The polymorphic marker DXS304 is within 5 centimorgans of the fragile X locus

The fragile X syndrome, which is the most common cause of inherited mental retardation, poses important diagnostic problems for genetic counseling. The development of diagnostic strategies based on DNA analysis has been impaired by the lack of polymorphic markers very close to the disease locus. Here we report that the polymorphic probe U6.2 (locus DXS304) is much closer to the fragile X locus than all the previously reported markers. A recombination fraction of 0.02 between DXS304 and the fragile X locus was estimated by multipoint linkage analysis (confidence interval 0.002 to 0.05). Our data suggest that DXS304 is distal to the fragile X locus. This marker thus represents a major improvement for carrier detection and prenatal diagnosis in fragile X families.     

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets

We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.     

Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells.

The bovine papillomavirus type 1 (BPV-1) genome replicates as a plasmid within the nuclei of BPV-1-transformed murine C127 cells at a constant multiple copy number, and spontaneous amplification of the viral DNA is rarely observed. We report here that a mutant BPV-1 plasmid within a contact-inhibited C127 cell line replicated as a stable multicopy plasmid in exponentially growing cells but amplified to a high level in confluent cell culture. In situ hybridization analysis revealed that most of the mutant viral DNA amplification occurred in a minor subpopulation of cells within the culture. These consisted of giant nondividing cells with greatly enlarged nuclei, a cell form which was specifically induced in stationary-phase cultures. These observations indicated that expression of a viral DNA replication factor was cell growth stage specific. Consistent with this hypothesis, considerable amplification of wild-type BPV-1 DNA associated with characteristic giant cell formation was observed in typical wild-type virus-transformed C127 cultures following a period of growth arrest achieved by serum deprivation. Further observations indicated that induction of the giant-cell phenotype was dependent on BPV-1 gene expression and implicated a viral E1 replication factor in this process. Moreover, heterogeneity in virus genome copy numbers within the giant-cell population suggested a complex regulation of induction of DNA synthesis in these cells. It appears that this process represents a mechanism employed by the virus to ensure maximal viral DNA synthesis within a growth-arrested cell. Fundamental questions concerning the integration of the virus-cell control circuitry in proliferating and resting cells are discussed.     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.