Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the -glucuronidase (GUS) gene, under the control of the doubled enhancer element (the –208 to –46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to –389 bp from ATG) promoter of wheat, -amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 10⁶ protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.     

Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants

We have isolated and analyzed a pre-ferredoxin gene from Arabidopsis thaliana. This gene encodes a 148 amino acid precursor protein including a chloroplast transit peptide of 52 residues. Southern analysis shows the presence of a single copy of this ferredoxin (Fd) gene in the A. thaliana genome. Its expression is tissue-specific and positively affected by light. Response times, both to dark and light conditions, are remarkably rapid.A chimeric gene consisting of a 1.2 kb Fd promoter fragment fused to the -glucuronidase reporter gene was transferred to tobacco. This fusion gene is expressed in a tissue-specific way; it shows high levels of expression in green leaves, as compared to root tissue.     

Calmodulin gene family in potato: developmental and touch-induced expression of the mRNA encoding a novel isoform

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca²⁺-binding area. The expression patterns of different genes were studied by northern analysis using the 3-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the -glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation

A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by two general promoters (cytomegalovirus and medaka -actin) and one muscle-specific promoter (zebrafish -actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, 34.6% (81/234), 10% (10/60), and 18% (38/212) showed homogenous GFP-expression for the derivative constructs of the cytomegalovirus, -actin, and -actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was (a) extended for an entire lifetime without degradation, and (b) transmitted as a genetic trait to F1 and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Effects of retinoic acid on the growth of cultured rabbit articular chondrocytes: Relation with alkaline phosphatase activity and beta receptor

The effects of retinoic acid (RA) on rabbit articular cartilage cells were studied for concentrations ranging from 5.10^-5 M to 10^-7 M; the treatment with RA over three days resulted in dose dependent inhibition of chondrocyte proliferation between 5.10^-5 and 10^-5 M with persistence of the inhibitory effect until 10^-6 M. RA until 10^-7 M induced a slight, but significant, enhancement of cell proliferation. This growth stimulating effect seems to be related to the Beta receptor system because Beta blockers, such as sotalol and DL propranolol, were able to suppress the stimulating action of agonist type isoprenaline. The activity of alkaline phosphatase (AP) was also determined. The highest dose of RA (5.10^-5 M) induced an increase (x 3) of AP activity, and 10^-7 M RA decreased it (x 0.4).     

Transient reporter gene (GUS) expression in creeping bentgrass (Agrostis palustris) is affected by in vivo nucleolytic activity

Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When -glucuronidase (GUS) reporter gene activity was evaluated in heparin-treated (0.6%), 14-month old callus following microprojectile bombardment, GUS activity increased 1000-fold compared to equivalent aged untreated Penn A4 callus. Similar enhancement from heparin pretreatment (0.6% or 1.2%) was not observed in 6-month old callus. This is likely due to much higher activities of nuclease in the younger callus.     

Modifications of the EEG Activity upon Lateralized Stimulation of the Visual Inputs to the Right and to the Left Brain Hemispheres by Light with Different Wavelengths

In 80 healthy humans, we studied changes in the frequency spectrum and values of the relative spectral coefficients for subsequent 1-Hz-wide frequency bands under conditions of simultaneous lateralized stimulation of the retinal zones, which form receptive fields for the right- and for the left-brain hemispheres; light of different colors was used for stimulation. We found that reactions of the right and left hemispheres to such stimulations demonstrated obvious specificity; spatial characteristics of these reactions were obtained. Changes in the values of most corresponding spectral coefficients in the hemispheres usually had similar directions and demonstrated similarity in their intensities, which is indicative of a complementary pattern of the interhemispheric interaction. Reciprocal changes in the corresponding spectral coefficients in the hemispheres were observed more rarely. Modifications of EEG upon red-green stimulations of different polarity were found to be similar, which can be a manifestation of the moderating influence of the right hemisphere on the left one.     

Sucrose-regulated expression of a chimeric potato tuber gene in leaves of transgenic tobacco plants

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato tubers. Class-I patatin genes encode 98 to 99% of the patatin mRNA in tubers, but are not normally expressed in other tissues. They are not totally tuber-specific; however, since they can be induced to express at high levels in other tissues under conditions of sink limitation or in explants cultured on medium containing elevated levels of sucrose. To examine the evolution of the mechanisms that regulate patatin gene expression, we introduced a chimeric patatin--glucuronidase (GUS) gene containing 2.5 kb of 5 flanking sequence from the Class-I potato patatin gene PS20 into tobacco plants. The construct was not expressed at significant levels in leaves of juvenile plants or plantlets cultured in vitro, but was expressed at high levels in explants cultured on medium containing 0.3 to 0.4 M sucrose. While there were differences in the expression of the chimeric gene between transgenic tobacco and potato plants, the pattern of sucrose induction was very similar. These results suggest that the mechanism that controls patatin gene expression in potato tubers evolved from a widely distributed mechanism in which gene expression is regulated by the level of available photosynthate.     

Protective role of curcumin against isoproterenol induced myocardial infarction in rats

The effect of curcumin on the biochemical changes induced by isoproterenol (ISO) administration in rats was examined. ISO (300 mg Kg^–1 administered subcutaneously twice at an interval of 24 h) caused a decrease in body weight and an increase in heart weight, water content as well as in the levels of serum marker enzymes viz creatine kinase (CK), lactate dehydrogenase (LDH) and LDH₁ isozyme. It also produced electrocardiographic changes such as increased heart rate, reduced R amplitude and ST elevation. Curcumin at a concentration of 200 mg.Kg^–1 when administered orally, showed a decrease in serum enzyme levels and the electrocardiographic changes got restored towards normalcy. Myocardial infarction was accompanied by the disintegration of membrane polyunsaturated fatty acids expressed by increase of thiobarbituric acid reactive substance (TBARS), a measure of lipid peroxides and by the impairment of natural scavenging, characterized by the decrease in the levels of superoxide dismutase, catalase, glutathione peroxidase, ceruloplasmin, alpha tocopherol, reduced glutathione (GSH) and ascorbic acid. The oral pretreatment with curcumin two days before and during ISO administration decreased the effect of lipid peroxidation. It was shown to have a membrane stabilizing action by inhibiting the release of -glucuronidase from nuclei, mitochondria, lysosome and microsome. Curcumin pre- and co-treatment decreased the severity of pathological changes and thus, could have a protective effect against the damage caused by myocardial infarction (MI).     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants

A genomic clone encoding the potato homolog of the yeast ubiquitin-ribosomal protein fusion gene ubi3 was isolated and characterized. Chimeric genes containing the ubi3 promoter (920 bp of 5 to the ubiquitin start codon) were constructed in which the reporter gene -glucuronidase (GUS) was either fused directly to the promoter, or introduced as a translational fusion to the ubiquitin-coding region. After introduction into the potato by Agrobacterium-mediated transformation, GUS activities were measured in leaves and in tubers of transgenic clones. GUS activity was 5- to 10-fold higher in clones expressing the ubiquitin-GUS translational fusion than in clones containing GUS fused directly to the ubi3 promoter. For both types of constructs, GUS activity was highest in meristematic leaves and declined during leaf expansion, then rose again to near the meristematic levels during senescence. GUS activity in tubers was similar to that in young leaves. In contrast to the native ubi3 genes, the chimeric ubi3-GUS transgenes were not activated in the tuber by wounding.