In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluoride membranes: Cleavage at tryptophan residues with 2-(2′-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine to obtain internal amino acid sequence

Juvenile nephronophthisis, an autosomal recessive cystic kidney disease, is the primary genetic cause for chronic renal failure in children. The gene (NPHP 1) for nephronophthisis type 1 has recently been identified. Its gene product, nephrocystin, is a novel protein of unknown function, which contains a src-homology 3 domain. To study tissue expression and analyze amino acid sequence conservation of nephrocystin, the full-length murine Nphp 1 cDNA sequence was obtained and Northern and in situ hybridization analyses were performed for extensive expression studies. The results demonstrate widespread but relatively weak NPHP 1 expression in the human adult. In the adult mouse there is strong expression in testis. This expression occurs specifically in cell stages of the first meiotic division and thereafter. In situ hybridization to whole mouse embryos demonstrated widespread and uniform expression at all developmental stages. Amino acid sequence conservation studies in human, mouse, and Caenorhabditis elegans show that in nephrocystin the src-homology 3 domain is embedded in a novel context of other putative domains of protein-protein interaction, such as coiled-coil and E-rich domains. It is concluded that for multiple putative protein-protein interaction domains of nephrocystin, sequence conservation dates back at least to Caenorhabditis elegans. The previously described discrepancy between widespread tissue expression and the restriction of symptoms to the kidney has now been confirmed by an in-depth expression study.

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Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA

Journal of Endocrinology ◽

10.1677/joe.1.06161 ◽

2005 ◽

Vol 186 (2) ◽

pp. 387-396 ◽

Cited By ~ 16

Author(s):

Kristien Vandenborne ◽

Simon A Roelens ◽

Veerle M Darras ◽

Eduard R Kühn ◽

Serge Van der Geyten

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Cdna Sequence ◽

Chicken Genome ◽

Amino Acid Sequence Homology ◽

Tertiary Structures ◽

Genome Database ◽

Releasing Hormone ◽

Translation Start

In this paper we report the cloning of the chicken preprothyrotropin-releasing hormone (TRH) cDNA and the study of its hypothalamic distribution. Chicken pre-proTRH contains five exact copies of the TRH progenitor sequence (Glu-His-Pro-Gly) of which only four are flanked by pairs of basic amino acids. In addition, the amino acid sequence contains three sequences that resemble the TRH progenitor sequence but seem to have lost their TRH-coding function during vertebrate evolution. The amino acid sequence homology of preproTRH between different species is very low. Nevertheless, when the tertiary structures are compared using hydrophobicity plots, the resemblance between chicken and rat prepro-TRH is striking. The cloning results also showed that the chicken preproTRH sequence includes neither a rat peptide spacer 4 (Ps4) nor a Ps5 connecting peptide. Comparison of the cDNA sequence with the chicken genome database revealed the presence of two introns, one in the 5′ untranslated region, and another downstream from the translation start site. This means that the gene structure of chicken preproTRH resembles the gene stucture of this precursor in mammals. Based on the cDNA sequence, digoxigenin-labelled probes were produced to study the distribution of preproTRH in the chicken brain. By means of in situ hybridization, preproTRH mRNA was detected in the chicken paraventricular nucleus (PVN) and in the lateral hypothalamus (LHy).

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The identification of tryptophan residues in proteins as oxidised derivatives during amino acid sequence determinations

FEBS Letters ◽

10.1016/0014-5793(79)81095-9 ◽

1979 ◽

Vol 104 (1) ◽

pp. 115-118 ◽

Cited By ~ 2

Author(s):

Adam S. Inglis ◽

Donald E. Rivett ◽

David T.W. McMahon

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Tryptophan Residues

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Human serum amyloid A protein. Complete amino acid sequence of a new variant

Biochemical Journal ◽

10.1042/bj2820615 ◽

1992 ◽

Vol 282 (2) ◽

pp. 615-620 ◽

Cited By ~ 31

Author(s):

C M Beach ◽

M C De Beer ◽

J D Sipe ◽

L D Loose ◽

F C De Beer

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Serum Amyloid A ◽

Allelic Variation ◽

Serum Amyloid ◽

Complete Amino Acid Sequence ◽

Amyloid A ◽

New Variant ◽

Serum Amyloid A Protein ◽

Tryptophan Residues

Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2′-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).

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Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2441 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2441-2453 ◽

Cited By ~ 74

Author(s):

D J Montell ◽

C S Goodman

Keyword(s):

Nervous System ◽

In Situ Hybridization ◽

Amino Acid ◽

Amino Acid Sequence ◽

Cdna Sequence ◽

Drosophila Embryogenesis ◽

Substrate Adhesion ◽

Genes Encoding ◽

Developing Nervous System

In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system.

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