W Harriman | PubMed Health

PubMed1995

A rapid assay for detecting cellular TdT enzymatic activity.

We have developed a solid-phase assay for the quantification of terminal deoxynucleotidyl transferase (TdT) enzymatic activity in crude cellular extracts. Affinity-purified, polyclonal anti-TdT antibodies are bound to the wells of a microtiter plate, and TdT in extracts is then bound to the immobilized antibodies. The enzymatic activity of the antibody-bound TdT is measured directly in the wells of the microtiter plate. This method yields highly reproducible values, even with samples of low activity. Because it is also technically very simple, it is ideal for determining enzymatic activity for large numbers of clones with limited numbers of cells.

3T3 Cells↗

PubMed1993

Immunoglobulin class switch recombination.

A B lymphocyte that produces the immunoglobulin heavy (H) chain mu may switch to the production of another heavy chain class: gamma, epsilon, or alpha. Since the new heavy chain retains the original variable (V) region, antigenic specificity is maintained. The switch is accompanied by a large deletion of DNA at the heavy chain locus. To explain how this deletion is generated, three models have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, both by-products of looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch. One requirement for switching may be transcription of the constant (C) region to which the cell switches. The switch rearrangement is catalyzed by a switch recombinase, and the isolation of the components of this putative enzyme system is in progress. Although the switch deletion is an accepted fact, the discussion is enlivened by scenarios for switching without DNA rearrangement; such suggestions include processing at the RNA level and trans-splicing.

Animals↗