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The following primers were used to amplify BLNK exon 1 and the associated flanking sequence for both the SSCP analysis and cloning of this region of the gene: 5′-GAACTGCTGACGTGACCA-3′ (5′ untranslated region of exon 1) and 5′-CCCTAAAAGCT-CAGTCCAC-3′ (intron 1). The products of two independent PCR reactions were cloned and sequenced.
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0344610915
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note
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DNA samples from five individuals demonstrated altered migration in comparison to the wild-type pattern. Sequencing showed a T to A substitution at the +113 position in intron 1 in three people, a G to A substitution at the +121 position in intron 1 in one individual and an A to G substitution 5 base pairs upstream of the start codon.
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15
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0345041950
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note
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The primers used to amplify BLNK cDNA were from the 5′ untranslated region [shown in (9)] and 5′-GTCGCTGTCAAAGTCATCGGA-3′ from exon 4. The Btk-specific primers were from exons 6 and 8 of that gene. The primers used to amplify TdT, λ5, VDJ-μ, and glyceraldehyde phosphate dehydrogenase (GAPDH) were as reported in (7).
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0344610914
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+ B cells (normal is 5 to 22%). Serum IgG in the father was 1110 mg/dl, IgA was 337 mg/dl, and IgM was 93 mg/dl. The mother's IgG was 1060 mg/dl, IgA was 115 mg/dl, and IgM was 127 mg/dl.
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Bone marrow mononuclear cells were stained with antibody to CD19 conjugated to phycoerythrin (PE) (Dako, Carpinteria, CA), antibody to CD34 conjugated to peridinin chlorophyll protein (PerCP) (Becton-Dickinson, San Jose, CA), and polyclonal antibodies against human light chains conjugated to fluorescein isothiocyanate (FITC) (Southern Biotechnology Associates, Birmingham, AL). Antibodies to nuclear TdT (Supertechs, Bethesda, MD) and cytoplasmic μ heavy chains (Southern Biotechnology Associates) conjugated to FITC and PE, respectively, were applied after cell permeabilization with OrthoPermeafix (Ortho Diagnostics, Raritan, NJ). Monoclonal antibody to BLNK (of IgG2a isotype) was generated against a glutathione S-transferase fusion protein encoding amino acids 4 through 205, as described in (1). This antibody was used in combination with an antibody to CD19 of IgM class (BLY3) (Research Diagnostics, Flanders, NJ), with fluorochrome-conjugated secondary antibodies specific for murine IgC and IgM. Antibody to BLNK was applied after cell permeabilization with OrthoPermeafix Isotype-matched nonreactive antibodies were from Becton-Dickinson. Immunofluorescence staining was analyzed with a FACScan flow cytometer equipped with CellQuest software (Becton-Dickinson).
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We thank M. D. Cooper for a helpful discussion, E. Boylin for technical assistance, and S. Saucier for administrative assistance. Supported by NIH grants AI25129, AI42787, CA71516, and CA58297; March of Dimes grant FY97-0384; the Assisi Foundation; National Cancer Institute CORE grant P30 CA21765; American Lebanese Syrian Associated Charities; the Human Frontiers Scientific Organization; and funds from the Federal Express Chair of Excellence. A.C.C. is a Pew Scholar in the Biomedical Sciences.
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