ABSTRACT. Our investigations have focused on localizing cis- elements responsible for the down regulation of the adult beta- like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic- fetal phenotype. Analyzed DNA sequences included delta and beta 5' flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5' flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcriptional activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor- mediated suppression early in development and then may modulate its expression at a later stage.

Keywords: Globin gene, transcription, silencer, mutagenesis, human erythroleukemia cells.

Reprint requests to: Griffin P. Rodgers, M.D., Molecular and Clinical Hematology Branch, NIDDK, National Institutes of Health, Bldg. 10, Room 9N318, 10 Center Drive, Bethesda, MD 20892-1822, phone: (301) 402-2418, fax: (301) 402-0101, email: gprod@helix.nih.gov e-mail: gprod@helix.nih.gov.

ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal blood disorder characterized by chronic hemolysis with hemoglobinuria and venous thrombosis. PNH clones arise through somatic mutations in the X-linked PIG-A gene that occur in early hematopoietic stem cells. Here we report 28 previously undescribed mutations; we confirm that somatic mutations are spread throughout the entire coding region of the PIG-A gene and that the majority are frameshift mutations producing a non- functional PIG-A protein [PIG-A(o)]. In addition, we found 1 total deletion of the PIG-A gene, and 2 short nucleotide duplications. Although mutations are spread throughout the entire coding region, we observe more missense mutations in exon 2 than in the other exons. The increasing number of identified missense PIG-A mutations should help elucidate structure-function relationships in the PIG-A protein.

Keywords: Aplastic anemia, somatic mutation, heteroduplex analysis, single strand conformation analysis.

Reprint requests to: Dr. Khèdoudja Nafa, Department of Human Genetics, Box 110, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, phone: (212) 639-5170, fax: (212) 717-3571, e-mail: k-nafa@ski.mskcc.org.

ABSTRACT: A study on blood cell damage after irradiation of fresh whole blood with 630 nm laser light was carried out in vitro. Various fluence rates of laser light were used with and without cooling of blood. Damage to the blood was assessed by blood cell counts, osmotic fragility measurements and examination of blood films. Exposure of a 1 mm blood layer to 630 nm laser light without cooling led to changes in blood counts first detected at fluence rates of 130 mW/cm2. Changes in osmotic fragility first became evident at 210 mW/cm2. Increasing cell damage with increasing fluence rates was evident in blood films. Using the cooling device changes in whole blood after irradiation first occurred at a fluence rate of 293 mW/cm2. Measurement of the fluence rates at which cell damage begins is important in laser induced fluorescence diagnostics and photodynamic therapy applications in blood or blood products using photosensitizers.

Keywords: Blood counts, blood films, laser induced fluorescence diagnostics, osmotic fragility,photodynamic therapy.

Reprint requests to: Frank Fischer, Ph.D., Department of Chemistry & Chemical Engineering, Royal Military College of Canada, P.O. Box 17000, Station Forces, Kingston, Ontario, Canada K7K 7B4, phone: (613) 541-6000 ext. 6360, fax: (613) 542-9489, e-mail: fischer-f@rmc.ca.

Communicated by Brian S. Bull, M.D., Loma Linda University, School of Medicine, Loma Linda, California.

Communicated by Frank Fischer, Ph.D., Royal Military College of Canada, School of Medicine, Kingston, Ontario, CANADA.