The evolution of paired domains

Pax genes are important developmental control genes. They are involved in nervous system development, organogenesis and oncogenesis. A DNA specific binding domain called the paired domain, which is well conserved during evolution, defines Pax genes. Furthermore, Pax genes are also conserved in terms of their functions. For example, the Pax-6 gene has been showed to be one of the master control genes for eye development both in Drosophila and vertebrates. All of these properties of Pax genes make them an excellent model for studying the evolution of gene function. ^ Molecular evolutionary studies of paired domain are carried out in this study. Five Pax genes from cnidarians, which are the most primitive organisms possessing a nervous system, were isolated and characterized for their DNA binding properties. By combining data obtained from Genbank and this study, the phylogenetic relationship between Pax genes was studied. It was found that Pax genes could be divided into five groups: Pax-1/9, Pax-3 /7, Pax-A, Pax-2/5/ 8/B, and Pax- 4/6. Furthermore, Pax-2/5/8/ B, Pax-A and Pax-4/6 could be clustered into a supergroup I, while Pax-1/9 and Pax-3/7 could be clustered into supergroup II. The phylogeny was also supported by studies on DNA binding properties of paired domains from different groups. A statistical method was applied to infer the critical amino acid residue substitutions between two supergroups and within the supergroup I. It was found that two amino acid residues were mainly responsible for the difference of DNA binding between two supergroups, while only one amino acid was critical for the evolution of novel DNA binding properties of Pax-4/6 group from ancestor. Evolutionary implications of these data are also discussed. ^

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Domains of expansin gene expression define growth regions in the shoot apex of tomato

Expansins are members of a multigene family of extracellular proteins, which increase cell wall extensibility in vitro and thus are thought to be involved in cell expansion. The major significance of the presence of this large gene family may be that distinctly expressed genes can independently regulate cell expansion in place and time. Here we report on LeExp9, a new expansin gene from tomato, and compare its expression in the shoot tip with that of LeExp2 and LeExp18. LeExp18 gene is expressed in very young tissues of the tomato shoot apex and the transcript levels are upregulated in the incipient primordium. LeExp2 mRNA accumulated in more mature tissues and transcript levels correlated with cell elongation in the elongation zone. In situ hybridization experiments showed a uniform distribution of LeExp9 mRNA in submeristematic tissues. When gibberellin-deficient mutant tomatoes that lacked elongation of the internodes were treated with gibberellin, the phenotypic rescue was correlated with an increase in LeExp9 and LeExp2, but not LeExp18 levels. We propose that the three expansins define three distinct growing zones in the shoot tip. In the meristem proper, gibberellin-independent LeExp18 mediates the cell expansion that accompanies cell division. In the submeristematic zone, LeExp9 mediates cell expansion at a time that cell division comes to a halt. LeExp9 expression requires gibberellin but the hormone is not normally limiting. Finally, LeExp2 mediates cell elongation in young stem tissue. LeExp2 expression is limited by the available gibberellin. These data suggest that regulation of cell wall extensibility is controlled, at least in part, by differential regulation of expansin genes.

Interaction of Plectin with Keratins 5 and 14: Dependence on Several Plectin Domains and Keratin Quaternary Structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.Journal of Investigative Dermatology advance online publication, 10 July 2014; doi:10.1038/jid.2014.255.

Remarks on Lipschitz domains in Carnot groups

In this Note we present the basic features of the theory of Lipschitz maps within Carnot groups as it is developed in [8], and we prove that intrinsic Lipschitz domains in Carnot groups are uniform domains.

On restricted families of projections in ℝ3

We study projections onto non-degenerate one-dimensional families of lines and planes in R 3 . Using the classical potential theoretic approach of R. Kaufman, one can show that the Hausdorff dimension of at most 12 -dimensional sets [Math Processing Error] is typically preserved under one-dimensional families of projections onto lines. We improve the result by an ε , proving that if [Math Processing Error], then the packing dimension of the projections is almost surely at least [Math Processing Error]. For projections onto planes, we obtain a similar bound, with the threshold 12 replaced by 1 . In the special case of self-similar sets [Math Processing Error] without rotations, we obtain a full Marstrand-type projection theorem for 1-parameter families of projections onto lines. The [Math Processing Error] case of the result follows from recent work of M. Hochman, but the [Math Processing Error] part is new: with this assumption, we prove that the projections have positive length almost surely.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.