Use of fluorescence threshold triggering and high-speed flow cytometry for rare event detection.

A simple rare event detection method utilizing dual-parameter flow cytometry is described, which allows quantitation of specific cellular events at the level of two cells in 10(7) total cells. Using a standard unmodified single laser flow cytometer sampling at a rate of 25,000 events/sec and a fluorescence discriminator, 10(7) total cells are processed in 7 min. The assay involves precise characterization of instrument flow rates to calculate total events processed by the cytometer rather than accumulate total events in computer memory. This method of detecting rare events is demonstrated by using a model system of breast cancer cells labeled with a metabolically activated dye and serially diluted into normal peripheral blood. Potential applications include validation of methods to detect minimum residual disease following myeloablative therapy, detection of any remaining tumor cells following purging methods, and validation of methods to detect circulating fetal cells in maternal blood.

The developmental switch in embryonic rho-globin expression is correlated with erythroid lineage-specific differences in transcription factor levels.

During chicken embryogenesis, the rho-globin gene is expressed only in the early developmental stages. We have examined the mechanisms that are responsible for this behavior. The transcription of the rho-globin gene is strongly correlated with the presence during development of primitive erythroid lineage cells, consistent with the idea that the expression of the rho-globin gene is restricted to that lineage. The "switching off" of rho-globin during development thus reflects the change from primitive to definitive cell lineages which occurs during erythropoiesis in chicken. We use transient expression assays in primary erythroid and other cells to show that the information for lineage- and tissue-specific expression of the rho-globin gene is contained in a 456 bp region upstream of the gene's translational start site. DNA-binding studies, coupled with analysis of the effect on expression of deletions and binding site mutations, were used to identify important control elements within this 456 bp region. We find that binding sites for the ubiquitous transcription factor Sp1, and the specific hematopoietic factor GATA-1, are crucial for expression of the gene in primitive erythroid cells. Quantitative analysis shows that nuclei of the primitive erythroid lineage contain 10-fold more of these factors than do the nuclei of definitive cells. We show that in principle these differences in factor concentration are sufficient to explain the lineage-specific behavior that we observe in our assays. We suggest that this may be an important part of the mechanism for lineage-restricted rho-globin expression during chicken erythroid development. Similar mechanisms may be involved in regulation of other (but not all) members of the globin family.

Developmental regulation of globin gene expression.

We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the alpha- and beta-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the rho-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.

A model system for peptide hormone action in differentiation: interleukin 2 induces a B lymphoma to transcribe the J chain gene.

Physiological levels of a purified T cell hormone, interleukin 2 (IL-2), were found to stimulate a cloned murine B cell line (BCL1) to secrete pentamer IgM antibody. The peptide hormone acts at the cell surface via specific IL-2 receptors and induces changes in the 5' chromatin of the J chain gene that correlate with its transcription and with the production of the J chain protein required for pentamer IgM assembly. There was no effect of IL-2 on cell proliferation nor on mu heavy chain gene transcription. These results define a specific function for IL-2 in B cell differentiation. In addition, the IL-2/BCL1 system provides a model for examining the mechanism by which signals generated by hormone-receptor interaction are transmitted to the nucleus and regulate gene expression.

Accessibility of the promoter sequence in the J-chain gene is regulated by chromatin changes during B-cell differentiation.

The gene for the immunoglobulin M (IgM)-polymerizing protein, the J chain, is activated when the mature B cell is triggered to secrete pentamer IgM. Activation of the gene was found to be associated with chromatin changes in a 240-base-pair region at the 5' end of the gene. Analyses of lymphoid lines showed that the 5' region was resistant to nuclease digestion at the immature B-cell stage; it became slightly more accessible in mature B cells and cells at an early stage in the IgM response and then displayed an open, hypersensitive structure in IgM-secreting cells. In addition, analyses of normal, mitogen-stimulated lymphocytes showed that the open hypersensitive structure was coinducible with J-chain gene expression. These results suggest that the 5' chromatin changes precede transcription, making control sequences within the site accessible to regulatory factors.

Endonucleolytic activity that cleaves immunoglobulin recombination sequences.

An endonucleolytic activity has been identified in nuclear extracts of chick embryo bursa and mouse fetal liver cells. The activity introduces a double-strand cut in the vicinity of the recombination site of immunoglobulin joining gene segments. The cleavage occurs at the dinucleotide pair TG-AC. This activity is a good candidate for the putative endonuclease involved in recombination of the immunoglobulin variable, diversity, and joining regions. It is distinct from the endonuclease activities previously reported by others.