A large deletion within the protein 4.1 gene associated with a stable truncated mRNA and an unaltered tissue-specific alternative splicing.

Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed a greater than 70-kb genomic deletion, beginning within intron 1 and ending within a 1.3-kb region upstream from exon 13. This deletion encompassed both erythroid and nonerythroid translation initiation sites. It accounts for the largest deletion known in genes encoding proteins of the red blood cell membrane. The corresponding mRNA was shortened by 1727 bases, due to the absence of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a similar pattern in lymphoblastoid cells as in reticulocytes. Differential splicing of exons within the undeleted region remained regulated in a tissue-specific manner. Exons 14, 15, and 17a were absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was present in reticulocytes but absent from lymphocytes. Thus, differential splicing on a local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.

Production of polyclonal and monoclonal antibodies for specific detection of nitrosation-proficient denitrifying bacteria in biological fluids.

Polyclonal and monoclonal antibodies were raised against the nitrosating enzyme previously isolated and purified from a denitrifying bacteria Pseudomonas aeruginosa. Using the polyclonal antibodies, a preliminary ELISA test was set up which allowed the detection of the nitrosating enzyme in 2 ml urine samples with a minimal total bacteria count of greater than or equal to 10(5) cells/ml. The use of such a rapid immunological screening test in clinical settings should ascertain whether individual subjects at higher risk for cancer of the stomach or bladder harbour more nitrosation-proficient microorganisms in their microflora. The availability of monoclonal antibodies provides a tool for studying the mechanisms of bacteria-mediated nitrosamine formation from precursor amines.

Effects of anti-alpha monoclonal antibodies on initiation and elongation by the Escherichia coli RNA polymerase.

Anti-alpha monoclonal antibodies have been used to investigate the role of the alpha subunit of RNA polymerase in initiation and elongation. The four inhibitory monoclonal antibodies studied strongly inhibit cAMP receptor protein-dependent initiation with lac P+ and partially inhibit initiation directed by the lac UV5 promoter. The data suggest that the epitopes to which each of the monoclonal antibodies bind may be proximal to the contact domain between cAMP receptor protein and RNA polymerase. Recycling of RNA polymerase through the initiation process is slower in the presence of an inhibitory monoclonal antibody. Once a 9-nucleotide-long transcript has formed, incubation with the anti-alpha monoclonal antibody does not affect subsequent elongation. The monoclonal antibodies still bind to the elongation complex as indicated by sedimentation of the complex formed after incubation with Staphylococcus aureus cells (immunoprecipitin). These results suggest that the resistance of the elongation complex to these antibodies is not a consequence of their inability to bind to RNA polymerase. Only one of the alpha subunits may be involved in the initial process of transcription, and the antigenic domain of this subunit appears to be occluded by the nascent transcript present in the elongation complex.