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

Abstract

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pte-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.Somatic-DNA recombination activates and diversifies variable-region genes of antigen receptors (29,51). This DNA rearrangement, known as V-(D)-J joining, occurs between two pairs of recombination signal sequences (RSS), i.e., a heptamer (5'-CACAGTG-3') and a nonamer (5'-GGTTTTTGT-3'), when one pair is separated by a 12-bp spacer and the other is separated by a 23-bp spacer (9,12,35,(42)(43)(44). Substrate requirements and the effects of base substitutions have been studied in detail for recombination signal sequences (2,18). For the enzymatic machinery, several nuclear proteins have been reported as having possible endonucleolytic activities (11,19,25) or as being DNA-binding components of the putative V-(D)-J recombinase (1,16,17,32,34).Two chromosomal locations have been reported for recombination functions associated with V-(D)-J joining. A defect in DNA recombination has been observed in the scid mouse, CB-17 scid (5), in which V-(D)-J joining is apparently impaired in both B and T cells. Extensive DNA deletions are found at recombination junctions in the scid animal (33, 39). The CB-17 scid gene has been mapped to mouse chromosome 16 (6). Recently, two genes that can activate V-(D)-J joining were isolated by introducing chromosomal DNA fragments into the fibroblast cell line NIH 3T3 (38,46,47 protein is much larger than other HMG members, the hydrophilic region of T160 has a strong homology to HMG1. In order to study whether the T160 gene has a genetic linkage to scid or rag, the chromosomal location for T160 was determined. Using restriction fragment length polymorphism segregation analysis, genetic linkage was established for T160 and RAG genes on the proximal end of chromosome 2. In this report, we characterize the T160 protein and discuss its possible role in V-(D)-J recombination.MATERIALS AND METHODS cDNA library. Total RNA was prepared from a pre-B-cell line, 38B9 (4), according to the manufacturer's protocol (Amersham) and passed through an oligo(dT)-cellulose column three times to obtain poly(A)+ RNA. cDNA was synthesized by the method of Gubler and Hoffman (15) using oligo(dT) and random he...

Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.

“…There is a continuous open reading frame of 2127 bases beginning at position 275. The sequence surrounding the methionine start codon conforms well with the initiation sites of other vertebrate cDNAs (30) and is conserved in homologs isolated from mouse (24) and Drosophila melanogaster (S.L.B., D.E.H., and S.J.L., unpublished work). A consensus polyadenylylation signal AATAAA is present within the 435 bases of the 3' untranslated sequence beginning at position 2800.…”

“…Initial experiments placed the gene on chromosome 11. Further refinement with a series of hybrid cell lines containing only small defined segments of human chromosome 11 on a rodent genomic background (33) (24). Previously, a syntonic relationship had been demonstrated only for mouse chromosome 2 and human chromosome lip (35).…”

A Drosophila single-strand DNA/RNA-binding factor contains a high-mobility-group box and is enriched in the nucleolus.

“…Sequence ¶ comparisons show that this protein is the homolog of the recently described human structure-specific recognition protein (hSSRP; ref. 6) and the presumed mouse thymus recombination factor T160 (7). Here we report its developmental expression pattern, its cytological localization, and initial characterization ofits nucleic acid binding activity.…”

Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors.

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