Cleavage

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RSSs
Synapsis
RAG1/RAG2
Cleavage
Coding ends
Signal ends
Joining
Coding joint
Signal joint
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Fig. 1.   Promoters and enhancers confer RAG
accessibility to RSSs. (A) Schematic depiction of
TCR miniloci. NotI (N) and XhoI (X) sites used
to introduce PD and iE are shown. (B)
Generation of SEs from chromosomal TCR miniloci.
Genomic DNA was harvested from independent pools
of 5B3 transfectants (letters above each lane)
containing the specified miniloci (lanes 3-14) or
untransfected 5B3 (lanes 1 and 2). All pooled
transfectants harbored an average of 5-6
substrate copies and are derived from 20 clones.
Cells were cultured in the absence or presence of
tetracycline (24 h) and subjected to LM-PCR
analysis (13) to measure induced cleavage of the
3'D RSS. Control assays for recombinase activity
(endogenous J 2/3-SEs) and DNA content (C ) are
shown in Middle and Bottom, respectively. The
linearity of each assay was confirmed by serial
dilution (lanes 15-18) of the RAG-induced PE
sample shown in lane 12. Similar results were
obtained with multiple independent subclones for
each construct (data not shown).
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Fig. 3.   Restriction endonuclease sensitivity is
independent of promoter location. (A) Diagram of
LM-PCR strategies for XmnI or EcoRV cleavage
products. BW-1/2 linkers are shown in bold, and
TCR -specific primers are indicated by arrows. (B
and E) Levels of RE cleavage products within
modified substrates. Nuclei from 5B3 clones
containing the indicated TCR substrate or
untransfected 5B3 cells were incubated with XmnI
(0, 0.5, 1, 5, and 10 units) or EcoRV (at
0, 0.1, 0.5, 1, and 5 units). Genomic DNA from
treated nuclei was subjected to nested LM-PCR for
cleavage products (A). The linearity of each
assay was confirmed by serial dilutions (lanes
21-24) of the maximally digested PE sample
(lane 15). (C, D, F, and G) Quantification of RE
sensitivity in TCR miniloci. LM-PCR signals for
XmnI (5 units) or EcoRV (5 units) treatment were
quantified by PhosphorImager analysis. Values
were normalized to signals obtained with PCR
assays for DNA content (C ), substrate copy
number (J 1.1), and cleavage of accessible loci
(C for XmnI or c-myc for EcoRV). Normalized
values are shown relative to data for PE (C and
E) or P-DJ (D and F).
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Fig. 4.   Histone hyperacetylation is
insufficient for recombinational accessibility.
Acetylation levels of histones H3 and H4
associated with the D 1 and J 1.1 gene segments
are shown for independent pools of 5B3
transfectants (Exp. 1 and Exp. 2). Chromatin
immunoprecipitation assays were performed on
mononucleosome preparations with antibodies
specific for acetylated lysines on H3 (black
bars) or H4 (white bars). Acetylation of histones
at the endogenous TEA element, which is inactive
in 5B3 cells, is shown as a negative control.
Acetylation values were calculated as described
in Materials and Methods.