Journal: The Journal of Experimental Medicine
Article Title: Direct in vivo VH to JH rearrangement violating the 12/23 rule
Figure Lengend Snippet: Analysis of IgH V region joints derived from B cells in ΔD mice. (A) Alignment of joints amplified from the RNA of 2-wk-old ΔD/ΔD and ΔD/JHT mice. The joints are shown from the codon immediately 5′ of the second cystein (position 104) of the V H gene and extending to the conserved glycine of the J H region. Sequences labeled with an asterisk use the putative D H element, DST4.2 (underlined). The sequences were analyzed using the http://www.DNAPLOT.de , http://www.imgt.cines.fr , or http://www.ncbi.nlm.nih.gov/igblast websites. In the analysis of bulk-sorted cells, some sequences were found repeatedly, as indicated by the superscripts next to the sequence numbers. As we did not observe repeated sequences in the single cell analyses, we consider the repeats in the bulk analysis an artifact resulting from the high number of amplification cycles. Two sets of sequences (1, 11 and 30, 31, 32) may represent hybrid sequences generated in the course of gene amplification by PCR (reference 27 ). Sequences were submitted through http://www.ncbi.nih.gov/Genbank/index.html in a consistent order (GenBank/EMBL/DDBJ accession nos. AY841948 , AY841949 , AY841950 , AY841951 , AY841952 , AY841953 , AY841954 , AY841955 , AY841956 , AY841957 , AY841958 , AY841959 , AY841960 , AY841961 , AY841962 , AY841963 , AY841964 , AY841965 , AY841966 , AY841967 , AY841968 , AY841969 , AY841970 , AY841971 , AY841972 , AY841973 , AY841974 , AY841975 , AY841976 , AY841977 , AY841978 , AY841979 ). (B) Analysis of joints from single cell sorted and bulk sorted or MACS B cells from 5- and 10-wk-old ΔD/ΔD or WT mice. Because of space limitations, only their productive versus nonproductive status is listed. (C) CDR3 length comparison of V H J H joints (excluding sequences using DST4.2) from ΔD/ΔD and ΔD/JHT B cells and V H D H J H joints from WT B cells isolated from 2-, 5-, and 10-wk-old mice. Gray bars represent average number of nucleotides in the CDR3 defined as starting after the cysteine in the 3′ end of the V H and ending with the last nucleotide before the conserved tryptophan of J H . Error bars represent standard deviations. To demonstrate that the difference in the CDR3 length of the joints from ΔD/ΔD and WT B cells is due to the absence of D H elements and only a single round of N and P nucleotide addition, the average length of D H sequence in WT V H D H J H joints (white bars) plus that of N/P nucleotides added in a single round (black bars) are shown on top of the CDR3 values for ΔD/ΔD sequences. The first group of bars represents a mix of sequences amplified from cDNA of 2-wk-old mice with a natural distribution of J H usage. The second and third group is from DNA of 5-wk-old mice sequenced using a J H 4 or J H 2 primer, respectively. Because J H element length varies, different J H elements contribute differently to overall CDR3 length. The last bar gives the average and standard deviation for sequences derived from DNA of single cells of a 10-wk-old mouse using J H 2 primer. Sequences from appropriately age-matched WT mice were are not available. The average D H element length in V H D H J H joints was calculated from the number of nucleotides of D H origin in the WT sequences of the corresponding group. To approximate the average number of nucleotides per one round of N/P nucleotide addition, the N/P nucleotides at the D H J H and V H D H border in the WT joints of a corresponding group were added and divided by the number of sequences and by a factor of two.
Article Snippet: To isolate the unknown sequence fused to the JH region in the nonproductively rearranged IgH allele of the LN1 mouse, a “pan”-PCR genome walking strategy was performed using adaptor AP1 and adaptor specific primers A1 and A2 (GenomeWalker Kit; CLONTECH Laboratories, Inc.) as described in the original publication by the inventors ( ).
Techniques: Derivative Assay, Mouse Assay, Amplification, Labeling, Sequencing, Generated, Polymerase Chain Reaction, Magnetic Cell Separation, Isolation, Standard Deviation