Cyclic nucleotide binding proteins in the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis> genomes

Background  

Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules.     

Determining the evolutionary potential of a gene

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base- substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta- galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.      

Oxygen equilibria and ligand-binding kinetics of erythrocruorins from two burrowing polychaetes of different modes of life,Marphysa sanguinea andDiopatra cuprea

Journal of Comparative Physiology A - Oxygen equilibria, ligand-binding kinetics and some other physicochemical properties are reported for erythrocruorins of two intertidal polychaetes:Marphysa...     

A phylogenetic survey of myotubularin genes of eukaryotes: distribution,protein structure,evolution, and gene expression

Background  

Phosphorylated phosphatidylinositol (PtdIns) lipids, produced and modified by PtdIns kinases and phosphatases, are critical to the regulation of diverse cellular functions. The myotubularin PtdIns-phosphate phosphatases have been well characterized in yeast and especially animals, where multiple isoforms, both catalytically active and inactive, occur. Myotubularin mutations bring about disruption of cellular membrane trafficking, and in humans, disease. Previous studies have suggested that myotubularins are widely distributed amongst eukaryotes, but key evolutionary questions concerning the origin of different myotubularin isoforms remain unanswered, and little is known about the function of these proteins in most organisms.     

Applied environmental stresses to enhance the levels of polyphenolics in leaves of hawthorn plants

In this investigation, two species of Crataegus (hawthorn) were chosen because their polyphenolic constituents have recently received greater attention for the treatment of patients with severe heart disease. One-year-old plants of hawthorn ( Crataegus laevigata and C. monogyna ) were subjected to water-deficit (continuous water deprivation), cold (4°C), flooding (immersion of roots of plants in water) or herbivory (leaf removal) stress treatments (each of 10 days duration) in order to assess their effects on levels of polyphenolics, namely (-)-epicatechin, catechin, chlorogenic acid, vitexin, vitexin-2'- O -rhamnoside, acetylvitexin-2'- O -rhamnoside, hyperoside, quercetin, and rutin in the leaves. The working hypothesis followed is that one or more of these stress treatment will elicit increases in the levels of these polyphenolics. Cold stress causes increases in levels of vitexin-2'- O -rhamnoside, acetylvitexin-2'- O -rhamnoside, hyperoside, and quercetin in both Crataegus species. Water-deficit stress increased the productivity of chlorogenic acid, catechin, and (-)-epicatechin in both hawthorn species. Flooding and herbivory caused no net increases, and in some cases, decreases in levels of polyphenolics. These studies indicate that either water-deficit stress or cold stress treatments, or a combination of the two, can be used to enhance the levels of desired polyphenolics in the leaves of these two hawthorn species in a photobioreactor system. These results may have significance for hawthorn in adapting to water-deficit or cold stress and are important considerations for the use of hawthorn in the treatment of heart disease in humans.     

The production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure.

In vitro shoot cultures of Hypericum perforatum derived from wild populations grown in Armenia have a wide variation of hypericin and pseudohypericin metabolite content. We found that a germ line denoted as HP3 produces six times more hypericin and fourteen times more pseudohypericin than a second line labeled HP1. We undertook a structural comparison of the two lines (HP1 and HP3) in order to see if there are any anatomical or morphological differences that could explain the differences in production of these economically important metabolites. Analysis by LM (light microscopy), SEM (scanning electron microscopy), and TEM (transmission electron microscopy) reveals that the hypericin/pseudohypericin-containing black glands located along the margins of the leaves consist of a peripheral sheath of flattened cells surrounding a core of interior cells that are typically dead at maturity. The peripheral cells of the HP3 glands appear less flattened than those of the HP1 glands. This may indicate that the peripheral cells are involved in hypericin/pseudohypericin production. Furthermore, we find that these peripheral cells undergo a developmental transition into the gland's interior cells. The fact that the size of the peripheral cells may correlate with metabolite production adds a new hypothesis for the actual site of hypericin synthesis.     

FokI method of gene synthesis

An accurate, fast and simple method is presented for synthesis of a gene, or any DNA fragment with a defined sequence. The method is based on the observation that large (approx. 100 bp long) inserts can be cloned into a plasmid using a technique of oligodeoxynucleotide (oligo)-directed double-strand (ds) break repair. The procedure involves transformation of Escherichia coli with a denatured mixture of an insert-carrying oligo and linearized plasmid DNA [Mandecki, Proc. Natl. Acad. Sci. USA 83 (1986) 7177-7181]. The nucleotide (nt) sequences are inserted between two FokI restriction nuclease sites in one of four pUC-derived plasmids. Since FokI makes a staggered ds break at a DNA site 9 and 13 nt away from its recognition site, upon cleavage of the plasmid DNA with FokI, a restriction fragment is liberated that by design contains unique 4-nt-long 5'-protruding ends. The uniqueness of ends permits efficient and directed simultaneous ligation of several restriction fragments to form a gene. The method offers flexibility due to the modular-type assembly and does not require any restriction sites within the constructed gene. The sequence error rate is low: about one error per 4000 bp of DNA cloned. Synthetic DNA for only one DNA strand needs to be provided. The method was applied to the synthesis of a gene fragment encoding the N-terminal 143 amino acid residues of the human immunodeficiency virus transmembrane protein (p41).