Biomedical subjects
Publications and source records attributed to F W Alt.
The ability of lymphocyte receptor V, D and J gene segments to rearrange generates much of the receptor diversity that is the hallmark of the immune system. Naturally, the mechanisms of immunoglobulin and T-cell receptor gene recombination are of enormous interest. Here, Fred Alt and colleagues review current understanding of the process and speculate on future findings.
Expression of both N-myc and c-myc is induced rapidly and dramatically in normal pre-B cells after stimulation with interleukin-7 (IL-7), a pre-B cell-specific growth factor. These IL-7-induced increases in N-myc and c-myc expression are mediated at the transcriptional level; for N-myc, a major portion of the induction results from release of an attenuation block between exons 1 and 2. Although c-myc expression has been shown to be regulated by many factors, these studies provide the first demonstration of N-myc regulation by a growth factor. Furthermore, mitogen-induced proliferation of more mature B-lineage cells is accompanied by induction of c-myc expression in the complete absence of N-myc expression. Thus, N-myc expression in normal B-lineage cells, as in transformed cells, is limited to cells that represent the precursor stages of this differentiation pathway. Together, these findings imply a specific function for N-myc in the early stages of B-cell development.
The N-myc gene encodes a putative transcription factor that is thought to function in the regulation of gene expression during cell differentiation and/or growth. To examine the role of N-myc during development, we have used targeted mutagenesis in embryonic stem cells to produce a mouse line that carries an N-myc null allele. Mice homozygous for the mutation died between 10.5 and 12.5 days of gestation. Histological analysis of mutant embryos revealed that organs and tissues expected at these stages of development were present. However, multiple defects were observed, primarily in tissues and organs that normally express N-myc. In particular, mutant hearts were underdeveloped, often retaining the S-shape more typical of 9-day-old embryos. In addition, cranial and spinal ganglia were reduced in size and/or cellularity. Most of the noted defects were more consistent with a role of N-myc in proliferation of precursor populations than with a block in differentiation per se, at least at these early stages. These results demonstrate that N-myc plays an essential role during development and clearly confirm that N-myc has a physiological function that is distinct from that of the other myc-family genes.
We have used nuclear run-on and DNase I sensitivity analyses to study the activity of the N-myc genes in cell lines that represent different stages of B-cell development. Both transformed pre-B-cell lines and a nontransformed pre-B-cell clone transcribe the N- and c-myc genes at substantial levels; in the nontransformed clone, transcription of these genes is regulated by the pre-B-cell growth factor interleukin-7. In contrast, transformed cell lines that represent the more mature stages of the B-cell pathway and mitogen-stimulated normal splenic B lymphocytes express the c-myc gene but do not express the N-myc gene at detectable levels. Down-regulation of N-myc expression in these cells occurs at the level of transcriptional initiation. Correspondingly, a set of DNase I-hypersensitive sites present in the 5' region of the N-myc promoter of pre-B-cell lines are absent in B-cell lines. To further elucidate this process, we have constructed fusion cell lines between an N-myc-expressing pre-B-cell line and a nonexpressing myeloma line; the hybrid cell lines transcriptionally down-regulate the pre-B copies of the N-myc gene. Lack of N-myc expression in a number of nonlymphoid cell lines also resulted from lack of N-myc transcription. Together, our findings demonstrate that the down-regulation of N-myc expression in the later stages of B-cell development is mediated primarily at the level of transcriptional initiation. They further show that dominant, trans-acting factors present in more mature B-lineage cell lines act to down-regulate the transcription of N-myc.
Mouse plasmacytomas induced by pristane oil alone, or in combination with Abelson murine leukemia virus (A-MuLV), regularly carry one of three alternative chromosomal translocations that juxtapose c-myc to immunoglobulin heavy- or light-chain loci. E mu-c-myc transgenic mice develop translocation-free plasmacytomas after induction by pristane oil and/or A-MuLV [Sugiyama, H., Silva, S., Wang, Y., Weber, G., Babonits, M., Rosen, A., Wiener, F. & Klein, G. (1990). Int. J. Cancer, 46, 845-852]. In order to test whether another member of the myc family, N-myc, could play a similar role as c-myc, we treated E mu-N-myc transgenic mice with pristane and helper-free A-MuLV. Of 20 mice that received a single pristane injection followed by A-MuLV, 17 developed plasmacytomas with a mean latency period of 54 +/- 20 days. In a corresponding group that only received a single pristane injection, five out of six transgenic mice developed plasmacytomas with a mean latency period of 142 +/- 32 days. However, after three monthly injections of pristane, all 15 transgenic mice developed plasmacytomas with a mean latency period of 128 +/- 20 days. All plasmacytomas expressed the N-myc transgene, while none of them expressed either c-myc or endogenous N-myc. None of the tumors carried the usual plasmacytoma-associated translocations.
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The human VH5 family consists of two functional genes and one pseudogene. We have found a novel 1.2-kb VH5 gene transcript in normal fetal liver and cord blood and in transformed B lineage cells. VH5-positive cDNA clones were isolated from precursor B acute lymphoblastic leukemia, B chronic lymphoblastic leukemia, Epstein-Barr Virus-transformed B cell lines, and cord blood, and were identified as transcripts of unrearranged VH5 genes (germline transcripts). The cDNA clones were derived from both functional and pseudo-VH5 genes. Most germline transcripts appear to initiate at the normal VH promoter and are cleaved and polyadenylated at sites several hundred bases downstream of the VH5 coding region. Correct splicing of the leader intron was observed in all clones. In functional and pseudo-VH5 cDNAs, an open translational reading frame extends from the leader to a termination codon in the nonamer. Only limited polymorphisms were observed in the coding as well as flanking regions of the VH5 transcripts. Functional and pseudo-VH5 transcripts and previously identified murine germline VHJ558 transcripts are discussed.
The NZB mouse is genetically predisposed to the development of autoimmune disease that resembles the human autoimmune systemic lupus erythematosus and autoimmune hemolytic anemia, with increased titers of anti-DNA, and Coombs' autoantibodies. The various autoimmune traits are controlled separately by a limited number of genes. Genetic studies have shown that several immune loci are involved in autoimmunity: T cell abnormalities, H-2 complex and immunoglobulin genes have been implicated. In this report, we present evidence for a significant correlation of NZB V kappa 1 haplotype defined by restriction fragment length polymorphism analysis with anti-erythrocyte autoantibodies in NZB x 129/J and NZB x SM/J recombinant inbred lines.
Preferential usage of JH-proximal VH genes has been demonstrated in immature murine B cell repertoires. To determine whether this phenomenon is also evident in human repertoires, we studied utilization of VH6, the most JH-proximal human VH gene. Examination of VH gene usage in a panel of precursor B cell acute lymphoblastic leukemia samples indicated that 15% of the IgH rearrangements utilized VH6. VH6 is a single-member family in a total repertoire of 100-200 VH genes; thus, if usage were purely random, one would expect VH6 rearrangement frequency to be less than 1%. Analysis of VH gene usage in normal lymphoid tissues also revealed biased usage of VH6. VH6 was preferentially utilized in 16- to 24-week-old fetal liver as compared to adult peripheral blood mononuclear cells or spleen. Possible implications of the conservation of preferential usage of JH-proximal genes in both immature murine and human repertoires are discussed.
X-linked agammaglobulinemia (XLA) is characterized by an arrest in early B lymphocyte differentiation. Precursor B cells are present in the bone marrow (BM), whereas peripheral blood B cell numbers are severely decreased. A series of Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) was established from peripheral blood of three XLA patients belonging to one pedigree. These BLCL manifested productive VHDJH rearrangements and a random utilization of the VH families. The CDR3 regions of the rearrangements varied in length from 12 to 47 nucleotides and included N regions in all cases. The results supported the conclusion that the few B lymphocytes in peripheral blood of XLA patients exhibit all mechanisms that generate immunoglobulin (Ig) heavy (H) chain diversity. However, no evidence for somatic mutation was found. Within the VH3 family 50% of the expressed VH gene segments belonged to a single subgroup and within the VH4 family a preferential utilization of one VH4 gene element was observed. The utilization of H chain joining (HH) elements was biased to JH4 and JH6 and a high percentage of the CDR3 regions was found to be generated by unconventional mechanisms, such as multiple D usage and the fusion of D elements to D segments with irregular recombination recognition signals. These unique features of the recombined and expressed VHDJH regions in XLA may explain the inability of XLA patients to respond to a variety of antigens. Alternatively, they could be secondary to a B lymphocyte maturation defect in XLA.
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X-linked agammaglobulinemia (XLA) patients manifest a very low production of immunoglobulins (Ig) of all classes and plasma cells are virtually absent. The XLA gene plays a crucial role in the transition of pre-B cells to later B cell stages, as hardly any slg-positive B lymphocytes can be detected. In the bone marrow almost normal numbers of pre-B lymphocytes are present. These cytoplasmatic C mu+ pre-B lymphocytes appear to express truncated M heavy chain molecules lacking the variable region segment. The T lymphocyte compartment is intact: the numbers of mature T cell receptor (TcR) alpha beta expressing T lymphocyte populations and their proliferative responses to antigens are normal. That the B cells are primary and exclusively affected was proven by X-chromosome inactivation studies. There is no evidence that the XLA gene is directly involved in the Ig gene rearrangements since B lymphoblastoid cell lines (BLCLs) established from peripheral blood of XLA patients were found to produce IgM molecules composed of complete Ig heavy and light chains and were shown to contain normal VHDJH recombinations. The data do not exclude the involvement of the XLA gene in a B cell specific process that makes the Ig loci accessible for recombination. Investigations on the degree of diversity of immunoglobulins generated by XLA patients exposed no limitations in the VH family usage. Sequence analysis of expressed VH3 and VH4 rearrangements however revealed that some genetic elements of the Ig locus might be over-represented and that a high portion of rearrangements was generated by unconventional mechanisms. By restriction length polymorphism (RFLP) and pulsed field gel electrophoreses analyses the XLA gene was mapped to an 8- to 12-Mb DNA fragment located in the Xq22 region. The known location of the XLA gene on the X-chromosome with closely linked RFLP markers and the availability of X-chromosome inactivation assays provides methods for carrier detection and prenatal diagnosis.
The immunoglobulin light chain V kappa 1 gene family is polymorphic in murine inbred strains and this family has been subdivided into five sub-groups (V kappa 1A-E). The V kappa 1A sub-group contributes to approximately 2% of the total serum immunoglobulin light chains in several mouse strains. However, it has been reported that this sub-group is absent in New Zealand Black (NZB) mouse serum. Amino acid sequencing of myeloma proteins from this inbred mouse has shown that they belong to the V kappa 1B sub-group. We report here the structure of nine functional germline genes from NZB mice that have high homologies to the V kappa 1A, V kappa 1B, V kappa 1C, and V kappa 1D sub-groups. In addition, a novel germline gene representing the prototype of a new sub-group (designated V kappa 1F) has been identified. We have isolated different V kappa 1 germline genes from a single restriction fragment length polymorphism (RFLP) fragment, as well as identical V genes from two different RFLP migrating bands. Therefore, the complexity of the genes encoding the immunoglobulin variable region cannot be determined solely by RFLP analysis. Nucleotide sequence analysis of 16 V kappa 1 genes which code for NZB autoantibodies indicate that they belong to five different V kappa 1 sub-groups with five hybridomas (31%) expressing the V kappa 1A sub-group. Comparison of the sequences of V kappa 1 genes expressed in hybridomas with corresponding germline genes show no somatic mutations.
Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process.
We found that the canary N-myc gene is highly related to mammalian N-myc genes in both the protein-coding region and the long 3' untranslated region. Examined coding regions of the canary c-myc gene were also highly related to their mammalian counterparts, but in contrast to N-myc, the canary and mammalian c-myc genes were quite divergent in their 3' untranslated regions. We readily detected N-myc and c-myc expression in the adult canary brain and found N-myc expression both at sites of proliferating neuronal precursors and in mature neurons.
To further analyze the VDJ recombination defect in lymphoid pre-B cells from mice with severe combined immune deficiency (scid mice), we have assayed the ability of Abelson murine leukemia virus (A-MuLV) transformed pre-B cells from scid mice to rearrange a recombination substrate in which inverted VH to DJH joins activate a selectable (gpt) gene. In unselected populations, substrate rearrangements occurred frequently, but were aberrant and probably analogous to the aberrant rearrangements observed at endogenous scid Ig gene loci. In contrast, populations of scid pre-B lines selected for gpt activity within the substrate contained mostly "normal" VH to DJH joins within the introduced substrate. These findings demonstrate that scid pre-B cells can make normal joins at low efficiency and are discussed with respect to the potential mechanism of the scid defect and the occurrence of Igs in leaky scid mice.
We have cloned and determined the nucleotide sequence of the Ig VH and VL region genes of an IgM kappa mAb that binds to denatured DNA and myelin from a patient (POP) with chronic lymphocytic leukemia and peripheral neuropathy. Sequence analysis indicates that the V region of the kappa L chain gene (PopVK) has 99% homology to a V kappa IIIa germ-line gene and the V region of the mu H chain gene (PopVH) has 96% homology to the VH26 germ-line gene that is a member of the VH3 gene family. It is likely the V kappa and VH genes arose from these respective germ-line genes via somatic mutation or from closely related genes. V kappa III genes have frequently been used by other IgMk mAb especially those with rheumatoid factor activity, and the VH26 gene with no somatic mutation has been used by several anti-DNA antibodies, suggesting the possibility of preferential association of these or related germ-line genes with autoantibodies. The minor differences between the sequences of POP's VH and V kappa genes and sequences used by other autoantibodies, may be responsible for this antibody's crossreactivity with myelin and, as a result, the autoimmune neuropathy.