The FlowMetrixTM System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95®-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.
DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these processes is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37. Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wild-type human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs. These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.
We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in “leaky” coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.
SummaryThe severe combined immunodeficiency (SCID) mutation has been postulated to affect a V(D)J recombinase activity involved in coding joint formation. Analysis of 38 joints from 34 distinct sequences of normally rearranged T cell receptor (TCR) 3' and ~ genes from adult, SCID thymocytes reveals coding joints with an increased number of P nucleotides. One-third of P sequences are >--4 nucleotides in length and P elements of up to 15 bases are observed. This suggests that the SCID defect deregulates P nucleotide addition. Consequently, essential V(D)J recombination intermediates may seldom be generated. y oung adult mice homozygous for the SCID mutation are severely deficient in mature B and T cells (1). Observation of abnormal antigen-receptor gene rearrangements in vitally transformed SCID bone marrow cells and spontaneous SCID thymic lymphocytes suggested initially that this might be due to defective V(D)J recombinase activity in these mice (2). Sequence analysis of numerous abnormal SCID Ig (3-7) and TCR (8) rearrangements, as well as studies with recombination substrates (9-11) have subsequently shown that C.B-17 scid/scid mice contain a V(D)J recombinase that recognizes recombination signal sequences adjacent to variable region gene segments (V, D, and J), cleaves the DNA normally at the borders of the variable region gene segments, ligates the signal sequences at a normal frequency, but fails to recombine the V, D, and J gene segments at any frequency comparable to that in normal cells. Further, V(D)Jjoints frequently result in deletion of one or both of the participating coding elements (2-7).Recently, while studying natural killer cells and T cell progenitors in the thymus of SCID mice, we obtained evidence for full-length, potentially productive, TCK 3" chain transcripts (12). Southern blot analyses revealed normal rearrangements at the TCK 3" locus and PCK amplification, cloning and DNA sequencing were performed to capture TCR % and partner chain,/~, gene junctional sequences with small deletions in the coding segments. Nucleotide sequences reveal that many characteristics of SCID V(D)J recombination are indistinguishable from normal V(D)J recombination (Kienker, L. J., W. A. Kuziel, B. A. Garni-Wagner, V. Kumar, and P. W. Tucker, manuscript submitted for publication), but P nucleotide addition is different. P nucleotides are newly distinguished inserted bases at coding joints that form inverted repeats of the neighboring gene segment termini (13). Presumably, there are up to 2 P nucleotides at the ends of junctional inserts and these occur when the associated gene segment appears in full in the coding joint (13). Here we show that SCID coding joints have an excessive number of P nudeotides in junctional inserts. This suggests that the SCID defect deregulates the addition of P nucleotides leading to the frequent failure to form this essential V(D)J recombination intermediate. Materials and MethodsMice. C.B-17/Icr scid/scid mice, originally obtained from Dr. Melvin Bosma, Fox Chase Cancer Center (Phi...
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