[Retropubic radical prostatectomy].
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Adenocarcinoma↗
Biomedical subjects
L Gagnier
Publications and source records attributed to L Gagnier.
Two novel mutations in the arylsulfatase A gene associated with juvenile (R390Q) and adult onset (H397Y) metachromatic leukodystrophy.
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Adult↗
A protocol for detection of mitochondrial DNA deletions: characterization of a novel deletion.
OBJECTIVES: To develop a protocol capable of identifying deletions in mitochondrial DNA and use it to identify the breakpoints of a mtDNA deletion in a patient with chronic progressive external ophthalmoplegia (CPEO). DESIGN AND METHODS: Deletions in mtDNA were identified by a combination of long range PCR and Southern blotting. The precise breakpoints were determined by automated DNA sequencing. RESULTS: A series of DNA samples from patients with suspected mitochondrial disease was subjected to a protocol, which combines long range PCR and Southern blotting. We found a unique deletion in a patient with CPEO and we identified the precise location of this deletion through DNA sequencing. CONCLUSIONS: Long range PCR has the advantages of speed, minimal samples requirements, and sensitivity. Southern blotting is better able to evaluate heteroplasmy and detect duplications. We suggest a protocol that enables us to identify precisely the breakpoints in a unique mutation of mtDNA in a patient with CPEO.
Adolescent↗
Two new polymorphisms in the arylsulfatase A gene and their haplotype associations with normal, metachromatic leukodystrophy and pseudodeficiency alleles.
Linkage disequilibrium exists between metachromatic leukodystrophy (MLD) and pseudodeficiency mutations and selected polymorphisms within the arylsulfatase A gene. We have identified 2 new polymorphic NlaIII sites, NlaIII1 and NlaIII2, in the gene that, when used in combination with the known BsrI and BamHI polymorphisms, extends the haplotype associations of the pseudodeficiency and the most common infantile onset MLD alleles. Fixed haplotypes have also been established for 3 other recurring MLD mutations, ala212val, pro426leu, and thr274met. The NlaIII2 site is relatively rare and was found only in association with the pseudodeficiency variant carrying the glycosylation site mutation alone.
Alleles↗
Metachromatic leucodystrophy in three families from Nova Scotia, Canada: a recurring mutation in the arylsulphatase A gene.
Metachromatic leucodystrophy (MLD) is a lysosomal storage disease resulting from a deficiency of arylsulphatase A. We have identified a child with infantile onset MLD who is homozygous for an A212V mutation, a mutation previously reported but not further characterised. We have introduced this mutation into an arylsulphatase A expression vector by site directed mutagenesis. Transient expression of this mutant plasmid in COS cells yields very low levels of arylsulphatase A activity consistent with the patient's phenotype. The arylsulphatase A pseudodeficiency also segregates in this family causing difficulty in interpreting enzyme levels in the absence of DNA data. Two other patients from the same province, also carrying the A212V allele, have juvenile and adult onset MLD and are heterozygous for P426L ("A" allele) and I179S alleles respectively, known late onset alleles.
Adult↗
Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.
The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.
Bacterial Proteins↗
P1 plasmid partition: binding of P1 ParB protein and Escherichia coli integration host factor to altered parS sites.
The Escherichia coli integration host factor (IHF) participates in P1 plasmid partition by assisting the interaction of P1 ParB protein with its specific site, parS. Together they form an extremely high-affinity protein-DNA complex, in which parS DNA is wrapped around a core of ParB and IHF protein in a precise three-dimensional conformation. We have investigated the interaction of ParB and IHF with mutant DNA sites, to examine protein specificity and cooperativity. The results indicate that ParB specifically recognizes two separate types of sequence repeats in its minimal binding site in one half of the parS site. The affinity of ParB or IHF for parS is much greater in the presence of the other protein. Mutations that decrease ParB or IHF binding to parS have relatively minor defects in vivo, because each protein still binds well to parS in the presence of the other protein. We observed that ParB acts better when provided in cis than in trans to parS in vivo. Our experiments suggest that in vivo, the local concentration of ParB protein near the plasmid is high, so that ParB can act reasonably well to promote partition in cells without IHF. However, this activity is lower than in wild-type cells, indicating that IHF is essential for long-term plasmid stability.
Bacterial Proteins↗
The P1 plasmid partition complex at parS. II. Analysis of ParB protein binding activity and specificity.
The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF). ParB binds to regions of parS that flank the IHF binding site. In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns. Methylation protection and interference experiments were performed on supercoiled plasmids. Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS. These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS. ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity. The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone. The stimulation by IHF is strongly influenced by helical phasing. These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site.
Bacterial Proteins↗