Importance of long-range interactions in protein folding.

Long-range interactions play an active role in the stability of protein molecules. In this work, we have analyzed the importance of long-range interactions in different structural classes of globular proteins in terms of residue distances. We found that 85% of residues are involved in long-range contacts.

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method.

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5.

The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria.

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. These cells showed typical characteristics of apoptosis, when they were cultured at 32 degrees C. In this process, p53 recovered the wild-type p53 function and the expression of the p21 (waf1/cip1/sdi1), cyclin G1 and gadd45 genes was increased.

Mapping of 25 drought-inducible genes, RD and ERD, in Arabidopsis thaliana.

We mapped 25 Arabidopsis thaliana drought-inducible genes. Responsive to Dehydration (RD) and Early Responsive to Dehydration (ERD), to the Arabidopsis genome and compared map positions with those of mutants that show environmental stress response. We hybridized CIC yeast artificial chromosome (YAC) library filters with the cDNAs and determined the map positions of 18 corresponding genes.

Structural organization and expression of the mouse gene for Pur-1, a highly conserved homolog of the human MAZ gene.

We have characterized the genomic structure and expression of the mouse gene for Pur-1. The cloned Pur-1 gene spans a 5-kb region encompassing the promoter, five exons, four introns and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule.

Structure-based prediction of DNA target sites by regulatory proteins.

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist.

ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-beta signaling.

Upon transforming growth factor-beta (TGF-beta) binding to its cognate receptor, Smad3 and Smad4 form heterodimers and transduce the TGF-beta signal to the nucleus. In addition to the Smad pathway, another pathway involving a member of the mitogen-activated protein kinase kinase kinase family of kinases, TGF-beta-activated kinase-1 (TAK1), is required for TGF-beta signaling. However, it is unknown how these pathways function together to synergistically amplify TGF-beta signaling.

Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis.

Helicase-related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase-related proteins make up several gene families. In this study, we have identified 134 ORFs that encode helicase-like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase-related proteins.

Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3.

Drosophila transcription factor cubitus interruptus (Ci) and its co-activator CRE (cAMP response element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response to hedgehog protein (Hh) signaling. In the anterior region, in contrast, the carboxyl-truncated form of Ci generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription factors (glioblastoma gene products (GLIs) 1, 2, and 3) were identified, but their functional difference in Hh signal transduction is unknown.

Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor.

The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and forms a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta).

Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.

We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity).

Poly(L-lysine)-graft-dextran copolymer promotes pyrimidine motif triplex DNA formation at physiological pH. Thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH.

Ovine MHC class II DRB1 alleles associated with resistance or susceptibility to development of bovine leukemia virus-induced ovine lymphoma.

For the further characterization of bovine leukemia virus (BLV)-induced leukemogenesis, we investigated the association between polymorphism of ovine leukocyte antigen (OLA)-DRB1 gene and tumor development after infection of sheep with BLV. We infected 28 sheep with BLV and cloned exon 2 of the OLA-DRB1 gene from asymptomatic animals and from animals with lymphoma Sequence analysis revealed that, among 12 healthy sheep without any evidence of tumor, ten (83.3%) carried DRB1 alleles encoding Arg-Lys (RK) at positions beta70/71 as compared with only 6 (37.

Viral ski inhibits retinoblastoma protein (Rb)-mediated transcriptional repression in a dominant negative fashion.

The mechanism by which the viral oncogene ski (v-ski) transforms chicken embryo fibroblasts is currently unknown. Recently, the c-ski gene product (c-Ski) was found to bind to N-CoR (nuclear hormone receptor co-repressor), an element implicated in transcriptional repression mediated by multiple transcriptional repressors including the nuclear hormone receptors and Mad. c-Ski is required for transcriptional repression mediated by Mad involved in negative regulation of cellular proliferation.

Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation of mouse melanoma cells in culture.

Malignant melanomas are tumors that are well known to respond poorly to treatment with chemotherapeutic reagents. We report here that mannosylerythritol lipid (MEL), an extracellular glycolipid from yeast, markedly inhibited the growth of mouse melanoma B16 cells in a dose-dependent manner. Exposure of B16 cells to MEL at 10 microM and higher concentrations caused the condensation of chromatin, DNA fragmentation, and sub-G1 arrest, all of which are hallmarks of cells that are undergoing apoptosis.

The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions.

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A.

Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis.

In plants, a number of MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) homologues have been reported. However, there have been no reports of protein-protein interactions between these kinases or molecular analysis of MAPK cascades in higher plants. To analyze a possible MAPK cascade in Arabidopsis thaliana, we took two molecular approaches.

Effect of tenascin-C deficiency on chemically induced dermatitis in the mouse.

Tenascin-C is a large extracellular matrix glycoprotein characterized by its spatiotemporal expression during embryogenesis, carcinogenesis, and wound healing. Many in vitro studies on tenascin-C have revealed its multifunctional properties; however, disruption of the tenascin-C gene did not reveal any obvious abnormalities during development, and its function in vivo remains unclear. Here, we investigated whether tenascin-C is involved in inflammatory dermatitis in adults by studying chemically induced dermatitis in tenascin-C knockout mice.

ProTherm: Thermodynamic Database for Proteins and Mutants.

The first release of the Thermodynamic Database for Proteins and Mutants (ProTherm) contains more than 3300 data of several thermodynamic parameters for wild type and mutant proteins. Each entry includes numerical data for unfolding Gibbs free energy change, enthalpy change, heat capacity change, transition temperature, activity etc., which are important for understanding the mechanism of protein stability.

Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate.

To identify stable RNA secondary structure causing band compression, 30 lambda DNA clones and four cDNA clones (about 10 kb in total length) were sequenced using Transcriptional Sequencing, which is based on the phage RNA polymerase chain termination reaction with fluorescent 3' deoxynucleoside triphosphate, using the canonical set of rNTPs for the substrate. Electrophoresis was performed on acrylamide gel containing 7 M urea at 50 degrees C using ABI 377 DNA sequencer. A total of 159 band compressions were identified, and most compression sites seem to be due to hairpin structures.

Role of two series of Ca2+ oscillations in activation of ascidian eggs.

Changes in [Ca2+]i are an essential factor regulating egg activation. Matured ascidian eggs are arrested at metaphase I, and two series of [Ca2+]i transients have been observed after fertilization: Ca2+ waves just after fertilization (Series I) and [Ca2+]i oscillation between the first and second polar body extrusion (Series II). We investigated mechanisms involved in the elevation of [Ca2+]i and the role of the [Ca2+]i transients during egg activation in Ciona savignyi.

Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants.

A possible MAP kinase (MAPK) cascade of Arabidopsis thaliana was identified on the basis of both yeast 2-hybrid analysis and complementation analysis of yeast mutants. Specific protein-protein interactions between ATMPK4 (a MAPK) and MEK1 (a MAPKK) and interactions between MEK1 and ATMEKK1 (a MAPKKK) were detected by using the 2-hybrid system. A growth defect of the yeast mpk1delta mutant was reversed by coexpression of ATMPK4 and MEK1.

CBP alleviates the intramolecular inhibition of ATF-2 function.

The transcription factor ATF-2 (also called CRE-BP1), whose DNA-binding domain consists of a basic amino acid cluster and a leucine zipper (b-ZIP) region, binds to the cAMP response element as a homodimer or as a heterodimer with c-Jun. The amino-terminal region of ATF-2 containing the transcriptional activation domain is phosphorylated by stress-activated kinases, which leads to activation of ATF-2. We report here that CBP, which was originally identified as a co-activator of CREB, directly binds to the b-ZIP region of ATF-2 via a Cys/His-rich region termed C/H2, and potentiates trans-activation by ATF-2.

High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper.

Full-length cDNAs are essential for functional analysis of plant genes. We constructed high-content, full-length cDNA libraries from Arabidopsis thaliana plants based on chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment, to select full-length cDNA. More than 90% of the total clones obtained were of full length; recombinant clones were obtained with high efficiency (2.