Hitoshi Sakano

T cell receptor β gene sequences in the circular DNA of thymocyte nuclei: Direct evidence for intramolecular DNA deletion in V-D-J joining

We have identified circular DNA containing T cell receptor (TCR) beta gene sequences in mouse thymocytes, thereby providing direct evidence for the intramolecular DNA deletion model of V-D-J joining in TCR beta genes. Two types of excision products of V-D-J joining have been identified. Type I, a circular reciprocal recombinant of normal V-D or D-J joining, contains a 7mer-7mer head-to-head structure expected from an excised product of normal V-D or D-J joining. Type II contains a D beta 2-J beta 1 structure on the circular DNA; the recombination event producing this molecule occurs between an upstream J and a downstream D segment, probably leaving the reciprocal 7mer-7mer structure on the chromosome. Some type I molecules seem to represent excision products of secondary joining after formation of the first D-J or V-D-J structure. The recombination mechanism that generates the circular DNA is discussed.

HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.

Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

ABSTRACT We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

Characterization of immunoglobulin enhancer deletions in murine plasmacytomas.

We have analyzed enhancer deletions found in murine plasmacytomas by DNA cloning. This analysis revealed that the deletions occurred between the JH region and the switch region, removing the Ig heavy chain enhancer. The loss of the enhancer did not significantly affect the level of heavy chain expression as determined by RNA blots. Nucleotide sequence analysis revealed that there are no characteristic or homologous sequences around the recombination site. Extra nucleotides were found at the recombination sites, in a manner analogous to Ig and T‐cell receptor V‐D‐J joining. The germline JH and switch sequences involved in the deletion were analyzed by the in vitro DNA cleavage system with an endonucleolytic activity purified from mouse fetal liver nuclear extracts. It was found that the germline JH DNA was strongly cleaved at the deletion recombination site.

The 3′ enhancer region determines the B/T specificity and pro-B/pre-B specificity of immunoglobulin Vκ-Jκ joining

Abstract Using transgenic substrates, we found that the immunoglobulin κ gene 3′ enhancer (E3′) acts as a negative regulator in V κ -J κ joining. Although the E3′ was originally identified as a transcriptional enhancer, it acts in a Buppressive manner for recombinational regulation. Base substitution analysis has shown that the PU. 1-binding site within the E3′ regulates the B/T specificity of V κ -J κ joining. In a substrate with a mutated PU. 1-binding site (GAGGAA to TCTTCG), V κ -J κ joining occurred not only in B cells, but also in T cells. The E3′ region is also responsible for determining the pro-B/pre-B specificity of V κ -J κ joining. When the E3′ region was deleted, κ gene rearrangement actively occurred at the early pro-B stage of B cell development: non-germline (N) nucleotides were common at recombination junctions.

Immunoglobulin V gene replacement is caused by the intramolecular DNA deletion mechanism

Circular DNA resulting from V gene replacement was studied with an A‐MuLV transformed cell line containing ablts. This cell line undergoes V gene replacement at elevated temperatures in the immunoglobulin (Ig) heavy chain (H) gene. Examination of circular DNA revealed that a heptamer‐related sequence (TACTGTG) within the coding region of VDJ was joined to the recombination signal sequence (RSS) of a germline VH segment. This provides direct evidence for a intramolecular DNA deletion mechanism for V gene replacement. In the pre‐B cell line as well as in in vivo lymphocytes, unusual circular DNAs were found which were structurally similar to the V gene replacement circles. They represented excision products of the deletion type recombination between one complete RSS and a heptamer‐like sequence in the Ig H region.

A pre-B cell nuclear protein that specifically interacts with the immunoglobulin V-J recombination sequences

DNA-nuclear protein interactions were studied with synthetic recombination signal sequences (RSSs) for immunoglobulin V-J joining. With a gel retardation assay, a DNA-binding protein that specifically interacts with RSSs was detected in nuclear extracts from a pre-B cell line, 38B9. This protein was found in all the recombination-competent pre-B cell lines tested in this study, but not in myeloma, mature T cell, monocyte, or fibroblast cell lines. DNA footprint analysis with dimethyl sulfate demonstrated that the 7-mer region of the RSS was strongly protected when complexed with the binding protein. Furthermore, a single base substitution in the 7-mer region totally abolished the binding. The molecular mechanism of V-J joining is discussed in the context of the RSS-binding protein.