The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein.Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TlG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.

The gene mutated in the autosomal recessive disorder ataxia telangiectasia (AT), designated ATM (for 'AT mutated'), is a member of a family of phosphatidylinositol-3-kinase-like enzymes that are involved in cell-cycle control, meiotic recombination, telomere length monitoring and DNA-damage response. Previous results have demonstrated that AT cells are hypersensitive to ionizing radiation and are defective at the G1/S checkpoint after radiation damage. Because cells lacking the protein tyrosine kinase c-Abl are also defective in radiation-induced G1 arrest, we investigated the possibility that ATM might interact with c-Abl in response to radiation damage. Here we show that ATM binds c-Abl constitutively in control cells but not in AT cells. Our results demonstrate that the SH3 domain of c-Abl interacts with a DPAPNPPHFP motif (residues 1,373-1,382) of ATM. The results also reveal that radiation-induction of c-Abl tyrosine kinase activity is diminished in AT cells. These findings indicate that ATM is involved in the activation of c-Abl by DNA damage and this interaction may in part mediate radiation-induced G1 arrest.

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We have reexamined the balance between cell birth, cell maturation, and cell death in the thymus by labeling dividing thymocytes and their progeny in vivo with [3H]-thymidine, isolating clearly defined subpopulations by fluorescence-activated cell sorting, and determining the distribution of label by autoradiography. When mature thymocytes were precisely defined (as CD4'CD8-CD3' or CD4-CD8' CD3+) and separated from immature single positives (CD4+CD8-CD3-and CD4-CD8+ CD3-), a lag was observed in the rate of entry of H3lIthymidine into mature cells. Thus, many of the mature thymocytes appear to derive from a small nondividing cortical thymocyte pool, rather than originating directly from the earliest dividing CD4+CD8+ blasts. There was little evidence for cell division during or after mature thymocyte formation, suggesting a one-for-one differentiation from cortical cells rather than selective clonal expansion. The rate of production of mature single positive thymocytes agreed closely with estimates of the rate of export of mature T cells from the thymus and was only 3% of the rate of production of doublepositive cortical thymocytes. This was compatible with a stringent selection process and extensive intrathymic cell death and suggested that no extensive negative selection occurred after the mature cells were formed.The majority of CD4+CD8+ (double positive) cortical thymocytes are believed to die in the thymus after a short life-span (1-5). However, some do mature into CD4+CD8-and CD4-CD8+ (single positive) medullary thymocytes through a process of positive selection by self major histocompatibility complex (MHC) antigens (6-15). The number entering the mature state is reduced by a process of negative selection, which eliminates cells with excessive direct reactivity with self-MHC and self-antigens (8,9,12,13,(16)(17)(18)(19). Cortical thymocytes that are not rescued by the positive selection process presumably also die intrathymically of neglect.Kinetic studies on T-lymphocyte development in the thymus originally presented the paradox of a very rapid rate of thymocyte birth (equivalent to one-third of all thymocytes per day) (1,4) compared to a slow rate of export to the periphery (equivalent to 1% of all thymocytes per day) (3, 4). The conclusion from the balance sheet was that most cells born in the thymus die in the thymus. A series of studies comparing the relative retention in the thymus of iododeoxyuridine and thymidine after their incorporation into thymocyte DNA had seemed to provide direct evidence for this intrathymic death (19-21); however, deiodination, rather than cell death and nucleoside reutilization, appears to have been the real explanation of these results (22). Since the balance sheet aspects remain the primary argument for extensive intrathymic death, it is important to recheck the evidence for thymocyte overproduction and to determine whether it is the mature or the immature subpopulations that are involved.The uptake of label into the DNA of cells in the S phase of the cell cyc...