily detectable in the gastrointestinal tract. AlthoughGPX2is a single gene in humans, there are two genes in the mouse genome with homology toGPX2.By analyzing a panel of mouse interspecies DNA from the Jackson Laboratory's backcross resource, we have chromosomally mapped these two genes. One was mapped to the central region of mouse chromosome 12 betweenD12Mit4andD12Mit5,nearfosandTgfb3.This region is homologous to human 14q24.1, where humanGPX2has been mapped, and most likely represents the functional mouseGpx2gene. The otherGpx2-like gene was mapped to mouse chromosome 7 betweenPcsk3andHbb.We have isolated the latter gene from a P1 phage library. Its pseudogene nature is revealed by the sequence analysis: (a) it is intronless; (b) it has a single nucleotide deletion in the coding region; and (c) it has a poly(A) tail at its 3′-untranslated region.     

Penner serotyping of Campylobacter isolates from poultry, with absorbed pooled antisera

The Penner serotyping system, based on detection of heat-stable antigens with a passive haemagglutination technique, was used in studies on Campylobacter epidemiology in poultry. Preparation of specific antisera by absorption allowed the use of pooled antisera. Over 80% of the Campylobacter isolates were typable with this modified Penner serotyping system. Typability of strains was clearly affected by storage of the strains before actual typing. Extracted antigens appeared to be stable for at least 6 months at 4°C. Therefore, it is advisable to store extracted antigens from freshly isolated Campylobacter strains instead of reculturing frozen-stored strains, when actual typing cannot be performed directly after primary isolation. Untypability of isolates may partly be explained by the detection of Campylobacter serovars not yet represented in the serotyping system. Experiments on repeated serotyping of several Campylobacter strains did not suggest any serovar instability within the strains.     

Specificity studies of 3-mercaptopyruvate sulfurtransferase

3-Mercaptopyruvate sulfurtransferase (E.C. 2.8.1.2; MST) is an enzyme believed to function in the endogenous cyanide (CN) detoxification system because it is capable of transferring sulfur from 3-mercaptopyruvate (3-MP) to CN, forming the less toxic thiocyanate (SCN). To date, 3-MP is the only known sulfur-donor substrate for MST. In an effort to increase the understanding of what chemical properties of 3-MP affect its utilization as a substrate, in vitro enzyme kinetic studies of MST were conducted using two mercaptic acids that are structurally related to 3-MP. Neither of these compounds was able to serve as a sulfur-donor substrate for MST. Inhibitor studies determined that 3-mercaptopropionic acid did not affect the K_m of MST for 3-MP but did decrease V_max and, thus, was determined to be a noncompetitive inhibitor. Alternatively, 2-mercaptopropionic acid 2-MPA decreased K_m and V_max and was determined to be an uncompetitive inhibitor of MST with respect to 3-MP. These data indicate that the α-keto group of 3-MP is necessary for its utilization as a substrate, and the inhibitor studies suggest that the position of the sulfur may also affect the binding of these compounds to the enzyme. These observations increase the understanding of what factors can affect the utilization of a compound as a sulfur-donor substrate for MST and may aid in the development of alternative sulfur-donor substrates for MST. © 1996 John Wiley & Sons, Inc.