Blackwell Tk

Ordered rearrangement of immunoglobulin heavy chain variable region segments.

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ‐line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B‐lymphoid cells as well as normal B‐lymphoid cells. Abelson murine leukemia virus (A‐MuLV) transformed fetal liver cells ‐ the most primitive B‐lymphoid cell analog available for analysis ‐ generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain‐producing tumor lines, representing more mature stages of the B‐cell pathway, and normal B‐lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B‐cell differentiation in which D‐to‐JH rearrangements generally occur first and on both chromosomes followed by VH‐to‐DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain‐producing lines and, especially, in normal B‐lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH‐to‐DJH rearrangement.

Separate elements control DJ and VDJ rearrangement in a transgenic recombination substrate.

We describe transgenic mice that carry an antigen receptor gene minilocus comprised of germline T cell receptor (TCR) beta variable gene elements (V, D and J) linked to an immunoglobulin (Ig) C mu constant region gene with or without a DNA segment containing the Ig heavy chain transcriptional enhancer (E mu). Transgenic constructs lacking the E mu‐containing segment did not undergo detectable rearrangement in any tissue of six independent transgenic lines. In contrast, transgenic constructs containing this DNA segment underwent rearrangement at high frequency in lymphoid tissues, but not other tissues, of four independent lines. Analyses of purified B and T cells, as well as B and T cell lines, from transgenic animals demonstrated that the E mu‐containing segment within the construct allowed partial TCR gene assembly (D to J) in both B and T cells. However, complete TCR gene rearrangement within the construct (V to DJ) occurred only in T cells. Therefore, we have demonstrated elements that can control two separate aspects of TCR beta VDJ rearrangement within this construct. One lies within the E mu‐containing DNA segment and represents a dominant, cis‐acting element that initiates lymphoid cell‐specific D beta to J beta rearrangement; various considerations suggest this activity may be related to that of the E mu element. The second element provides T cell‐specific control of complete (V beta to DJ beta) variable region gene assembly; it correlates in activity with expression of the unrearranged V beta segment.

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre‐) B cell differentiation, we find that most Abelson murine leukemia virus (A‐MuLV)‐transformed pre‐B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A‐MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre‐B cell lines from normal mice, attempted V kappa‐to‐J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back‐to‐back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site‐specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

Regulation of the Assembly and Expression of Immunoglobulin Genes: Variable Region Assembly and Heavy Chain Class Switching

To elucidate further the general mechanisms which control the assembly and expression of Immunoglobulin variable region gene segments and heavy-chain class switching, we have exploited Abelson murine leukemia virus (A-MuLV) transformed pre-B cells as a model system. The Abelson virus is a replication defective retrovirus which is unique in its capacity to transform very early B-lineage cells in vitro (1). A-MuLV transformants generated from murine fetal liver or adult bone marrow have provided both a static representation of various pre-B cell differentiation stages, as well as a dynamic view of pre-B cell immunodifferentiation events (2,3). Our analyses of these lines have provided a variety of new and sometimes surprising findings regarding the early stages of B cell differentiation; and importantly, all of these novel aspects first observed in A-MuLV transformants were found to reflect events that occur early in normal B lineage cells in vitro. Furthermore, we have utilized gene transfer technology to gain additional insight into the molecular events involved with the regulation of immunogloblin and T cell receptor variable region (V) gene assembly (3).