scholarly journals The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA.

1989 ◽  
Vol 109 (4) ◽  
pp. 1389-1397 ◽  
Author(s):  
M Ohtsubo ◽  
H Okazaki ◽  
T Nishimoto
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Synthetic Peptides ◽  
Protein A ◽  
Chromosome Condensation ◽  
Dnase I ◽  
Isolated Nuclei ◽  
Mrna Maturation ◽  
Almost All ◽  
Cellulose Column

The RCC1 gene, a regulator for the onset of chromosome condensation was found to encode a protein with a molecular mass of 45 kD, determined using the antibody against the synthetic peptides prepared according to the amino acid sequence of the putative RCC1 protein. The p45 located in the nuclei was released from the isolated nuclei, either by DNase I digestion or by treatment with 0.3 M NaCl. Consistently, p45 bound to the DNA-cellulose column was eluted with 0.3 M NaCl. After sequential treatment with DNase I and 2 M NaCl, almost all of the RCC1 protein were released from the nuclei. Thus, RCC1 protein locates on the chromatin and is not a component of the nuclear matrix. In mitotic cells, p45 is dispersed into the cytoplasm. Presumably, RCC1 protein plays a role in regulating the onset of chromosome condensation, at the level of transcription or of mRNA maturation.

scholarly journals Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

scholarly journals Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system

1985 ◽  
Vol 230 (1) ◽  
pp. 133-141 ◽  
Author(s):  
L P Chung ◽  
D R Bentley ◽  
K B Reid
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Protein A ◽  
Classical Pathway ◽  
Amino Acid Residues ◽  
Cloned Cdna

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3′ non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.

scholarly journals Strain-specific and common epitopes of gonococcal pili.

1984 ◽  
Vol 160 (1) ◽  
pp. 208-221 ◽  
Author(s):  
J B Rothbard ◽  
R Fernandez ◽  
G K Schoolnik
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Synthetic Peptides ◽  
Cyanogen Bromide ◽  
Sequence Variation ◽  
Antigenic Structure ◽  
Carboxyl Terminus ◽  
Specific Epitope ◽  
Absorption Studies ◽  
Strain Ms11

The antigenic structure of gonococcal pilin, strain MS11 (Tr), was investigated by assaying the binding of antisera engendered by intact pili from strains MS11 and R10 and their two major cyanogen bromide-generated fragments, CNBr-2 (residues 9-92) and CNBr-2 (residues 93-159), to synthetic peptides corresponding to the amino acid sequence of MS11 pilin. Four peptides were synthesized corresponding to regions of sequence variation between MS11 and R10 gonococcal pilin. Antisera against the homologous pilus filament and against its CNBr-3 fragment bind peptides equivalent to residues 121-134 and 135-151, which comprise the 30 amino acid disulfide loop near the carboxyl terminus of the protein. Heterologous pili antisera did not bind these peptides. Absorption studies proved that each peptide contained an independent, strain-specific epitope. Synthetic peptides corresponding to regions of identical sequence between MS11 and R10 pilin were used in similar binding experiments to localize a weakly immunogenic, common determinant between residues 48 and 60. less than 15% of the antibodies raised against intact pili were directed at this site. Antisera raised against MS11 or R10 CNBr-2 bind a separate peptide, residues 69-80. This region is immunogenic only as a fragment, not in the intact pilus filament.

scholarly journals Biochemical and Immunological Characterization of MP65, a Major Mannoprotein Antigen of the Opportunistic Human PathogenCandida albicans

2000 ◽  
Vol 68 (2) ◽  
pp. 694-701 ◽  
Author(s):  
Maria J. Gomez ◽  
Bruno Maras ◽  
Alessandra Barca ◽  
Roberto La Valle ◽  
Donatella Barra ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Synthetic Peptides ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Amino Acid Sequences ◽  
Antigenic Determinants ◽  
Cell Mediated Immunity ◽  
Antigenic Peptides

ABSTRACT In the search of the antigenic determinants of a 65-kDa mannoprotein (MP65) of Candida albicans, tryptic fragments of immunoaffinity-purified MP65 preparations were tested for their ability to induce lymphoproliferation of human peripheral blood mononuclear cells (PBMC). Five major peptides (T1 to T5) were shown to induce a vigorous proliferation of PBMC from the majority of the eight healthy human subjects tested. With the use of synthetic peptides, critical amino acid sequences of the two most immunoactive (T1 and T2) peptides were determined. Similar to what was found for the MP65 molecule, no PBMC multiplication was induced by the antigenic peptides in cultures of naive cord blood cells. The amino acid sequence analysis of tryptic and chymotryptic peptides of MP65 demonstrated a substantial homology with the deduced sequences of two cell wall proteins ofSaccharomyces cerevisiae, encoded by the genesYRM305C and YGR279C. However, the antigenic peptides were those showing the least similarity with the corresponding regions of the above proteins. In particular, the lymphoproliferation-inducing sequence of the T1 peptide scored only 20% identity with the homologous regions of S. cerevisiaeproteins. Besides disclosing the amino acid sequence of MP65, this study provides an initial characterization of some of its antigenic determinants, as well as of synthetic peptides of potential use to detect specific immune responses against MP65, a major target of anticandidal cell-mediated immunity in humans.

Occludin confers adhesiveness when expressed in fibroblasts

1997 ◽  
Vol 110 (9) ◽  
pp. 1113-1121 ◽  
Author(s):  
C.M. Van Itallie ◽  
J.M. Anderson
Keyword(s):  
Cell Adhesion ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tight Junctions ◽  
Synthetic Peptides ◽  
Integral Membrane Protein ◽  
Cell Contact ◽  
Suspended Cell ◽  
Extracellular Loops ◽  
Cell Cell

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

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