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Biomedical subjects

F W Alt

Publications and source records attributed to F W Alt.

At least 145 records · Page 8Linked to original sources
PubMed1993

Impairment of V(D)J recombination in double-strand break repair mutants.

Cells maintain the integrity of their genome through an intricate network of repair systems that recognize and remove lesions from DNA. The only known site-directed recombination process in vertebrates is the V(D)J recombination of lymphocyte antigen receptor genes. A large panel of cell lines deficient in DNA repair were tested for the ability to perform V(D)J recombination after introduction of the RAG-1 and RAG-2 genes. Two mutants failed to generate normal V(D)J recombination, and further analysis provided evidence for two distinct nonlymphoid-specific genes that encode factors involved in both DNA repair and V(D)J recombination.

Animals↗
PubMed1993

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.

Aging↗
PubMed1993

Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

Animals↗
PubMed1993

LH-2: a LIM/homeodomain gene expressed in developing lymphocytes and neural cells.

A screen for early markers of B-lymphocyte differentiation has identified a homeobox gene, denoted LH-2, that has a pattern of expression distinct from that of other related genes. The LH-2 cDNA sequence encodes a polypeptide of 426 amino acids that contains a homeodomain and two repeats of a cysteine-rich domain referred to as a LIM domain. The homeodomain of the LH-2 protein is related to that of other LIM/homeodomain proteins, most strikingly with that of the Drosophila apterous protein. Expression of LH-2 was found in B- and T-lymphoid cell lines. Expression in B-cell lines was highest in lines that represent early stages of differentiation, whereas in T-cell lines there was no clear correlation with the stage of differentiation. In embryonic and adult tissues, the highest level of LH-2 expression was found in discrete regions of the developing central nervous system, primarily in diencephalic and telencephalic structures, and in a subset of lymphoid tissues. The expression pattern and structural characteristics of the LH-2 gene suggest that it encodes a transcriptional regulatory protein involved in the control of cell differentiation in developing lymphoid and neural cell types.

Aging↗
PubMed1993

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.

Animals↗
PubMed1993

A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon.

LPS stimulation of B lymphocytes induces germline transcription of and subsequent switching to the gamma 2b gene. Mature germline transcripts contain an I exon (non-coding) spliced to the C gamma 2b exons. To investigate the role of germline transcription and/or transcripts in heavy chain class switching, we have replaced the germline I gamma 2b promoter and I exon in ES cells with an expressed neomycin resistance gene. The mutated chromosome retains the downstream target sequence for switch recombination (S regions) and all sequences necessary for expression of a switched gamma 2b gene. Wild-type or mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all lymphocytes were ES-cell derived. Chimeras derived from injection of heterozygous mutant ES cells had normal levels of serum IgG2b, but their splenic B cells showed a partial decrease in ability to switch to gamma 2b. Strikingly, B lymphocytes from chimeras derived by injection of homozygous mutant ES cells were deficient in IgG2b production both in vivo and in vitro, but normal with respect to production of other Ig heavy chain isotypes. Additional studies demonstrated that lack of ability to produce IgG2b by the mutant B cells correlated with lack of germline transcription and resulted from a specific defect in class-switch recombination to S gamma 2b. Together, these studies demonstrate that the I region is an important regulatory element for control of class-switch recombination.

Animals↗
PubMed1993

Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus.

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (E mu) in generating accessibility of the JH locus for VDJ recombination, we generated ES cells in which E mu or its flanking sequences were mutated by replacement with or insertion of an expressed neor gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell lines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core E mu [which includes part of the 3' matrix associated region (MAR) and the I mu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neor gene into the JH-C mu intron does not affect JH accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the E mu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated JH locus. Surprisingly, insertion of the neor gene into the 5' MAR sequence approximately 100 bp upstream of the core E mu also dramatically decreased recombination of the linked JH locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the E mu and upstream flanking sequences is required for efficient rearrangement of the JH locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.

Animals↗
PubMed1993

Gene rearrangement and B-cell development.

The differentiation of B lymphocytes from their progenitors progresses through a series of successive stages that are defined by sequential rearrangement of Ig loci and surface expression of various stage-specific markers, including Ig heavy and light chain proteins. Considerable evidence suggests that the appearance of cells with an orderly progression of Ig gene rearrangements is linked to the expression of the rearranged Ig gene products. Recent experiments have clarified our understanding of mechanisms by which rearrangement of Ig gene segments is controlled and how Ig gene products participate in the regulation of the B-cell differentiation program.

Animals↗
PubMed1993

Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus.

B lymphocyte differentiation is characterized by an ordered series of Ig gene assembly and expression events. In the majority of normal B cells, assembly and expression of Ig heavy (H) chain genes precedes that of light (L) chain genes. To determine the role of the Ig heavy chain protein in B cell development and L chain gene rearrangement, we have generated mice that cannot assemble Ig H chain genes as a result of targeted deletion of the JH gene segments in embryonic stem cells. Mice homozygous for this deletion are devoid of slg+ B cells in the bone marrow and periphery. B cell differentiation in these mice is blocked at the large, CD43+ precursor stage. However, these precursor B cells do assemble kappa L chain genes at a low level in the absence of mu H chain proteins. These data demonstrate that rearrangement and expression of the mu H chain gene is not absolutely required for kappa L chain gene rearrangement in vivo. Expression of mu chains may facilitate either efficient L chain gene rearrangement or the survival of cells that have rearranged light chain genes by promoting the differentiation of large, CD43+ to small, CD43- pre-B cells.

Animals↗
PubMed1993

Comparison of RAG gene expression in normal and transformed precursor lymphocytes.

Analyses of mechanisms that regulate V(D)J recombination have relied heavily on the use of transformed precursor lymphocyte cell lines. We now show that such lines have highly variable and frequently low levels of recombination activating genes (RAG)-1 and -2 gene expression. We also show that expression levels of the RAG genes can vary > 100-fold between different subcloned cells of an individual pre-B line. We discuss these findings in the context of normal regulation of RAG gene expression and the implication for the use of transformed pre-B cell lines as models for studying control of V(D)J recombination activity.

Animals↗
PubMed1993

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

Animals↗
PubMed1993

Binding of myc proteins to canonical and noncanonical DNA sequences.

Using an in vitro binding-site selection assay, we have demonstrated that c-Myc-Max complexes bind not only to canonical CACGTG or CATGTG motifs that are flanked by variable sequences but also to noncanonical sites that consist of an internal CG or TG dinucleotide in the context of particular variations in the CA--TG consensus. None of the selected sites contain an internal TA dinucleotide, suggesting that Myc proteins necessarily bind asymmetrically in the context of a CAT half-site. The noncanonical sites can all be bound by proteins of the Myc-Max family but not necessarily by the related CACGTG- and CATGTG-binding proteins USF and TFE3. Substitution of an arginine that is conserved in these proteins into MyoD (MyoD-R) changes its binding specificity so that it recognizes CACGTG instead of the MyoD cognate sequence (CAGCTG). However, like USF and TFE3, MyoD-R does not bind to all of the noncanonical c-Myc-Max sites. Although this R substitution changes the internal dinucleotide specificity of MyoD, it does not significantly alter its wild-type binding sequence preferences at positions outside of the CA--TG motif, suggesting that it does not dramatically change other important amino acid-DNA contacts; this observation has important implications for models of basic-helix-loop-helix protein-DNA binding.

Amino Acid Sequence↗
PubMed1993

DNA binding by N- and L-Myc proteins.

N- and L-Myc, like c-Myc, contain adjacent basic region (BR), helix-loop-helix (HLH) and leucine zipper (LZ) motifs, which characterize a family of DNA-binding proteins. We have used a polymerase chain reaction (PCR)-based binding site selection technique to demonstrate that the most highly preferred binding site for both N- and L-Myc fusion proteins contains a CACGTG motif, the core binding sequence previously identified for c-Myc. Further analysis identified other N-Myc binding sequences, including asymmetric sequences such as CAT-GTG. N-Myc, like c-Myc, preferentially forms heterodimeric DNA-binding complexes with Max protein. Mutational analyses of N-Myc basic region (BR), helix-loop-helix (HLH) and leucine zipper (LZ) regions revealed that all three regions are necessary for DNA binding by N-Myc-Max complexes, and that dimerization requires both HLH and LZ motifs, while BR sequences are needed only for DNA binding. Our findings support the notion that the LZ motif is a critical element in dimer formation by bHLH-LZ proteins.

Amino Acid Sequence↗
PubMed1992

Function and control of recombination-activating gene activity.

The RAG-1 and RAG-2 genes synergistically confer VDJ recombinase activity to nonlymphoid cell lines. To unequivocally test RAG gene function, we created lines of mice that lack functional copies of these genes. Consistent with the possibility that RAG gene encode the tissue-specific components of VDJ recombinase, RAG-2-deficient mice are viable but have a severe combined immune deficiency due to inability to initiate VDJ recombination and thereby generate mature lymphocytes. RAG-2-deficient mice have no obvious defect in any tissue or lineage other than lymphocytes, indicating that VDJ recombinase activity and RAG-2-gene function is required only for lymphocyte development. Levels of RAG-1 and RAG-2 expression in primary murine lymphoid tissues and lymphoid bone marrow cultures generally are much higher than those of transformed precursor B-cell lines. Low-level RAG gene expression in permanent cell lines results from a decline during propagation due to outgrowth of cells with lower RAG expression levels. The low and variable level of RAG gene expression in transformed pre-B cell lines correlates with low and variable rates of endogenous VDJ recombination; therefore, such lines are not reliable models for experiments aimed at studying mechanisms that target this activity to particular variable region gene segments. To generate such a system, we introduced RAG genes into B-lineage lines under the control of a heat shock-inducible promoter; heat-shock treatment induces extremely high-level but transient RAG expression accompanied by parallel induction of VDJ recombinase activity. Such cells efficiently rearrange transfected VDJ recombination substrates in a regulated manner that is dependent on the activity of transcriptional control elements associated with the target V gene segments.

Animals↗
PubMed1992

High frequency of myelomonocytic tumors in aging E mu L-myc transgenic mice.

Transgenic mice that contain constructs of the L-myc gene under the transcriptional control of the immunoglobulin heavy chain enhancer (E mu) develop thymic hyperplasia and are predisposed to T cell lymphomas. Here we describe a second form of malignancy that occurs in aging E mu L-myc transgenic mice. The mean latency period for the development of this malignancy is longer compared with the E mu L-myc T cell lymphomas but the overall incidence is increased threefold. The histopathological morphology is that of a highly malignant mesenchymal neoplasm that closely resembles human fibrous histiocytoma. The tumor cells were classified as myelomonocytic on the basis of several lineage-specific markers and the lack of rearrangements of the immunoglobulin heavy chain and the T cell receptor beta loci. Cultured tumor cells produce macrophage colony-stimulating factor (M-CSF) protein and express the M-CSF receptor, suggesting the involvement of an autocrine loop in this malignancy. Similar to the E mu L-myc T cell lymphomas, these tumors show high-level transgene expression but no detectable levels of endogenous c-myc mRNA, directly implicating the deregulated expression of L-myc in the generation of this malignancy. E mu L-myc myelomonocytic tumors show consistent trisomy of chromosome 16, implicating this as a secondary event in the development of this tumor. In the light of recent findings that L-myc is expressed in human myeloid leukemias and in several human myeloid tumor cell lines, the results described here might implicate L-myc in the development of naturally occurring myeloid neoplasias.

Aging↗
PubMed1992

Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines.

Transgenic mice carrying either the c-myc or N-myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre-B and B cell lymphomas (E mu-c-myc and E mu-N-myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v-myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre-B stage of development-including the recombinase activating genes RAG-1 and RAG-2. Continued RAG-1 and RAG-2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross-linking of the surface immunoglobulin on these lines with an anti-mu antibody leads to rapid, specific and reversible down-regulation of RAG-1 and RAG-2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG-1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre-B to B cell transition.

Animals↗
PubMed1992

A novel regulatory myosin light chain gene distinguishes pre-B cell subsets and is IL-7 inducible.

We describe a novel regulatory myosin light chain gene (termed precursor lymphocyte-specific regulatory light chain or PLRLC) that is expressed specifically in precursor B and T lymphocytes. PLRLC is the first example of a regulatory myosin light chain gene which displays specific expression in non-muscle cells. PLRLC is expressed in adult bone marrow derived normal and transformed pre-B cells; in the former, PLRLC expression levels are induced by the pre-B cell specific growth factor interleukin-7 (IL-7). PLRLC is not expressed in either transformed pre-B cells derived from fetal liver or in normal fetal liver pre-B clones grown in the presence of IL-7. Therefore this gene provides the first marker that clearly distinguishes these two pre-B subsets. Finally, several of the different PLRLC transcripts potentially encode regulatory myosin light chains with unique structural features. The unique distribution, regulation and structural features of the PLRLC gene products suggest an important role for PLRLC during lymphocyte development.

Amino Acid Sequence↗