A recently silenced, duplicate PgiC locus in Clarkia

Previous electrophoretic analysis showed that 17 diploid species of the wildflower Clarkia (Onagraceae) have two cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9), whereas 15 other diploid species have a single PGIC. Molecular studies revealed that the two isozymes in the former species are encoded by duplicate genes, PgiC1 and PgiC2, whereas the single isozyme in the latter is always encoded by PgiC1. Phylogenetic analysis of the nucleotide sequences implied that PgiC2 was silenced four times independently in the genus. Here we describe a psi PgiC2 from C. mildrediae, a species in which only PgiC1 is expressed. The discovery of the psi PgiC2 is significant because it confirms a formal prediction of the phylogenetic analysis. The psi PgiC2 includes 5,039 nucleotides corresponding to 18 of the 23 exons of PgiC, as well as the intervening introns and 3' nontranslated region. The absence of an increase of nucleotide substitutions in its "exons" suggests that the gene was silenced recently. The present study appears to be the first to establish that a specific duplicate gene locus regularly expressed in a group of related plant species has been silenced in one of them. The multiple independent silencings of PgiC2 suggest that it remained functional but inessential in ancestral lineages. We discuss the possibility that PgiC2 may have been preserved in these lineages by selection against mutants causing defective PGIC1- PGIC2 heterodimers.      

Colocalization of eNOS and the catalytic subunit of PKA in endothelial cell junctions: a clue for regulated NO production.

Localization and coordinate phosphorylation/dephosphorylation of endothelial nitric oxide synthase (eNOS) are critical determinants for the basal and stimulated production of nitric oxide. Several phosphorylation sites in eNOS have been identified as targets of the cAMP-dependent protein kinase A (PKA). Basal eNOS activity is also regulated by interaction with caveolin-1, the major coat protein of caveolae. In the present study we have examined in rat aorta endothelium the subcellular steady-state distribution of eNOS, the catalytic subunit of PKA (PKA-c), and caveolin-1. Basal eNOS expression was found in two distinct locations, the endothelial cell surface and the Golgi complex. Cell surface eNOS was equally distributed over caveolar and non-caveolar membranes but was 2.5-fold enriched on luminal lamellipodia located at endothelial cell contacts. PKA-c colocalized with eNOS in the lamellipodia, whereas caveolin-1 was absent from these membrane domains. PKA-c was also found associated with cell surface caveolae and with tubulovesicular membranes of Golgi complex and endosomes. The topological proximity of eNOS with the catalytic subunit of PKA in restricted intracellular locations may provide mechanisms for differential PKA-mediated eNOS regulation.     

A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli

The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.     

Preformation and pre-existence in the seventeenth century: A brief analysis

Conclusion It is beyond the scope of this paper to describe in detail the rise to popularity of the emboîtement theories during the last decades of the seventeenth century.⁵¹ Eventually the theories did gain great influence, but some points emerging from the above discussion indicate that the rise to popularity was not, perhaps, quite as rapid as has sometimes been assumed.⁵² Although the earlier preformation theories were sometimes regarded as the ancestors of the later ideas,⁵³ there was little intellectual continuity between the two movements, based as they were upon such divergent motivations. Nor can the preformation theories be regarded as the origin of the belief that a miniature can actually be seen within the egg, since the existence of metamorphosis as a perfectly valid alternative to epigenesis meant that the work of Malpighi and others, usually described as preformationist, was not always taken in this latter sense at the time it was published. The pre-existence theories developed in response to particular philosophical problems, and were themselves responsible for the reinterpretation of the observations. In France, the thoughts of Malebranche and Perrault were probably already exerting influence before their written support for pre-existence appeared, but elsewhere the idea was not taken up so rapidly, and ovism, for instance, could develop without associating itself with emboîtement. Malpighi always seems to have remained opposed to pre-existence,⁵⁴ but by the last decade of the century, the idea had become sufficiently powerful to influence Ray and Garden in Britain, and was receiving support from as influential a thinker as Leibniz.⁵⁵ But Garden and Hartsoeker were responsible for dividing emboîtement between two schools, just as the concept itself was becoming popular. The work of both Malpighi and Leeuwenhoek served as the basis of the animalculist version, illustrating how the microscopic discoveries served as much to disrupt the intellectual development of the emboîtement concept as they did to promote it. *** DIRECT SUPPORT *** A8402051 00002     

A LIGHT- AND ELECTRON-MICROSCOPIC SURVEY OF ALGAL CELL WALLS. I. PHAEOPHYTA AND RHODOPHYTA

Dawes , Clinton J., Flora M. Scott , and E. Bowler . (U. California, Los Angeles.) A light- and electron-microscopic survey of algal cell walls. I. Phaeophyta and Rhodophyta. Amer. Jour. Bot. 48(10): 925–934. Illus. 1961.—An introductory survey of the structure of the cell walls of brown, red, and green algae, as seen under light and electron microscopes, has been completed. In the present paper (Part I) the structure of the thalli of the Phaeophyta and Rhodophyta is compared, and the occurrence of intercellular spaces, pitting, and microfibrillar patterns is discussed. A detailed comparison of the cell-wall structure and growth of a brown alga, Dictyota flabellata, and of a red alga, Helminthocladia californica, is also presented. In Dictyota, typical of the brown algae, the microfibrillar pattern in the apical cells and in the adjacent cells of the thallus tip is reticulate. In mature cells, the microfibrils are dominantly parallel in orientation. Pits, which are fields of closely set pores, are distinctive. The microfibrils in the pit areas are masked by non-fibrillar material. Helminthocladia, with a cell wall characteristic of the red algae, differs from Dictyota in that the microfibrillar pattern is reticulate at all ages of the cell and throughout the thallus. In the pit areas, the microfibrils are not masked by amorphous material. Pit connections, characteristic of the Florideae, can be divided into 2 major groups. Either the pit connection is an open channel between 2 adjacent cells, or it is composed of numerous plasmodesmata traversing a continuous, loose, microfibrillar wall. The techniques of the survey are emphasized in that fragmented cell walls were studied, and, also, chemically cleared material was constantly compared with fresh material under light and electron microscopes. It is concluded that the cell wall, as a taxonomic character, is of value only in delimiting the Phaeophyta and Rhodophyta.     

ADP-ribosylation is involved in the integration of foreign DNA into the mammalian cell genome.

The most commonly used DNA transfection method, which employs the calcium phosphate co-precipitation of the donor DNA, involves several discrete steps (1,2). These include the uptake of the donor DNA by the recipient cells, the transport of the DNA to the nucleus, transient expression prior to integration into the host cell genome, concatenation and integration of the transfected DNA into the host cell genome and finally the stable expression of the integrated genes (2,3). Both the concatenation and the integration of the donor DNA into the host genome involve the formation and ligation of DNA strand-breaks. In the present study we demonstrate that the nuclear enzyme, adenosine diphosphoribosyl transferase (ADPRT, E.C. 2.4.2.30), which is dependent on the presence of DNA strand breaks for its activity (4,5) and necessary for the efficient ligation of DNA strand-breaks in eukaryotic cells (4,6), is required for the integration of donor DNA into the host genome. However, ADPRT activity does not influence the uptake of DNA into the cell, its episomal maintenance or replication, nor its expression either before or after integration into the host genome. These observations strongly suggest the involvement of ADPRT activity in eukaryotic DNA recombination events.     

Expression of ricin B chain in Escherichia coli

DNA encoding ricin B chain was fused to that encoding the E. coli OmpA signal peptide using the expression secretion vector pIN-111-ompA. When induced, E. coli cells transformed with the recombinant plasmid express ricin B chain. The recombinant product accumulates in the periplasmic space in a soluble, biologically active form.