Naomi Rosenberg

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.

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.

The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

Continuing kappa-gene rearrangement in a cell line transformed by Abelson murine leukemia virus

A cell line transformed by Abelson murine leukemia virus, called PD, is capable of carrying out kappa-gene rearrangement while growing in culture. Subclones of PD have diverse kappa-gene structures, and some derivatives show evidence of continued joining activity after as many as three subclonings. Analysis of PD sublineages has shown that a rearranged chromosome can undergo secondary kappa-gene rearrangements, producing either a new rearrangement or a deletion of C kappa. Although the PD line actively rearranges its kappa genes, its rearranged heavy-chain genes show little variation, and there is no rearrangement of lambda genes. In PD subclones, DNA fragments representing the reciprocal product of kappa-gene rearrangement are often evident, and they may undergo either further rearrangement or deletion. The implications of multiple rearrangements on a single chromosome and of the maintenance of reciprocal fragments are considered in the context of a model that postulates that the V kappa and J kappa segments are not all organized in the DNA in the same transcriptional direction, leading to inversions rather than deletions during joining.

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus‐transformed pre‐B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site‐specific join between the open reading frame of a LINE‐1 repetitive element and a remaining JH segment. We discuss this event in the context of the ‘accessibility’ model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre‐B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination‐deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre‐B cell lines and early in normal development. We propose that the predisposition of pre‐B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased ‘accessibility’ of the germline gamma 2b locus to switch‐recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.

Immunoglobulin synthesis by lymphoid cells transformed in vitro by Abelson murine leukemia virus

The majority of cell lines derived by infection of murine bone marrow cells with Abelson murine leukemia virus (A-MuLV) synthesize a mu chain but no detectable light chain. Aside from this mu-only phenotype, lines that make only light chain, both chains or no immunoglobulin-related polypeptides have also been found. Two lines have been studied in detail: one that makes only mu chain and one that makes only kappa light chain. Synthesis of both polypeptides can be increased by modifying the culture conditions so as to decrease the growth rate of the cells. Although some kappa chain secretion was observed, neither secreted nor surface mu was detected. We suggest that the mu- only phenotype may be an early normal step in the pathway of B lymphocyte maturation.

Multiple immunoglobulin heavy-chain gene transcripts in Abelson murine leukemia virus-transformed lymphoid cell lines.

Lymphoid cells transformed by Abelson murine leukemia virus (A-MuLV) contain three classes of RNA transcripts from immunoglobulin mu genes. P mu-mRNAs (productive) correspond to the normal 2.7-kilobase (kb) membrane (mu m) and 2.4-kb secreted (mu s) mu mRNA species both in size and coding capacity and occur at approximately equal abundance in most mu-positive (pre-B-like) A-MuLV transformants. A mu-mRNAs (aberrant) generally fall into one of two categories--aberrantly small 2.3-kb mu m and 2.0-kb mu s mRNAs which encode aberrantly small mu polypeptide chains, or normal-sized, V H-containing mu RNAs which do not encode immunologically identifiable mu polypeptide chains. In one case, the latter type of A mu-mRNA was demonstrated to result from an in-phase termination codon in the D segment of the mu mRNA. Also, most, if not all, A-MuLV transformants express members of a 3.0 to 1.9-kb set of C mu-containing, but V H-negative S mu-RNAs (for sterile), the expression of which may occur simultaneously with but independently of P mu-mRNAs or A mu-mRNAs. The S mu-RNA sequences do not encode immunologically identifiable mu chains and can be produced by cells with unrearranged heavy-chain alleles, such as T-lymphocytes, although the structure of the S mu-RNAs from T-lymphoid cells appears to be different from that of B-lymphoid cell S mu-RNAs. Certain A-MuLV transformants also express gamma-RNA sequences that are probably analogous to the three different forms of mu RNA. These data support the concept that heavy-chain allelic exclusion, like that of light chains, is not mediated by control at the DNA or RNA levels but is probably a consequence of feedback control from cytoplasmic mu chains.

The viral and cellular forms of the Abelson (abl) oncogene.

The precision of molecular biology has allowed a better definition of the components of the Abelson system. We know the gene structures and gene products for the cellular and viral forms of this family of related tyrosine kinases. However, many basic issues first identified in the early biological observations of Abelson, Rabstein, and others remain unanswered. The precise pathway for transformation in biochemical terms remains unknown for Ab-MLV and all of its relatives. Relatively little can be said to explain the preferential growth stimulation for certain hematopoietic cell types by the viral and other altered forms of the oncogene, and no clear insights into the function of the normal cellular forms of the abl oncogene are available. Future progress will certainly depend on the intensive efforts by many workers in the broader field of cellular growth control mechanisms.

Immunoglobulin heavy-chain expression and class switching in a murine leukaemia cell line

A cell line that switches from μ to γ2b synthesis during growth in culture uses the same VH region for both heavy chains but retains two copies of the Cμ gene. This suggests that the μ to γ2b class switch can occur, at least in part, by an RNA processing mechanism. Regulatory variants of this cell line lose constitutive μ-chain synthesis but simultaneously acquire lipopolysaccharide(LPS)-inducible synthesis of that chain. This co-variation is allele-specific and is correlated to a large deletion of DNA in the JH–Cμ intron.

Differences in oncogenic potency but not target cell specificity distinguish the two forms of the BCR/ABL oncogene.

Two forms of activated BCR/ABL proteins, P210 and P185, that differ in BCR-derived sequences, are associated with Philadelphia chromosome-positive leukemias. One of these diseases is chronic myelogenous leukemia, an indolent disease arising in hematopoietic stem cells that is almost always associated with the P210 form of BCR/ABL. Acute lymphocytic leukemia, a more aggressive malignancy, can be associated with both forms of BCR/ABL. While it is virtually certain that BCR/ABL plays a central role in both of these diseases, the features that determine the association of a particular form with a given disease have not been elucidated. We have used the bone marrow reconstitution leukemogenesis model to test the hypothesis that BCR sequences influence the ability of activated ABL to transform different types of hematopoietic cells. Our studies reveal that both P185 and P210 induce a similar spectrum of hematological diseases, including granulocytic, myelomonocytic, and lymphocytic leukemias. Despite the similarity of the disease patterns, animals given P185-infected marrow developed a more aggressive disease after a shorter latent period than those given P210-infected marrow. These data demonstrate that the structure of the BCR/ABL oncoprotein does not affect the type of disease induced by each form of the oncogene but does control the potency of the oncogenic signal.