Beta-galactosidase and chloramphenicol acetyltransferase assays in 96-well plates.
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Animals↗
Biomedical subjects
C Bignon
Publications and source records attributed to C Bignon.
cDNA cloning and genomic analysis of a new multigene family sharing common phylogenetic and expression profiles with the laminin receptor gene.
We have isolated a human laminin receptor (LR) cDNA which bears a different sequence in its 5' end with regard to the corresponding region in the regular LR mRNA. This different sequence hybridizes to a 1 kb mRNA. We have cloned a 740 bp cDNA for this transcript (cDNA 48-1). Search on sequence data bases revealed no sequence homology with known messengers or proteins. Using cDNA 48-1 in a simplified version of the protocol with which we had previously characterized the LR gene as a member of a retrogene family in mammals, we show in the present paper that the gene of this new transcript exhibits phylogenic, expression and amplification features that strickingly recall those of the LR gene.
Amino Acid Sequence↗
Laminin expression by two clones isolated from the colon carcinoma cell line LoVo that differ in metastatic potential and basement-membrane organization.
In the present report we describe the characteristics of 2 clones, E2 and C5, isolated from the human colon adenocarcinoma cell line LoVo. When grafted to immunosuppressed newborn rats, these clones formed tumors that varied with regard to differentiation rate, basement-membrane organization and lung metastatic potential. Production and distribution of laminin by E2, C5 and related tumors was studied by immunohistochemistry with an anti-laminin monoclonal antibody 4C12 (MAb 4C12). In lowly metastatic E2-derived tumors, strong regular stainings were observed which were strictly peri-tumoral and corresponded to the basal lamina. Since the antibody interacted with human laminin (the graft) but not with rat laminin (the host), this result indicated that basement-membrane laminin was supplied mainly by tumor-cell synthesis. In highly metastatic C5-derived tumors, the staining obtained with MAb 4C12 was peri-cellular and unorganized. Laminin synthesis by E2 and C5 cells in sub-cultures or soon after dissociation from explanted tumors was studied by metabolic labelling with 35S-methionine under steady-state conditions followed by immunoprecipitation and SDS-PAGE. High-molecular-weight laminin comprised by disulfide-linked A and B chains, i.e., heterotrimeric laminin, was found in cell lysates and in the secretion medium of cell lines and tumor cells. In addition, B1B2 dimers and free B chains were observed in cell lysates. Quantitatively, laminin expression by E2 and C5 clones or tumor cells was not significantly different. These findings suggest that basement-membrane defects in invasive clone LoVo C5 were not due to laminin under-expression.
Animals↗
A simple vacuum transfer device for nucleic acids.
We present a new vacuum device intended for transferring nucleic acids from agarose gels onto hybridization membranes. The apparatus is drawn so that plastic sheets serve as substitutes for the elaborate, but cumbersome and unnecessary, locking systems mounted on all the commercial blotters. This substitution results in (1) a much more easy and rapid set-up of the transfer and (2) a very simple framework allowing the possibility of low-cost manufacturing.
Blotting, Northern↗
Improved sequencing of "mini-prep" double-stranded templates using a multiwell microplate system.
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DNA↗
Genomic analysis of the 67-kDa laminin receptor in normal and pathological tissues: circumstantial evidence for retroposon features.
We have cloned two cDNAs for the human 67-kDa laminin receptor (LR). In the present report we show that these clones hybridize to many restriction fragments in Southern experiments in human. This particular pattern is accounted for by the presence of up to 16 and 21 copies of the laminin receptor gene per haploid genome in human and mouse, respectively. In contrast, a single gene copy is found in chicken. Chromosomal localization reveals four main loci: LAMRP1, laminin receptor pseudogene 1 (Chr 3); LAMRP2, laminin receptor pseudogene 2 (Chr 12); LAMRP3, laminin receptor pseudogene 3 (Chr 14); LAMRP4, laminin receptor pseudogene 4 (Chr X). Comparison of our experimental results to the known features of processed retropseudogenes enabled us to conclude that the LR gene belongs to a retroposon family in mammals.
Animals↗
Laminin biosynthesis in the extracellular matrix-producing cell line PFHR9 studied with monoclonal and polyclonal antibodies.
The biosynthesis of the basement-membrane glycoprotein laminin in the mouse teratocarcinoma cell line PFHR9 was studied by immunoelectron microscopy and pulse-chase experiments using monoclonal and polyclonal antibodies. By immunoelectron microscopy, most of the protein was found to be aggregated on the outer cell surface. Cytoplasmic stainings were rare and were located next to the intracellular side of the plasma membrane. Sequential immunoprecipitations of cell extracts with a monoclonal antibody (4C12) sensitive to the laminin native conformation and with a polyclonal antibody enables laminin, the B1 subunit and a 410 kDa molecule to be distinguished. Most of the laminin is of the A(B1B2) type, and the 410 kDa molecule appears to be a B1B2 heterodimer. The assembly of laminin from subunits is completed in less than 1 h, and B chains are incorporated via the formation of the B heterodimers. The B2 and A chains are not found as free forms, so their levels appear to be the rate-limiting factors for the assembly of the dimers and laminin respectively. The formation of an uncross-linked A(B1B2) complex as a short-lived intermediate in the biosynthetic process is possible. Together with immunoelectron microscopy, the present study suggests that the protein is rapidly exported after assembly to accumulate on the outer side of the cell membrane. The biosynthesis of laminin in the PFHR9 cell line appears to be similar to that in other matrix-producing cell lines.
Animals↗
[Laminin: biosynthesis, structure and functions].
Biosynthesized by epithelial, endodermal and Swann type cells, laminin (Lam) is a cross shaped multifunctional glycoprotein formed by the multimeric assembly of subunits which result from the activation of several genes. In vivo, depending on its location, because of its adhesive properties and multivalent affinities, Lam is in association as a part of supramolecular complexes together with compounds of the plasma, the basement membrane and the cell coat. In the basement membrane (MB) Lam has structural and functional roles. It may also be adsorbed on the cell coat or secreted. It is interacting with epithelial cells by the way of a plasma membrane receptor and has a role to play in cellular differentiation and proliferation. Lam is a molecular link of epithelial cells to MB. These features implicate the molecule in organogenesis, embryogenesis and post-traumatic healing. As a structural component of MB and as an attachment factor, Lam is involved: 1 in tumoral invasion which allows metastatic spreading, 2 in homing because metastatic cell display an increased receptivity to the molecule. The study of Lam expression, Lam receptivity and their factors of control should lead to a better understanding of the biochemical and molecular basis of differentiation, embryogenesis, organogenesis and metastasis.
Basement Membrane↗
Quantitative in situ hybridization of 3H-labeled complementary deoxyribonucleic acid (cDNA) to the messenger ribonucleic acid of thyroglobulin in human thyroid tissues.
Thyroglobulin (Tgb) mRNA content was studied in human thyroid tissues using liquid hybridization and in situ hybridization. Liquid hybridization revealed no differences in mRNA content, except in the case of colloid adenoma in which a lower amount of Tgb mRNA was found. Conditions for quantitative in situ hybridization of [3H]DNA complementary to the mRNA of Tgb are described. In situ hybridization allowed correlation of the morpho-functional state of the follicles and their content of Tgb mRNA.
Adenoma↗
Modulable Southern and Northern blot hybridization apparatus for routine screening of gene amplification and expression.
An apparatus for Northern and Southern blot hybridization is described. It allows from one to twenty-four blots to be processed at the same time, with different probes. All the pre-, post- and hybridization steps are performed without handling the filters and the experimenter is totally protected from beta radiations. The development of such modulable materials has become necessary since Southern and Northern techniques are becoming routine assays in hospitals, particularly in the field of oncology, in prognosis and for hereditary diseases, as an antenatal diagnosis procedure.
Blotting, Northern↗