Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed. Received: 1 March 1997 / Accepted: 13 May 1997     

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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Conditionality of phase resetting by inhibitors of 80 S translation inGonyaulax polyedra

Summary Gonyaulax polyedra was subjected to a cold treatment of 18 h at 10 °C leading to arrhythmicity. Subsequently, the circadian rhythm of bioluminescence was investigated at the permissive temperature of 20 °C. 1-h pulses of 10 M cycloheximide or 2 M anisomycin, when given after the temperature step-up, resulted only in a very weak resetting of the circadian oscillator, in marked contrast to the behaviour of cells kept continuously in oscillatory conditions at 20 °C. The extremely reduced sensitivity to 80 S inhibition was characteristic for the first cycle after the temperature step-up, whereas cells treated with cycloheximide in the second cycle after re-initiation of rhythmicity showed a gradual recovery of resettability, though the phase response curve was still atypical; treatment in the 3rd cycle after step-up led to a relatively normal phase response curve. The observed insensitivity in the 1st cycle was neither a consequence of insufficient drug action, nor of a transient non-oscillatory behaviour after temperature step-up. Already in the first hours after transfer to 20 °C, 80 S translation was strongly suppressed by cycloheximide, and the cells were also efficiently reset by changes of the light-dark zeitgeber. Resettability of the circadian oscillator by 80 S inhibitors is, therefore, conditional.Abbreviations Ani anisomycin - Chx cycloheximide - CT circadian time - LD light-dark cycle - LL constant light - TCA trichloroacetic acid This work was supported by the Deutsche Forschungsgemeinschaft     

Suppression of growth defects of α-amylase secreting Escherichia coli by signal sequence fusion

Abstract In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae . In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.     

Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts.

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.     

Localization of α-amylase isozymes within the endomembrane system of barley aleurone

Summary The localization of -amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts was studied using electron microscope immunocytochemistry. Antibodies were raised against total barley -amylase, i.e., -amylase containing both highisoelectric point (high-pI) and low-pI isoforms, as well as against purified high- and low-pI isoforms. All antibodies localized -amylase to the endoplasmic reticulum (ER) and Golgi apparatus (GApp) of the aleurone cell, and various controls showed that the labeling was specific for -amylase. Labeling of protein bodies and spherosomes, which are the most abundant organelles in this cell, was very low. There was no evidence that -amylase isoforms were differentially distributed within different compartments of the endomembrane system. Rather, both high- and low-pI isoforms showed the same pattern of distribution in ER and in the cis, medial, and transregions of the GApp. We conclude that in the Himalaya cultivar of barley, all isoforms of -amylase are transported to the plasma membrane via the GApp.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - GApp Golgi apparatus - PBS phosphate buffered saline - PCR partially coated reticulum - PM plasma membrane - TBS Tris buffered saline - TGN trans-Golgi network     

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI₂) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI₂ (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical ₁-adrenoceptor densities and affinities (as calculated from [³H]-CGP binding studies), G_i and G_s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI₂, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI₂ is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.     

Substrate specificities of lipases in view of kinetic resolution of unsaturated fatty acids

Several commercially available lipases have been evaluated with regard to their substrate specificity in the esterification of fatty acids having specific positions of cis double bonds, e.g. petroselinic acid (n-12 18:1), alpha-linolenic acid (n-3 18:3), gamma-linolenic acid (n-6 18:3), stearidonic acid (n-3 18:4), dihomogamma-linolenic acid (n-6 20:3), eicosapentaenoic acid (n-3 20:5) and docosahexaenoic acid (n-3 22:6), with n-butanol. A common feature of most lipases, e.g. those from Penicillium cyclopium, Candida cylindracea, Mucor miehei, Rhizopus arrhizus and Penicillium sp. is that fatty acids having the first double bond from the carboxyl end as a cis-4 (n-3 22:6), cis-6 (n-12 18:1, n-6 18:3, n-3 18:4) or a cis-8 (n-6 20:3) double bond are strongly discriminated against compared to the other fatty acids, such as myristic acid (14:0), the reference standard, and n-3 18:3. In the case of the lipase from porcine pancreas, however, the discrimination against the above fatty acids is not as strong as with the other lipases. In contrast, the lipase from Chromobacterium viscosum shows a preference for n-12 18:1, n-6 18:3 and n-3 18:4. The observed substrate specificities can be utilized for enrichment of particular fatty acids by lipase-catalysed kinetic resolution from fatty acid mixtures, derived from naturally occurring fats and other lipids.Dedicated to Prof. David A. Walker, Robert Hill Institute, Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK, on the occasion of his sixty-fifth birthday on 18 August 1993 Correspondence to: K. D. Mukherjee