Andrew Beavis

The Molecular Characterization of the Fetal Stem Cell Marker AA4

We have identified and characterized the stem cell antigen AA4. This molecule is a type I transmembrane protein whose overall structure suggests a role in cell adhesion. During fetal ontogeny (days 9-14 of development), AA4 is expressed in three major cell types: vascular endothelial cells, aorta-associated hematopoietic clusters, and primitive fetal liver hematopoietic progenitors. In the adult, AA4 is abundant in lung, heart, and whole bone marrow. In the adult hematopoietic compartment, aa4 transcripts are present in bone marrow CD34(-/lo) Lin- Sca-1+ c-Kit+ and CD34hi Lin- Sca-1+ c-Kit+ stem and progenitor cell subsets. Our observations suggest that AA4 plays a role in cell-cell interactions during hematopoietic and vascular development.

Use of a membrane-localized green fluorescent protein allows simultaneous identification of transfected cells and cell cycle analysis by flow cytometry

The simultaneous detection of the green fluorescent protein (GFP) and DNA content using propidium iodide (PI) by flow cytometry is made difficult because of the unique nature of these 2 fluorogenic reagents. For PI to enter cells efficiently and to stain DNA quantitatively, the cells must first be permeabilized; ethanol treatment is a routine method to achieve this. However, this permeabilization step causes GFP, which is normally found in the cytoplasm, to leach out of the cells. Although the use of paraformaldehyde-based fixatives allows GFP to be maintained in cells and retain its fluorescence even after ethanol permeabilization, the protocol we commonly employ results in inefficient PI staining and poor quality DNA histograms. To circumvent these difficulties, we have employed a GFP-fusion protein which localizes to the cellular membrane and as such is retained in cells upon ethanol permeabilization without prior fixation. This allows the GFP signal to be detected in cells treated with ethanol in preparation for PI staining and cell cycle analysis. This property facilitates the use of GFP as a marker for transfected cells in experiments designed to characterize the effects of ectopic expression of cellular or viral genes on cell cycle progression.

A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism.

ABSTRACT A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.

TCR Specificity Dictates CD94/NKG2A Expression by Human CTL

Activating and inhibitory CD94/NKG2 receptors regulate CTL responses by altering TCR signaling, thus modifying antigen activation thresholds set during thymic selection. To determine whether their expression was linked to TCR specificity, we examined the TCR repertoire of oligoclonal CTL expansions found in human blood and tissues. High-resolution TCR repertoire analysis revealed that commitment to inhibitory NKG2A expression was a clonal attribute developmentally acquired after TCR expression and during antigen encounter, whereas actual surface expression depended on recent TCR engagement. Further, CTL clones expressing sequence-related TCR, and therefore sharing the same antigen specificity, invariably shared the same NKG2A commitment. These findings suggest that TCR antigenic specificity dictates NKG2A commitment, which critically regulates subsequent activation of CTL.

Complementation of Myc-dependent cell proliferation by cDNA expression library screening.

The targeted knockout of the c-myc gene from rat fibroblasts leads to a stable defect in cell proliferation. We used complex cDNA libraries expressed from retroviral vectors and an efficient sorting procedure to rapidly select for cDNAs that can restore the growth rate of c-myc deficient cells. All of the biologically active cDNAs contained either c-myc or N-myc, suggesting that no other cellular genes can effectively bypass the requirement for c-myc in fibroblast proliferation. This approach provides a powerful screening method for cell cycle changes in genetically defined systems.

Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm

BACKGROUND Development of spectrally distinct green fluorescent protein (GFP) variants has allowed for simultaneous flow cytometric detection of two different colored mutants expressed in a single cell. However, the dual-laser methods employed in such experiments are not widely applicable since they require a specific, expensive laser, and single-laser analysis at 488 nm exhibits considerable spectral overlap. The purpose of this work was to evaluate detection of enhanced cyan fluorescent protein (ECFP) in combination with the enhanced green (EGFP) and enhanced yellow (EYFP) fluorescent proteins by flow cytometry. METHODS Cells transfected with expression constructs for EGFP, EYFP, or ECFP were analyzed by flow cytometry using excitation wavelengths at 458, 488, or 514 nm. Fluorescence signals were separated with a custom optical filter configuration: 525 nm shortpass and 500 nm longpass dichroics; 480/30 (ECFP), 510/20 (EGFP) and 550/30 (EYFP) bandpasses; 458 nm laser blocking filters. RESULTS All three fluorescent proteins when expressed individually or in combination in living cells were excited by the 458 nm laser line and their corresponding signals could be electronically compensated in real time. CONCLUSIONS This method demonstrates the detection of three fluorescent proteins expressed simultaneously in living cells using single laser excitation and is applicable for use on flow cytometers equipped with a tunable argon ion laser.

Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins

Flow cytometric analysis of fluorescent protein expressing cells is of particular interest to researchers in many areas. The detection of fluorescent proteins in cells allows one to monitor gene expression, determine intracellular protein localization, and identify transfected cells. Wild‐type green fluorescent protein has limited utility as its spectral properties are not suitable for standard cytometers. Site‐directed mutations have produced enhanced variants with improved extinction coefficient and quantum yield with standard 488‐nm excitation. Other variants have been constructed with shifted excitation and emission maxima and high quantum yield. It is now possible to monitor multiple processes in a single cell and detect enhanced green, yellow, and cyan fluorescent proteins using a single excitation beam at 458 nm. The authors carefully describe the custom filter setup required to accomplish this and the Boolean gating logic for analysis of the various subpopulations expressing any given combination of fluorescent proteins.