Abstract. We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of ,~7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at ,',,10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five-to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.ITTLE information exists about the structural components, assembly, and regulation of the tight junction or its developmental relationship to other cell structures. As a characteristic feature of polarized, transporting epithelial cells, the tight junction forms the intercellular seal which limits the movement of water and solutes between cells (14, 18, 34, 40) and may play a role in establishing and maintaining the separation of biochemically distinct apical and basolateral membrane components (2, 43). By thin-section electron microscopy the junction appears as a set of close membrane contacts positioned apically to the other intercellular junctions; i.e., desmosomes and the adherens and gap junctions (15). In freeze-fracture electron micrographic images the membrane appositions are revealed to correspond to interconnecting fibrils in the plane of the membrane. Fibrils appear composed of rows of intramembrane particles (16, 37) giving rise to the notion that the junction is composed of rows of transmembrane proteins which seal the extracellular space by their association with like proteins on adjacent cells.We have recently identified (38) and characterized ZO-1 (...

The intraepithelial cells of the murine small intestine include a significant number of CD3+ T cells that use T-cell receptor ygenes rather than T-cell receptor (3 genes. As with other sites of T-cell receptor 'y expression, combinatorial diversity is limited, but there is junctional diversity, and this, together with the specific variable region Eygene segments used, distinguishes ygene expression in the gut epithelium from that in cells derived from the dermal epithelium. The restriction of productive y gene expression largely to one V-J-C (V, variable; J, joining; C, constant) gene combination may result from nonproductive joining of other V-J combinations and from productively rearranged genes rendered nonfunctional by incorrect splicing.

Citation: Nishizaki, M. T., S. Barron, and E. Carew. 2015. Thermal stress increases fluctuating asymmetry in marine mussels: environmental variation and developmental instability. Ecosphere 6(5):85. http://dx.doi.org/10.1890/ ES14-00399.1Abstract. Faced with rising environmental temperatures, there is growing evidence that species are exhibiting shifts in ecological distribution, physiological performance, and behavioral strategy. Less is understood, however, about links between environmental conditions and the precision with which organisms are able to fulfill their developmentally programmed phenotype. Here, we report that developmental instability, assessed by the fluctuating asymmetry (FA) of right versus left valves in intertidal mussel shells, increases under elevated thermal stress. In a growth experiment, mussels that were exposed to elevated aerial temperatures (21.58 6 0.18C) for three hours each day displayed higher levels of FA compared to mussels exposed to cooler aerial temperatures (12.68 6 0.18C). Reciprocal field transplant experiments revealed that FA increased under higher aerial temperatures (e.g., on a south facing surface [19.68 6 0.28C]) compared to individuals living in cooler habitats (e.g., on a north facing surface [15.28 6 0.28C] or lower in the intertidal zone [14.18 6 0.68C]). Together, these results imply that the precision of developmental processes can be perturbed by environmental conditions and raise developmental instability as a potential impact of future environmental variability alongside shifts in physiology, behavior and biogeographic distribution.

No abstract

We report here the DNA sequence of the alcohol dehydrogenase gene (Adh) cloned from Drosophila willistoni. The three major findings are as follows: (1) Relative to all other Adh genes known from Drosophila, D. willistoni Adh has the last intron precisely deleted; PCR directly from total genomic DNA indicates that the deletion exists in all members of the willistoni group but not in any other group, including the closely related saltans group. Otherwise the structure and predicted protein are very similar to those of other species. (2) There is a significant shift in codon usage, especially compared with that in D. melanogaster Adh. The most striking shift is from C to U in the wobble position (both third and first position). Unlike the codon-usage-bias pattern typical of highly biased genes in D. melanogaster, including Adh, D. willistoni has nearly 50% G + C in the third position. (3) The phylogenetic information provided by this new sequence is in agreement with almost all other molecular and morphological data, in placing the obscura group closer to the melanogaster group, with the willistoni group farther distant but still clearly within the subgenus Sophophora.