Analysis of replication fork direction in the region downstream of the 3′ regulatory region by N/A 2D gel electrophoresis. The first dimension of the agarose gel is run in neutral conditions in which restriction enzyme-digested DNA molecules enriched in replication intermediates are separated according to their mass. The second dimension is run in alkaline conditions to separate parental and nascent strands. The direction of replication fork movement (indicated by arrow) is determined by hybridization to probes (solid rectangles) located at the 5′ or 3′ end of a specific restriction fragment (probes 199M11:34 and 40, 73 and 76, and 84 and 88); or from 5′ and 3′ ends of two adjacent restriction fragments (probes 199M11:53 and 57, 60 and 65, 81 and 82, and 92 and 93); or by using a single probe located at the ends of two overlapping fragments (probes 199M11:79 and 113). The size of the restriction fragment is indicated. Note that 199M11:34 and 199M11:117 were previously termed Cα-45 and Cα-100, respectively. Each segment (indicated by gray lines) was analyzed by two or more 2D gels with similar findings. The topmost restriction fragment and the topmost arrow (if there are two) always refer to the left of the two panels. The horizontal line (a) represents the parental strands of the replication intermediates, which run at a single position in the second dimension because they have a single molecular weight. The diagonal line (b) emanating from the parental strands represents nascent strands of different sizes, ranging from very small to as large as the parental strands. If replication forks move from 3′ to 5′, a probe located at the 3′ end of a fragment will detect nascent strands of sizes ranging from small to parental-sized, whereas probes located at the 5′ end will detect nascent strands only of a relatively large size. If forks move in both directions through a segment, then nascent strands of multiple sizes will be detected with probes for both 5′ and 3′ ends of this segment. Because of limitations in the resolution of N/A 2D gel electrophoresis, we cannot rule out the possibility that <10% of the forks progress from the opposite direction in the region upstream of 199M11:76. The spots present on the nascent strand arcs and the linear molecules on the autoradiogram probed with 199M11:84 or :88 are caused by the accumulation of a single-stranded break in this segment, produced by EcoRI digestion, which is released during alkaline treatment in the 2nd dimension. No spots were detected on neutral/neutral (N/N) 2D gels. Therefore, this single-strand break does not appear to form any replication barrier. H, HindIII; E, EcoRI; B, BamHI; X, XbaI.

Structural features of the 125 kb DNA insert of 199M11. (a) Schematic of sequence features of BAC199M11 (GenBank accession no. AF450245). Position 1 in 199M11 is the telomeric end of the chromosome segment cloned in this BAC and corresponds to position 1242 of GenBank accession no. K00691 (Cα membrane exon). The Igh locus is in inverted transcriptional orientation on chromosome 12 so that the 5′ end of the locus is distal (telomeric). “Downstream” with respect to Igh is proximal on the chromosome, and we will use upstream, or 5′, and downstream, or 3′, to describe a position relative to Igh, not the chromosome. V, D, J, and C sequences are located upstream of BAC199M11 sequences, Although there are two small gaps in the 199M11 sequence (indicated by vertical arrows at 26,423, between hs3b and hs4 enhancers, and at position 71,578), each estimated to be <500 bp, we present the data as a continuous sequence 1 to 124,151 (GenBank Accession no. AF450245). Repetitive DNA sequences identified by Repeat Masker and by individual searches of DNA segments are shown with vertical black bars and specifically identified in GenBank accession no. AF450245. A higher density of repetitive sequences is apparent closer to the Igh gene cluster and a relatively lower density toward the downstream end of the BAC. Igh regulatory elements, L7a pseudogene, Crip, Crp2, and Mta1 genes and CpG islands (at 64180–65703; 67270–67539; 96721–97360; and 112328–113229) are indicated. Transcription direction is indicated by horizontal arrows above the sequence, and direction of replication fork movement is indicated by horizontal arrows below the sequence. A region within which the direction of replication fork movement changes is indicated by “ori”. An A/T-rich segment is located at 77,700–77,746. Locations in the 199M11 sequence are given as distance from the Igh end; e.g., 199M11:99 is 99 kb from the IgA membrane exon (5′) end of 199M11. ESTs are indicated by rectangular boxes containing nos. 1–5. EST region 1 (61,000–61,484) matches a single EST from heart-AA646361 (5′ end sequence of IMAGE clone 1021178; the 3′ sequence of this clone has not been determined). Much of EST region 2 (62,082–67,432) is contained in AK010454, which has been termed a full insert mRNA sequence that encodes a gene. Multiple ESTs matching this region have been identified as part of UniGene Cluster Mm. 133306 Mus musculus. EST region 3 (77,453–81,342) matches two IMAGE clones that appear to be identical, 641181 (5′ sequence, AI605834, and 3′ sequence, AI450404) and 3418382 (5′ sequence, BE848059, and 3′ sequence, BE853477). This region, identified in UniGene Cluster Mm. 32319 Mus musculus, contains the replicative boundary in MEL [199M11:76–79 and a previously unidentified L7a pseudogene (199M11:79)]. The gene encoding the L7a protein (Surf-3) (32) appears to be present in a single copy within the mouse surfeit locus on chromosome 2 (52); and there are 15–20 L7a-related family members, three of which have been analyzed and found to have the hallmarks of processed pseudogenes (53), i.e., lack of introns, presence of multiple defects that would prevent translation, and flanked by direct repeats of 13–15 bp. Immediately 3′ of the L7a homology region in 199M11, there is a B2/SINE element followed by a short poly(A) tract. EST region 4 extends ≈9 kb from 87,921–96,874, within which several ESTs have been arranged in two UniGene Clusters: Mm 87616 Mus musculus (87,921–88,550) and Mm. 90118 Mus musculus (88,807–95,766). Within UniGene Cluster Mm. 90118 is BC022617, which has been identified as an mRNA/gene sequence, although a predicted protein sequence is not clearly specified. Comparison of BC022617 to 199M11 suggests that the gene encoding BC022617 contains at least five exons. EST region 5 (97,382–98,337) shows three matches, including to the 5′ (BE624876) and 3′ (BE629214) ends of IMAGE clone 3370299. (b) DotPlot analysis (54) (http://bio.cse.psu.edu/pipmaker) of 199M11 to identify internal repetitive sequences shows unique features associated with the 3′ Igh regulatory region, namely an extensive palindrome and multiple repetitive sequences. Parallel lines indicate direct repeats and perpendicular lines indicate inverted repeats.