Faculty Bibliography

Mammalian genomes are densely populated with long duplicated sequences. In this paper, we demonstrate the existence of doublets, short duplications between 25 and 100 bp, distinct from previously described repeats. Each doublet is a pair of exact matches, separated by some distance. The distribution of these intermatch distances is strikingly nonrandom. An unexpectedly high number of doublets have matches either within 100 bp (adjacent) or at distances tightly concentrated approximately 1,000 bp apart (nearby). We focus our study on these proximate doublets. First, they tend to have both matches on the same strand. By comparing nearby doublets shared in human and chimpanzee, we can also see that these doublets seem to arise by an insertion event that produces a copy without markedly affecting the surrounding sequence. Most doublets in humans are shared with chimpanzee, but many new pairs arose after the divergence of the species. Doublets found in human but not chimpanzee are most often composed of almost tandem matches, whereas older doublets (found in both species) are more likely to have matches spaced by approximately 1 kb, indicating that the nearly tandem doublets may be more dynamic. The spacing of doublets is highly conserved. So far, we have found clearly recognizable doublets in the following genomes: Homo sapiens, Mus musculus, Arabidopsis thaliana, and Caenorhabditis elegans, indicating that the mechanism generating these doublets is widespread. A mechanism that generates short local duplications while conserving polarity could have a profound impact on the evolution of regulatory and protein-coding sequences

In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Q(o) site inhibitor of the cytochrome bc(1) complex from Saccharomyces cerevisiae. Natural stigmatellin A induced clear shifts in the redox-induced FTIR difference spectra. For data interpretation a stigmatellin derivative (UST) with the conjugated trienes replaced by an aliphatic tail was synthesized, and the carbonyl group shown in crystal structures to interact with His181, the [2Fe-2S] ligand of the Rieske, was specifically (13)C labeled. Electrochemically induced FTIR difference spectra of the inhibitors in CH(3)OD were obtained and revealed signals characteristic for the oxidized and reduced forms of the labeled and unlabeled compounds. On the basis of signals from the inhibitors alone, the binding of the inhibitor to the bc(1) complex was monitored. Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the nu(C=O) vibrational mode of about 10 cm(-1). In addition, redox-dependent reorganizations of the protein were identified, including protonation changes of acidic residues at 1746 and 1734 cm(-1). The conformational changes observed upon inhibitor binding are discussed with respect to the crystal structures and proposed mechanistic models [Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669-684; Palsdottir, H., Lojero, C. G., Trumpower, B. L., and Hunte, C. (2003) J. Biol. Chem. 278, 31303-31311].

A culture model has been established to study the gradual development of tight junctions during the embryogenesis of the chick retinal pigment epithelium. This study asks how closely the culture model reflects normal development and how the composition, structure and function of embryonic tight junctions are affected by the apical and basal environments. The study focused on the expression of claudins, the fine-structure of tight junctional strands and the transepithelial electrical resistance. Between embryonic days 7 and 14, patches of junctional strands gradually expanded and coalesced to form a continuous junction, in vivo. Although there was a corresponding increase in claudin expression, different claudins appeared at different times. In culture, the apical and basal environments acted synergistically to promote a continuous network of tight junctions with higher electrical resistance. Independently, pituitary extract or the secretory products of either embryonic fibroblasts or the retina promoted the formation of tight junctions. In combination, three effects were identified. With basally placed fibroblast conditioned medium, apical retinal medium increased transepithelial electrical resistance by affecting structure alone. With basally placed pituitary extract, apical retinal conditioned medium increased transepithelial electrical resistance by affecting structure and by modulating claudin expression in a manner that was consistent with development in vivo. Although embryonic day 7 and 14 cultures in retinal medium exhibited similar structure, the transepithelial electrical resistance of the embryonic day 14 cultures was higher. This higher transepithelial electrical resistance correlated with differences in claudin expression and localization. Therefore, this experimental model can isolate the effects of retinal secretions on structure and claudin expression, and can help us to determine how claudins affect function when structure is held constant.

Mammalian eggs are surrounded by a zona pellucida (ZP) that regulates egg-sperm teractions during fertilization. The ZP consists of long filaments composed of two glycoproteins, ZP2 and ZP3, that are crosslinked by a third glycoprotein, ZP1. The presence of both ZP2 and ZP3 is essential for assembling a ZP around growing oocytes, as well as for fertility of females. Acrosome-intact sperm recognize and bind to O-linked oligosaccharides linked to Ser residues at the sperm combining-site of ZP3. Structural differences in oligosaccharides on ZP3 from different species may account for whether or not sperm are able to bind to the ZP. Bound sperm undergo the acrosome reaction, penetrate the ZP, and can then fuse with egg plasma membrane. Following fertilization, sperm are unable to bind to either ZP3 or the ZP of one-cell embryos.

Organogenesis requires the specification of a variety of cell types and the organization of these cells into a particular three-dimensional configuration. The embryonic vertebrate heart is organized into two major chambers, the ventricle and atrium, each consisting of two tissue layers, the myocardium and endocardium. The cellular and molecular mechanisms responsible for the separation of ventricular and atrial lineages are not well understood. To test models of cardiac chamber specification, we generated a high-resolution fate map of cardiac chamber progenitors in the zebrafish embryo at 40% epiboly, a stage prior to the initiation of gastrulation. Our map reveals a distinct spatial organization of myocardial progenitors: ventricular myocardial progenitors are positioned closer to the margin and to the dorsal midline than are atrial myocardial progenitors. By contrast, ventricular and atrial endocardial progenitors are not spatially organized at this stage. The relative orientations of ventricular and atrial myocardial progenitors before and after gastrulation suggest orderly movements of these populations. Furthermore, the initial positions of myocardial progenitors at 40% epiboly indicate that signals residing at the embryonic margin could influence chamber fate assignment. Indeed, via fate mapping, we demonstrate that Nodal signaling promotes ventricular fate specification near the margin, thereby playing an important early role during myocardial patterning

Previous studies have demonstrated that the carboxyl terminus of the gap junction protein Cx43 (Cx43CT) can act as an independent, regulatory domain that modulates intercellular communication in response to appropriate chemical stimuli. Here, we have used NMR, chemical cross-linking, and analytical ultracentrifugation to further characterize the biochemical and biophysical properties of the Connexin43 carboxyl terminal domain (S255-I382). NMR-diffusion experiments at pH 5.8 suggested that the Connexin43 carboxyl terminus (CX43CT) may have a molecular weight greater than that of a monomer. Sedimentation equilibrium and cross-linking data demonstrated a predominantly dimeric state for the Cx43CT at pH 5.8 and 6.5, with limited dimer formation at a more neutral pH. NMR-filtered nuclear Overhauser effect studies confirmed these observations and identified specific areas of parallel orientation within Cx43CT, likely corresponding to dimerization domains. These regions included a portion of the SH3 binding domain, as well as two fragments previously found to organize in alpha-helical structures. Together, these data show that acidification causes Cx43CT dimer formation in vitro. Whether dimer formation is an important structural component of the regulation of Connexin43 channels remains to be determined. Dimerization may alter the affinity of Cx43CT regions for specific molecular partners, thus modifying the regulation of gap junction channels

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are poorly understood. The cartilage oligomeric matrix protein gene (COMP) encodes a noncollagenous extracellular matrix protein whose expression pattern correlates with chondrocyte differentiation and arthritis. We have used the COMP promoter as a model to identify regulatory sequences necessary for chondrocyte-specific expression and to identify cell type-specific proteins that bind these sequences. We have previously cloned 1.9 kilobases of the 5(') flanking promoter sequence of the murine COMP gene and by deletion analysis have identified two spatially distant chondrocyte-specific regulatory regions. One element is situated proximally (-125 to -75), and a second region is located distally (-1925 to -592) relative to the transcription start site. In the present study, we performed a finer deletion analysis of the region of the COMP promoter from -1925 to -592 and identified a silencer region situated between -1775 and -1725. This silencer binds sequence-specific protein complexes; the intensity of these complexes is greater in two different fibroblast cell lines (NIH3T3 and 10T1/2) than in chondrocytic RCS cells. Competition experiments localized the binding site of these protein complexes from -1775 to -1746; deletion of this 30-bp site results in a selective increase in COMP promoter activity in fibroblasts. Four tandem repeats of this 30-bp site are sufficient to confer negative transcriptional regulation on a heterologous promoter (SV40) in NIH3T3 fibroblasts. These results suggest that negative regulation of transcription is an important mechanism for chondrocyte-specific expression of the COMP gene

Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. We show that this phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and that it requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. We suggest that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration

A genetic screen for mutations affecting embryogenesis in the medaka, Oryzias latipes, identified a mutant, whiteout (who), that exhibited hypochromic anemia. The who mutant initially had the normal number of blood cells, but it then gradually decreased during the embryonic and larval stages. The blood cells in the who mutants show an elongated morphology and little hemoglobin activity. Genetic mapping localized who to the vicinity of a LG12 marker, olgc1. By utilizing the highly conserved synteny between medaka and pufferfish, we identified a gene for delta-aminolevulinic acid dehydratase (ALAD), which is the second enzyme in the heme synthetic pathway, as a candidate for who. We found a missense mutation in the alad gene that was tightly linked to the who phenotype, strongly suggesting that the hypochromic anemia phenotype in the who mutant is caused by a loss of the alad function. Thus, who mutants represent a model for the human disease ALAD-deficiency porphyria.