Biomedical subjects

O Hyrien

Publications and source records attributed to O Hyrien.

23 records · Page 2Linked to original sources

PubMed1989

Large inverted duplications in amplified DNA of mammalian cells form hairpins in vitro upon DNA extraction but not in vivo.

I have analysed the duplex-to-hairpin transition of large inverted duplications with a short asymmetric center which are found in the amplified DNA of two mammalian cell lines resistant to cytotoxic drugs. Psoralen crosslinking experiments establish that this transition does not occur in vivo, but takes place in a significant portion of the palindromes during genomic DNA purification, at the phenol-chloroform extraction step. The introduction of single strand nicks in the DNA by gamma irradiation prior to its purification does not prevent hairpin formation but instead facilitates it. These results show that the rate-limiting step of the duplex-to-hairpin transition does not require negative supercoiling, and that transient melting of large segments of cellular DNA occurs during phenol-chloroform extraction. I also show, and discuss the fact, that only cellular DNA, and not cloned palindromic DNA, is able to undergo hairpin formation by this mechanism. These results bear practical implications for the study of inverted repeated DNA sequences in eukaryotic cells.

AMP Deaminase↗

PubMed1988

The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

The amplified DNA of HC50474, a Chinese hamster fibroblast cell line selected in three steps for high resistance to coformycin, consists chiefly of 150 copies of a large inverted duplication including the adenylate deaminase gene. Most if not all of these units are more than 2 x 120 kb long. The inverted duplication was first detected in the cells recovered from the second selection step, at the same chromosomal location as the first step amplified units. Its formation and amplification appear to be coupled since the second step cell line already contained 40 copies of this novel structure. Reamplification of the inverted duplication occurred at the third step of selection concomitant with the loss of amplified DNA acquired during the first step. The head-to-head junction has been formed by recombination within a recombinational hotspot described previously [Hyrien, O., Debatisse, M., Buttin, G. and Robert de Saint Vincent, B. (1987) EMBO J., 6, 2401-2408]. Sequences at the joint and in the corresponding wild-type region reveal that the crossover sites, one of which occurs in the putative promoter region of B2 repeat, are located at the top of significant stem-loop structures and that patchy homologies between the parental molecules on one side of the breakpoints allow alignment of these crossover sites. We present a model which explains the formation and amplification of this and other large inverted duplications by errors in DNA replication.

Animals↗

PubMed1987

A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA.

We have identified, in the amplified domain of adenylate deaminase (AMPD) overproducing Chinese hamster fibroblasts, a 2.6 kb recombinogenic DNA region which is frequently involved in amplification-associated rearrangements. The nucleotide sequence reveals a mosaic organization of four Alu-equivalent repeats of the B1 and B2 families and eight long A + T-rich DNA segments. Part of this region is enriched with long imperfect palindromes. The center of one palindrome contains a putative topoisomerase I cleavage site and this site defines the position of a novel junction which was formed by illegitimate recombination with anther A + T-rich DNA sequence located far apart on the amplified DNA. These findings and their significance are discussed in the context of related data from other systems and in the light of current models for eukaryotic DNA recombination, replication and organization.

AMP Deaminase↗

PubMed1986

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito.

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Animals↗

PubMed1986

Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase.

Four genes encoding proteins designated as W, X, Y1, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Y1 gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes--in a particular order--in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.

AMP Deaminase↗