Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster

A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.      

Effect of Chronic Ethanol Treatment on the γ-Aminobutyric Acid-Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.     

Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and beta h3 loci in wild mice populations

The presence of the L1 sequences, L1Md4 next to the pseudogene beta h3 and I12 found in the twelfth intron of the albumin gene, in certain strains of laboratory mice but not of others has led to the suggestion that these sequences were recent insertions into the Mus mus domesticus genome. To be sure that they are really recent insertions and not relics of an ancestral chromosome, we investigated the presence or absence of these sequences in populations of wild mice belonging to the semispecies M. m. domesticus and M. m. musculus as well as in other species of the genus Mus and in related murids. The sequence I12 in the albumin gene was found in 34% of the chromosomes of the wild mice belonging to M. m. domesticus and to a lesser extent (6%) in M. m. musculus. Of 114 M. m. domesticus chromosomes, L1Md4 was found in only nine, seven of which came from the same locality. Its presence was associated with the haplotype Hbbp, which is relatively rare in European populations of M. musculus. Since there was no evidence for the presence of these two L1 sequences in more distantly related species, we conclude that they are recent insertions in the M. musculus genome.      

Protein and m-RNA expression of farnesyl-transferases,RhoA and RhoB in rat liver hepatocytes: action of perillyl alcohol and vitamin A <Emphasis Type="BoldItalic">in vivo</Emphasis>.

Summary We analysed the action, in rats in vivo, of the protein isoprenylation inhibitor perillyl alcohol (POH) and that of vitamin A, alone or in association, on m-RNA and protein expression of farnesyltransferases (FTases α and β subunits) and their protein substrates RhoA and RhoB, in isolated hepatocytes. Combined administration of POH and vitamin A induced a sharp decrease in FTase α protein after 96 h, suggesting an involvement not only of farnesyltransferases but also of geranylgeranyltransferases, which share the FTase α protein. FTase β protein did not decrease. POH plus vitamin A, in contrast with POH or vitamin A alone, induced a decrease in RhoB protein, probably because of different cleavages. No modification was observed in RhoA protein. Vitamin A alone increased RhoB m-RNA and protein expression. As one of the functions of RhoB is cell polarisation, these data support our previous hypothesis of a polarised transport of vitamin A from hepatocytes to hepatic stellate cells. As the behaviours of m-RNAs and proteins in this study were often different, cytoplasmic metabolic pathways must be considered for the parameters studied. The behaviour of Rho B, which is thought to have an antioncogene function, is discussed in view of its isoprenylated forms in the membranes. These preliminary findings stress the need, when studying the association of two isoprenoids in cancer therapy, to consider normal as well as tumour-bearing animals.     

Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast

In contrast to animal and fungal cells, green plant cells contain one or multiple chloroplasts, the organelle(s) in which photosynthetic reactions take place. Chloroplasts are believed to have originated from an endosymbiotic event and contain DNA that codes for some of their proteins. Most chloroplast proteins are encoded by the nuclear genome and imported with the help of sorting signals that are intrinsic parts of the polypeptides. Here, we show that a chloroplast-located protein in higher plants takes an alternative route through the secretory pathway, and becomes N-glycosylated before entering the chloroplast.     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO₂peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm⁻²) vs. AB. (41.0 ± 8.1 glands · cm⁻²). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l⁻¹) vs. AB (26.8±11.07 mmol · l⁻¹). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.