Biomedical subjects

B Lapeyre

Publications and source records attributed to B Lapeyre.

36 records · Page 2Linked to original sources

PubMed1987

RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence.

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.

Amino Acid Sequence↗

PubMed1987

Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence.

Nucleolin (also called C23) is the major nucleolar protein of exponentially growing eukaryotic cells. It is found associated with intranucleolar chromatin and preribosomal particles. Through use of a polyclonal antiserum, nucleolin cDNA clones were isolated from a Chinese hamster ovary cell library constructed in the expression vector lambda gt11. The isolated cDNAs encoded a polypeptide containing 679 residues of the 713 amino acids of nucleolin. The amino acid sequence presents several unusual features: in particular, repetitive sequences are found at both ends of the molecule. A repeat, Hy-Thr-Pro-Hy-Lys-Lys-Hy-Hy, in which Hy is a nonpolar residue, is found six times in the NH2-end proximal portion, followed by three acidic stretches containing 25, 25, and 33 glutamic acid or aspartic acid residues. Four potential phosphorylation sites (serines) are also observed in this region. The COOH-terminal proximal portion of the protein carries a glycine-rich region with fairly regularly interspersed phenylalanine and dimethylarginine residues. The two terminal portions of the molecule exhibit unique potential secondary structures: alpha-helix (NH2 terminus) and extended (COOH terminus). The central region exhibits alternating hydrophobic and hydrophilic stretches. Five potential N glycosylation sites are detected. The structure of this protein may reflect two functions in preribosome biogenesis: interaction with chromatin (NH2 terminus) and with preribosomes (COOH terminus).

Amino Acid Sequence↗

PubMed1987

[Rupture of the anterior cruciate ligament: a frequently unrecognized cause of failure of unicompartmental knee prostheses. Apropos of a series of 79 Lotus prostheses with a follow-up of more than 5 years].

Out of 386 unicompartmental prostheses inserted in the Orthopaedic Centre in Dracy Le Fort between 1977 and 1986, 79 cases in 68 patients with a follow-up equal to or more than five years and a mean of seven years were able to be reviewed. This study showed that 75 per cent of knees had a satisfactory result and 25 per cent were failures. Apart from well-recognised causes of failure such as rheumatoid arthritis, overcorrection or the use of a six millimetre tibial plateau, the authors discovered a frequent cause not clearly recorded in the literature--that is anterior cruciate laxity. This situation was found retrospectively in the pre-operative weight-bearing radiographs. Out of 15 knees showing a pre-operative laxity equal to or greater than 10 mm in the lateral radiograph, 13 were failures and 10 were operated on again after a mean of three-and-a-half years. An investigation of anterior cruciate laxity should be made systematically by weight-bearing lateral radiographs, if necessary with stress views, before recommending a unicompartmental prosthesis.

Aged↗

PubMed1986

Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa).

By a combination of protein chemistry and recombinant DNA methods a glycine-rich region was found to be located near the carboxyl terminus of the nucleolar specific phosphoprotein, nucleolin, from Novikoff hepatoma (protein C23) and Chinese hamster ovary cells (100-kDa nucleolar protein). A sequence of 192 amino acid residues was derived from partial sequences of cyanogen bromide and N-bromosuccinimide fragments of protein C23 and deduced protein sequence from Chinese hamster ovary cell 100-kDa cDNA sequences. The 66 residues sequenced by protein methods were identical to the corresponding residues deduced by DNA sequencing. The multiple residues of NG,NG-dimethylarginine (DMA) contained in the nucleolin polypeptide were found to be limited to a segment of less than 10 kDa near the carboxyl-terminal end of the protein. This segment also contained internally repeated sequences (e.g. 7 copies of the sequence Gly-Gly-Arg-Gly-Gly were found) which were unrelated to sequences closer to the amino-terminal end. Most arginine residues in this region were surrounded by 2 or 3 glycine residues and were relatively close in sequence to phenylalanine residues.

Amino Acid Sequence↗

PubMed1986

Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence.

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.

Amino Acid Sequence↗

PubMed1985

Cloning of cDNA encoding a 100 kDa nucleolar protein (nucleoline) of Chinese hamster ovary cells.

Nucleoline (100 kDa) is the major nucleolar protein in exponentially growing cells that behaves like a nucleolar organizer protein and plays a key role in rDNA transcription and prerRNA processing. We reported the isolation of 5 cDNA clones by probing a cDNA library, constructed in the expression vector lambda gt11, with a polyclonal serum raised against nucleoline. A new immunoassay, using hybrid proteins (beta gal-cDNA encoded protein) was developed to establish that the isolated cDNAs encoded parts of nucleoline. A further confirmation resulted from the sequence comparison between the cDNA encoded peptide and a 42 aa peptide isolated from rat nucleoline (1). The 5 cDNAs overlapped extensively and covered more than 90% of a full length cDNA. By probing a Northern blot with the 100 kDa cDNA, a 2650 nucleotide polyA+ RNA was detected that contained just enough information to code for nucleoline.

Amino Acid Sequence↗

PubMed1985

A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein.

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.

Animals↗

PubMed1983