Molecular analysis of human X-linked diseases.
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Chromosome Mapping↗
Biomedical subjects
S M Forrest
Publications and source records attributed to S M Forrest.
Molecular analysis of X-linked diseases.
Explore the source record for details and available documents.
Fragile X Syndrome↗
Characterization of an osteoblast-like clonal cell line which responds to both parathyroid hormone and calcitonin.
The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.
Acid Phosphatase↗
Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12.
The tyrP gene which codes for a component of the tyrosine-specific transport system of Escherichia coli has been cloned on a 2.8-kilobase insert into plasmid pBR322. Transposon mutagenesis, using Tn1000, indicates that the tyrP+ gene is at least 1.1 kilobase in length. Labeling of the tyrP protein in maxicells with [35S]methionine indicates an apparent molecular weight of ca. 24,500. Sedimentation analysis reveals that the tyrP protein is associated with the cell membrane and is not free in the cytoplasm or periplasm. Strains with many copies of the tyrP+ gene show an enhanced uptake of tyrosine, but the expression of the system is still modulated by tyrosine and phenylalanine in the presence of the tyrR+ regulator protein. Accumulated radioactive tyrosine is rapidly effluxed by the addition either of energy uncouplers or of excess nonradioactive tyrosine, indicating that the transport system is energized by the proton motive force and that the internal pool is readily exchangeable. The effect of increasing expression of the tyrP gene on the steady-state level of tyrosine accumulated by cells indicates that although the transport system may be dependent on the proton motive force to drive uptake, the system never reaches thermodynamic equilibrium with it.
Base Sequence↗
Atomic force microscopy imaging of DNA-cationic liposome complexes optimised for gene transfection into neuronal cells.
BACKGROUND: Cationic liposomes represent an important gene delivery system due to their low immunogenicity, but are relatively inefficient, with optimisation of DNA-liposome complexes (lipoplexes) for transfection necessary for each cell type of interest. There have been few studies examining optimisation in neuronal cell types or determining how the structure of lipoplexes affects transfection efficiency. METHODS: Four commercially available cationic liposome formulations were used to optimise transfection efficiency in neuronal cells. The DNA to liposome ratio and the amount of DNA used in transfections were varied. Transfection efficiency was determined by the percentage of cells positive for the micro-galactosidase reporter gene product. The structure of lipoplexes was studied using atomic force microscopy. Lipoplexes were characterised further using dynamic light scattering to determine size and fluorescence techniques to show DNA compaction. RESULTS: Optimal transfection conditions were found to differ between immortalised cell lines and primary cells. High transfection efficiencies in immortalised cell lines were achieved predominantly with multivalent cationic liposomes while primary neuronal cells showed optimal transfection efficiency with monovalent cationic liposomes. The structure of lipoplexes was observed with atomic force microscopy and showed globular complexes for multivalent cationic liposomes, while monovalent liposomes gave less compact structures. In support of this finding, high levels of DNA compaction with multivalent liposomes were observed using fluorescence quenching measurements for all DNA to liposome ratios tested. One monovalent liposome showed increasing levels of compaction with increasing liposome amount. Dynamic light scattering showed little change in complex size when the different lipoplexes were studied. CONCLUSIONS: Optimisation of transfection efficiency was different for cell lines and primary neurons. Immortalised cells showed optimal transfection with multivalent liposomes while primary neurons showed optimal transfection with monovalent liposomes. The charge ratio of the monovalent liposome was below one, suggesting a different mechanism of lipoplex binding and uptake in primary neurons. The structure of lipoplexes, as
Animals↗
CpG hotspot causes second mutation in codon 408 of the phenylalanine hydroxylase gene.
A new mutation has been identified in exon 12 of the gene encoding phenylalanine hydroxylase at codon 408. The single base change from guanine to adenine changes the amino acid arginine to glutamine; thus, the mutation is defined as R408Q. This codon is the site of a mutation known to causes phenylketonuria. Both these mutations are located at the same CpG site.
Codon↗
Preferential deletion of exons in Duchenne and Becker muscular dystrophies.
Duchenne and Becker muscular dystrophy (DMD and BMD) genes are located in Xp21 on the short arm of the X chromosome. DMD patients display a much more severe clinical course than BMD patients, and yet about 10% of cases of each have been reported to have deletions for parts of the gene. Using a complementary DNA subclone of the DMD gene we have screened 66 DMD and BMD patients who had not previously shown deletions with the probes then available. Fifteen patients have a deletion of this part of the gene, indicating a higher deletion frequency in this region (22%). Exons were deleted in five severely affected DMD patients and in ten BMD patients. Significantly, most of these deletions begin in the same region of the cDNA, which implies that there is a common mechanism for the generation of many of these mutations. An apparently identical deletion in one family gave classical BMD in two brothers (presenting in their teens) and only very mild muscle weakness in their 86-year-old great-great-uncle. Taking these data together with data using the probes previously published, we are able to detect deletions directly in 40% of our families requiring antenatal diagnosis or carrier detection.
Adolescent↗