Genetic assay of misincorporation

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.     

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

Models of seasonal growth of the equatorial carp Labeo dussumieri in response to the river flood cycle

Synopsis The phenology of Labeo dussumieri, an omnivorous carp common to South Asia, was investigated in a population inhabiting a flood plain anabranch of the Mahaweli Ganga, Sri Lanka. The Mahaweli Ganga exhibited a bimodal discharge pattern typical of many equatorial rivers, with a minor peak during the S.W. monsoon and a major peak during the N.E. monsoon. Seasonal changes in several lotic variables were measured in an attempt to correlate changes in environmental conditions to reproduction and growth in L. dussumieri. The onset of gonad recrudescence and spawning were synchronized with the increased river discharge during the S.W. and N.E. monsoons: gonad development followed one monsoonal discharge peak and spawning took place at the beginning of the other. Most fish spawned at the beginning of the major discharge peak in October and November, following the September dry season. Increased discharge was concomitant with a fall in temperature, light intensity, pH and conductivity. Growth was shown to be seasonal, exhibiting an annual bimodal pattern with peaks coincident with S.W. and N.E. monsoonal rains. Seasonal changes in growth were expressed by two models in terms of: (a) change of somatic weight or fork length with time, (b) change of specific growth rate in response to river discharge, modified by somatic weight. Gonad recrudescence and spawning stress did not appear to influence growth rate.     

Resumption of cellular activity induced by cytokinin and gibberellin treatments in tomato flowers targeted for abortion in unfavorable light conditions

Mitotic activity and nuclear DNA synthesis in tomato ( Lycopersicon esculentum Mill., cv. King plus) flowers targeted for abortion under unfavorable light conditions are completely stopped 6 days after macroscopic appearance of the inflorescence. Ovular cells are arrested at the G₁ (80%) and G₂ (20%) stages of the cell cycle. Exogenous applications of a mixture of N⁶-benzyladenine (BA) and gibberellins A₄₊₇ (GA) directly on the inflorescence may prevent its failure. Nuclear DNA synthesis and mitoses resume in ovules of the flower 16 to 20 h after the BA+GA treatment. When applied alone, BA and GA are able to mimic the effect of the mixture upon the progression of ovular cells through their cycle. Sporogenesis processes are also set in motion by the exogenous plant growth regulators. The mechanism of action of cytokinins and gibberellins in the control of floral development is discussed.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [¹⁴C]glycerol 3-phosphate and [¹⁴C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C₁₈-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.Abbreviation BSA bovine serum albumin