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M J Bosma

Publications and source records attributed to M J Bosma.

At least 19 recordsLinked to original sources
PubMed2000

Unexpected rearrangement and expression of the immunoglobulin lambda1 locus in scid mice.

In severe combined immunodeficient (scid) mice, V(D)J recombination is severely impaired due to a recessive mutation (scid). Thus, we were surprised to find in this study that Vlambda1-Jlambda1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH-DJH and Vkappa-Jkappa rearrangements. Particularly surprising, we found the level of Vlambda1-Jlambda1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid Vlambda1-Jlambda1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, approximately 15% of Vlambda1-Jlambda1 rearrangements lacked abnormal deletions. Productive lambda1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect lambda1-expressing cells by flow cytometry in the spleens of bcl-2-transgenic scid mice. Strikingly, lambda1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of Vlambda1 and Jlambda1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid lambda1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of Vlambda1 and Jlambda1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged Vlambda1 and Jlambda1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.

Animals↗
PubMed1999

Differential effect of an Ig mu transgene on development of pre-B cells in fetal and adult SCID mice.

Progression of pro-B lymphocytes to the pre-B stage depends on the expression of a pre-B cell receptor (pre-BCR), consisting of an Ig mu H chain, Ig surrogate light chain, and associated signal transducing chains. Mice that are unable to express a pre-BCR show an arrest of B cell development at the pro-B stage. Such is the case for severe combined immune deficient (SCID) mice in which mu chains are not made because of a defect in V(D)J recombination. When mu chains are made, as in SCID mice bearing a functional mu transgene, then B cell differentiation can proceed to the pre-B stage. However, as reported here, a mu transgene (M54) that promotes development of SCID pre-B cells in adult bone marrow fails to do so in fetal liver. We suggest that a pre-BCR containing the M54 mu chain cannot signal progression of pro-B cells to the pre-B stage in the fetal liver microenvironment.

Age Factors↗
PubMed1999

Extended duration of DH-JH rearrangement in immunoglobulin heavy chain transgenic mice: implications for regulation of allelic exclusion.

Here we show that suppression of VH-DJH rearrangement in mice bearing a mu heavy (H) chain transgene (mu-tg mice) is associated with an extended period of DH-JH rearrangement, the first step of Immunoglobulin H chain gene rearrangement. Whereas DH-JH rearrangement is normally initiated and completed at the pro-B cell stage, in mu-tg mice it continues beyond this stage and occurs most frequently at the small (late) pre-B stage. Despite ongoing DH-JH rearrangement in late pre-B cells of mu-tg mice, VH-DJH rearrangement is not detectable in these cells. We infer that the lack of VH-DJH rearrangement primarily reflects tg-induced acceleration of B cell differentiation past the stage at which rearrangement of VH elements is permissible. In support of this inference, we find that the normal representation of early B lineage subsets is markedly altered in mu-tg mice. We suggest that the effect of a productive VH-DJH rearrangement at an endogenous H chain allele may be similar to that of a mu-tg; i.e., cells that make a productive VH-DJH rearrangement on the first attempt rapidly progress to a developmental stage that precludes VH-DJH rearrangement at the other allele (allelic exclusion).

Alleles↗
PubMed1997

Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

Animals↗
PubMed1997

Effect of different Ig transgenes on B cell differentiation in scid mice.

In this study we show that the ability of bone marrow pre-B cells to differentiate into B cells in H/L chain transgenic scid mice correlates with the ability of the transgenes to inhibit initiation of endogenous kappa gene rearrangement. Initiation of rearrangement was scored by assaying for DNA double-strand breaks (DSB) at the recombination signal/coding borders of J kappa 1 and J kappa 2. In H/L chain transgenic scid mice that develop B cells, we found little or no DSB; whereas in H chain only transgenic scid mice, in which pre-B cells are unable to give rise to B cells, we found a normal level of DSB but no VJ kappa coding joints. As scid mice are deficient in the repair of DSB, we suggest that initiation of kappa gene rearrangement in H chain transgenic scid mice causes B lineage cells to die at the late pre-B stage. In one transgenic scid line (Y-Sp6), which falls to generate B cells despite containing a H and L chain transgene, we found evidence for loss of B lineage cells at two stages of development: the pro-B to pre-B transitional stage and the late pre-B stage.

Animals↗
PubMed1997

Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus.

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.

Animals↗
PubMed1995

Development of B cells in scid mice with immunoglobulin transgenes: implications for the control of V(D)J recombination.

The inability of scid pro-B cells to progress to the pre-B and B cell stages is believed to be caused by a defective recombinase activity that fails to resolve chromosomal breaks resulting from attempted V(D)J recombination. In support of this model, we report that certain immunoglobulin transgenes, specifically those which strongly inhibit endogenous VH-to-DJH and V kappa-to-J kappa rearrangement in wild-type mice, allow scid pro-B cells to progress to the pre-B and B cell stages. This rescue of scid B cell differentiation is associated with a dramatic reduction in expression of the recombination activation genes, RAG1 and RAG2, and with reduced transcription of the kappa locus.

Animals↗
PubMed1995

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage.

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.

Animals↗
PubMed1994

Abnormal deletions in the T-cell receptor delta locus of mouse thymocytes.

Separate genetic elements (V, D, and J) encode the variable regions of lymphocyte antigen receptors. During early lymphocyte differentiation, these elements rearrange to form contiguous coding segments (VJ and VDJ) for a diverse array of variable regions. Rearrangement is mediated by a recombinase that recognizes short DNA sequences (signals) flanking V, D, and J elements. Signals flank both the 5' and 3' sides of each D element, thereby allowing assembly of a functional VDJ gene. However, in rearrangements involving the D delta 2 and J delta 1 elements of the mouse T-cell receptor delta (TCR delta) locus, we unexpectedly found that the D delta 2 element and a portion of its 5' signal are often deleted. Approximately 50% of recovered D delta 2 to J delta 1 rearrangements from thymocytes of adult wild-type mice showed such deletions. An additional 20% of the rearrangements contained standard D delta 2-J delta 1 coding junctions but showed some loss of nucleotides from the 5' D delta 2 signal. This loss was clearly associated with another event involving a site-specific cleavage at the 5' signal/coding border of D delta 2 and rejoining of the modified signal and coding ends. The abnormal loss of D delta 2 and a portion of the 5' D delta 2 signal was infrequently observed in D delta 2-to-J delta 1 rearrangements recovered from neonatal mice. The possible basis and significance of this age-dependent phenomenon are discussed.

Aging↗
PubMed1993

V(D)J recombination in peritoneal B cells of leaky scid mice.

Developing lymphocytes in immune-deficient severe combined immunodeficient (scid) mice express a defective recombinase activity and rarely succeed in making an antigen receptor; those cells that do succeed account for the known B and T cell leakiness in this mutant mouse strain. To gain more insight into the nature of the scid defect, we assessed the status of heavy (H) and light (L)k, chain genes in immunoglobulin (Ig)Mk-secreting B cells from the peritoneal cavity of old leaky scid mice, the only lymphoid site where scid B cells have been routinely detected. We found these cells to be unusual in that their nonexpressed H chain alleles were either abnormally rearranged or in germline configuration (wild-type B cells generally show normal rearrangements at both H chain alleles). The VDJH junctions of the expressed alleles showed little or no nontemplated (N) addition, similar to neonatal B cells from wild-type mice. About half of the V(D)J junctions lacking N additions contained nucleotides that could have been encoded by either of the participating coding elements (VDH, DJH, or VJk), indicating that the recombination occurred between short stretches of homology. Unusually long templated (P) additions were seen in both VDJH and VJK junctions, and many recombinations appeared to involve P-based homologies. These findings suggest that: (a) B cell leakiness results from a low frequency of coding joint formation in cells expressing the defective scid recombinase activity; (b) joining of scid coding ends is facilitated when the ends contain short stretches of sequence homology, where in many cases, one of the homologous sequences results from a P addition; and (c) scid peritoneal B cells may arise early in ontogeny.

Aging↗
PubMed1993

B-lineage cells in mu-transgenic scid mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain gene rearrangement.

The severe combined immunodeficiency (scid) mouse mutation impairs the recombination of Ig and TCR genes. Mice homozygous for this mutation (scid mice) lack pre-B, B, and T lymphocytes. Earlier we introduced a functionally rearranged mu-heavy chain gene into the scid mouse genome and found that this resulted in the development of pre-B cells in the bone marrow of these mice; however, sIgM+ B cells were not detected. We have now investigated the growth properties and rearrangement status of Ig genes in early B-lineage cells arising in mu-transgenic scid mice. We find that the presence of a functional mu-transgene allows pro-B cells from these mice to proliferate in short-term culture with IL-7. Nevertheless, rearrangements of Ig light chain genes are not detected in the bone marrow of such mice. Furthermore, the frequency of rearrangement detected at the endogenous Ig heavy chain locus in scid pro-B and pre-B cells is reduced relative to that in wild-type cells.

Animals↗
PubMed1993

Scid mouse Pre-B cells with intracellular mu chains: analysis of recombinase activity and IgH gene rearrangements.

Four Pre-B cell clones with intracellular mu chains were recovered from individual leaky scid mice by transformation of bone marrow or peritoneal cells with Abelson murine leukemia virus. Three clones were derived from independent bone marrow cell cultures. These express the defective scid recombinase activity and contain truncated mu chains resulting from abnormal and/or incomplete (D to J only) gene rearrangements. A fourth clone was obtained from a peritoneal cell culture and may represent a revertant. It expresses a recombinase activity indistinguishable from that of wild-type cells; one of its rearranged IgH alleles (VDJ+) encodes a normal size mu chain, the other is non-productively rearranged (VDJ-).

Alleles↗
PubMed1992

V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.

Animals↗
PubMed1992

V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals.

In the murine T cell receptor delta locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination.

Animals↗
PubMed1992

B and T cell leakiness in the scid mouse mutant.

Recombination of antigen receptor gene elements (V, D and J) that code for Ig and TCR variable regions is defective in the scid mouse mutant. However, the defect is not absolute. Developing scid lymphocytes occasionally succeed in forming a productive VDJ and VJ rearrangement at two critical loci (e.g., IgH/IgL or TCR beta/TCR alpha), and given the appropriate stimuli, differentiate into clones of functional B or T cells. Scid mice with detectable B and/or T lymphocytes are referred to as leaky. This review considers the nature and possible basis of B and T cell leakiness in scid mice.

Animals↗
PubMed1991

Coding joint formation of endogenous T cell receptor genes in lymphoid cells from scid mice: unusual P-nucleotide additions in VJ-coding joints.

The mouse mutation scid adversely affects the process of VDJ recombination. Attempts to form coding joints, that is, to joint V or D to J gene segments generally fail in developing scid lymphocytes. It has been proposed that the scid mutation results a defective VDJ recombinase system. Here we describe five scid T cell lymphomas containing one or two TcR gamma coding joints each, even though the majority of the multiple TcR gamma chain gene rearrangements and all TcR beta rearrangements in these cells were abnormal with the deletions typically found in scid lymphoid cells. One of the five T cell lymphomas was shown to have an active VDJ recombinase system; however, this activity was defective indicating that the scid phenotype has been retained. We conclude that the scid VDJ recombinase system has not completely lost the ability to form coding joints. P-nucleotide additions of unusual length or composition were found at the junctional border in five of the eight TcR gamma coding joints. This might reflect a defect in the activity of a component of the VDJ recombinase system involved in the generation of P-nucleotide additions. In one of the observed rearrangements, a V gamma 5-J gamma 3 coding joint was formed. This establishes the transcriptional orientation of J gamma 3-C gamma 3 and confirms a previously proposed organization of the TcR gamma genes.

Animals↗
PubMed1991

T-lymphocyte development in scid mice is arrested shortly after the initiation of T-cell receptor delta gene recombination.

Scid mice lack functional lymphocytes because they carry a mutation that impairs rearrangement of immunoglobulin and T-cell receptor (TCR) genes. Rearrangement of TCR delta, but not gamma and beta genes, was routinely observed in DNA of scid thymocytes and thymocyte hybridomas. TCR delta gene rearrangements appeared to involve D delta 1, D delta 2, and J delta 1 elements only; rearrangement of elements upstream of D delta 1 (e.g., V delta 1) was not observed, and transcripts corresponding to fully assembled TCR delta genes (VDJ delta or VDDJ delta) were not detected in RNA from scid thymocytes. These findings suggest that D delta 1, D delta 2, and J delta 1 may be among the first TCR gene elements to undergo recombination and that scid T-lineage cells are developmentally arrested during or shortly after this stage of differentiation. One class of TCR delta recombination fragments (D delta 2-J delta 1) was amplified by the polymerase chain reaction (PCR) and cloned, and the recombination junctions were sequenced. Most fragments showed normal coding joints. Interestingly, five of seven coding joints that lacked N insertions showed evidence of recombination between short stretches (2-3 bp) of homologous sequence. As discussed, the general absence of V delta-, J gamma-, and J beta-associated rearrangements, despite the occurrence of normal D delta 2-J delta 1 rearrangements, raises the possibility that the scid mutation may cause premature cessation of TCR gene recombination and thereby arrest early T-cell development.

Animals↗