Comparison of the orotidine 5'-monophosphate decarboxylase sequences of eight species

Predicted amino acid sequences of the enzyme orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) from eight different organisms are compared. The comparisons are made on the basis of primary structural differences, primary amino acid sequence, hydropathy profiles, and secondary structure predictions. The organisms compared are Mus musculus, Aspergillus nidulans, Neurospora crassa, Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli, and Salmonella typhimurium.     

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.     

Neurotrophin-3- and norepinephrine-mediated adrenergic differentiation and the inhibitory action of desipramine and cocaine

In the presence of neurotrophin-3 (NT-3), high-affinity norepinephrine (NE) uptake by quail neural crest cells was significantly increased as judged by in vitro colony assay of adrenergic differentiation. In the presence of the related neurotrophins nerve growth factor (NGF) or brain-derived neurotrophic (BDNF) factor, or of basic fibroblast growth factor (bFGF), there were no significant changes. When NE was added to the culture medium in addition to NT-3, more colonies contained dopamine-β-hydroxylase (DBH)-immunoreactive cells, an enzyme that is characteristic for adrenergic cells. The NE-mediated increase in the portion of colonies that contained DBH-immunoreactive cells was prevented by the tricyclic antidepressant desipramine (DMI) and by cocaine, two types of drug that block cellular transport of NE. To further examine whether NE acts via uptake, colony assays were performed in the presence and absence of adrenergic antagonists and agonists. These would be expected to mimic the DMI and NE effects, respectively, if the mechanism of action involved activation of adrenergic autoreceptors. Neither class of drug showed a detectable effect within a wide range of concentrations. Immunocytochemistry using antibodies against β1 and β2 adrenergic receptors further supported the notion that DMI action and β-receptor expression are not causally related. Ratio imaging was subsequently used in an attempt to elucidate the mechanism of NE action. Within a few minutes of addition of NE to the culture medium, there was an increase in intracellular free calcium in a subset of neural crest cells. Taken together, our data indicate that NT-3 is involved in the appearance of the NE transporter (NET) during embryonic development; internalized NE directly or indirectly increases adrenergic differentiation as measured by immunoreactivity of the adrenergic biosynthetic enzyme DBH; and norepinephrine uptake inhibitors have teratogenic potential. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 262–280, 1997.     

Plastid transformation of high‐biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV‐1) p24 antigen

Chloroplast transformation of the high‐biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV‐1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast‐codon‐optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 µg/g fresh weight or ~2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5‐yellow) with detectable p24 accumulation (up to approximately 450 µg/g fresh weight or ~4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5‐yellow leaves was reduced by ~40%. The pZF5‐yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3′ untransformed region in the plastid genome. Chloroplast‐expressed p24 was recognized by a conformation‐dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5‐yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion‐exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N‐terminal sequencing and mass spectrometry.     

Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken

Background  

Domestication of chicken is believed to have occurred in Southeast Asia, especially in Indus valley. However, non-inclusion of Indian red jungle fowl (RJF), Gallus gallus murghi in previous studies has left a big gap in understanding the relationship of this major group of birds. In the present study, we addressed this issue by analyzing 76 Indian birds that included 56 G. g. murghi (RJF), 16 G. g. domesticus (domestic chicken) and 4 G. sonneratii (Grey JF) using both microsatellite markers and mitochondrial D-loop sequences. We also compared the D-loop sequences of Indian birds with those of 779 birds obtained from GenBank.     

Liposome-mediated gene therapy in the kidney

Gene therapy directed to the kidney has been attempted to improve renal disorders such as inherited kidney diseases and common renal diseases that cause interstitial fibrosis, tubular atrophy, and glomerulosclerosis. Viral and non-viral vectors have been tried and been modulated to obtain sufficient transgene expression. However, gene delivery to the kidney is usually difficult because of characteristics of renal cell biology. Among non-viral vectors, the liposome system is a promising procedure for kidney-targeted gene therapy. Using cationic liposome, tubular cells were effectively transduced by retrograde injection of liposome/cDNA complex. Although transgene expression was reportedly modest using cationic liposomes, this method improved renal disease models such as carbonic anhydrase II deficiency and unilateral ureteral obstruction. In contrast, HVJ-liposome system is an effective transfection method to glomerular cells using intra-renal arterial infusion and improved glomerular disease models such as glomerulonephritis and glomerulosclerosis. In addition, intra-renal pelvic injection of DNA by HVJ-liposome system showed transgene expression in interstitial fibroblasts. In kidney-targeted gene therapy, liposome-mediated gene transfer is an attractive method because of its simplicity and reduced toxicity. In spite of modest transgene expression, several renal disease models were successfully modulated by liposome system. Although one limitation of liposome-mediated gene delivery is the duration of transgene expression, the liposome/cDNA complex can be repeatedly administered due to the absence of an immune response.     

Restoring lakes by using artificial plant beds: habitat selection of zooplankton in a clear and a turbid shallow lake

1. Return of large‐bodied zooplankton populations is of key importance for creating a shift from a turbid to a clear‐water state in shallow lakes after a nutrient loading reduction. In temperate lakes, recovery is promoted by submerged macrophytes which function as a daytime refuge for large zooplankton. However, recovery of macrophytes is often delayed and use of artificial plant beds (APB) has been suggested as a tool to enhance zooplankton refuges, thereby reinforcing the shift to a clear‐water state and, eventually, colonisation of natural plants. 2. To further evaluate the potential of APB in lake restoration, we followed the day–night habitat choices of zooplankton throughout summer in a clear and a turbid lake. Observations were made in the pelagic and littoral zones and in APB in the littoral representing three different plant densities (coverage 0%, 40% and 80%). 3. In the clear lake, the zooplankton (primarily Daphnia) were mainly found in the pelagic area in spring, but from mid‐May they were particularly abundant in the APB and almost exclusively so in mid‐June and July, where they appeared in extremely high densities during day (up to 2600 ind. L⁻¹). During night Daphnia densities were overall more equally distributed between the five habitats. Ceriodaphnia was proportionally more abundant in the APB during most of the season. Cyclopoids were more abundant in the high APB during day but were equally distributed between the five habitats during night. 4. In the turbid lake, however, no clear aggregation was observed in the APB for either of the pelagic genera (Daphnia and Bosmina). This may reflect a higher refuge effect in the open water due to the higher turbidity, reduced ability to orient to plant beds and a significantly higher fish density (mainly of roach, Rutilus rutilus, and perch, Perca fluviatilis) in the plant beds than in the clear lake. Chydorus was found in much higher proportions among the plants, while cyclopoids, particularly the pelagic Cyclops vicinus, dominated in the pelagic during day and in the pelagic and high density plants during night. 5. Our results suggest that water clarity is decisive for the habitat choice of large‐bodied zooplankton and that introduction of APB as a restoration measure to enhance zooplankton survival is only a useful tool when water clarity increases following loading reduction. Our results indicate that dense APB will be the most efficient.     

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.