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Biomedical subjects

U Storb

Publications and source records attributed to U Storb.

At least 19 recordsLinked to original sources
PubMed1992

N region diversity of a transgenic substrate in fetal and adult lymphoid cells.

The rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes requires the activity of an as yet undefined V(D)J recombinase. One component of the recombinase appears to be a terminal transferase which may be involved in the addition of untemplated nucleotides (N regions) to the V(D)J joints. It has been observed that rearranged Ig and TCR genes isolated from fetal liver have few if any N regions, whereas in the adult mouse, these genes have a large number of untemplated nucleotides. The presence of N regions greatly alters the composition of the third hypervariable, complementarity determining region of the respective proteins, thus playing a major role in the conformation of the binding site. It was possible that, for functional reasons, N region-containing Ig and TCR genes were not permissible at the fetal stage of development. We have produced transgenic mice with a rearrangement test gene which, after V-J recombination, does not result in the production of functional Ig or TCR proteins. We report here that the rearrangement products have no N regions in fetal liver, but that the majority of joints in adult lymphoid tissues have N additions. The study is also an interesting demonstration of the randomness of rearrangements and the enormous variability that can be created from a single pair of V and J sequences.

Animals↗
PubMed1992

Analysis of a T cell receptor gene as a target of the somatic hypermutation mechanism.

In an effort to identify cis-acting elements required for targeting of the somatic hypermutation process in mice, we examined whether a T cell receptor (TCR) transgene under the control of the immunoglobulin (Ig) heavy (H) chain intron enhancer would be mutated in antigen-stimulated B cells. Hybridomas were established from splenic B cells of mice carrying two copies of the TCR transgene after hyperimmunization with phosphorylcholine keyhole limpet hemocyanin. Northern analysis revealed that all of the transgene-containing hybridomas expressed the TCR mRNA. Multiple somatic point mutations were found in seven of eight endogenous Ig VH genes examined. In contrast, 29 of 32 TCR genes examined contained no mutations. One potential mutation was seen in each of the three other TCR genes. Our data indicate that although the TCR transgene is expressed in B cells, it is not efficiently targeted by the mutator mechanism. Furthermore, the presence of an Ig H chain enhancer is itself not sufficient for targeting of the somatic hypermutation mechanism.

Animals↗
PubMed1992

Rearrangement and expression of immunoglobulin genes in transgenic mice.

Transgenic mice are discussed which carry a rearrangement test transgene. The methylation status of the transgene varies, depending on the background mouse strain. When the transgene is bred into the C57BL/6 strain, it is completely methylated and not rearranged in lymphoid organs. After several generations of crossing into DBA/2 or SJL the transgene becomes unmethylated and rearranges at high frequency. A strain specific modifier of DNA methylation (Ssm-1) was mapped close to the Friend virus susceptibility locus (Fv-1) on mouse chromosome 4. Rearranged transgenes from spleen, bone marrow and thymus of adult mice or fetal liver were cloned and sequenced. A great variety of joints was found, with about 1/3 being in the correct reading frame. Small deletions into the V- and J-coding ends as well as N region additions contributed to the variability. The fetal joints showed no N regions. Since no functional immunoglobulin (Ig) gene can be created from this artificial test gene, the data indicate that the rearrangement mechanism of the fetus differs from that of the adult.

Animals↗
PubMed1992

Two conserved essential motifs of the murine immunoglobulin lambda enhancers bind B-cell-specific factors.

Two highly homologous enhancers associated with the two murine immunoglobulin lambda constant-region clusters were recently identified. In order to better understand the molecular basis for the developmental stage- and cell-type-restricted expression of lambda genes, we have undertaken an analysis of the putative regulatory domains of these enhancers. By using a combination of DNase I footprinting, electrophoretic mobility shift assay, and site-specific mutations, four candidate protein binding sites have been identified at analogous positions in both enhancers. A mutation of any of these sites decreases enhancer activity. Two of the sites, lambda A and lambda B, are essential for enhancer function, and both of these sites appear to bind both B-cell-specific and general factors. Nevertheless, isolated lambda A and lambda B sites show no evidence of inherent transactivating potential, alone or together, even when present in up to three copies. We suggest that the generation of transactivating signals from these enhancers may require the complex interaction of multiple B-cell-specific and nonspecific DNA-binding factors.

Animals↗
PubMed1992

Fusion of a scid pre-B cells with a wild type (myeloma) B cell results in correct rearrangement of a V(D)J recombination substrate.

Mice with the scid mutation have a defect in the V(D)J recombinase. In order to determine whether the SCID product is normally present in mature B cells that do not have the recombinase activity, scid pre-B cells were fused with myeloma cells. It was found that in the hybrid cells, a rearrangement test gene was correctly joined immediately after fusion. The same test gene was aberrantly rearranged in the scid pre-B cells. Stable hybrids between the scid pre-B and the myeloma cells had lost the expression of RAG-1 and RAG-2 genes, supporting the previous finding of an inhibitor of rearrangement in myeloma cells that acts shortly after fusion. Thus, mature B cells apparently contain the SCID product, the wild type SCID function is not competitively interfered with by products present in scid pre-B cells, and the SCID product seems not to be a target for the recombinase inhibitor.

Animals↗
PubMed1991

Identification and localization of an enhancer for the human lambda L chain Ig gene complex.

A strong transcriptional enhancer was identified for the human lambda L chain Ig gene complex. Enhancer activity was measured by activation of the chloramphenicol acetyl transferase (CAT) gene in a transient assay using both mouse and human B lymphoid cell lines. The smallest fragment identified with enhancer activity was 111 bp, which resides 11.7 kb downstream (3') of C lambda 7, a constant region gene we have recently isolated and identified as functional in the human population. Enhancer activity is orientation independent, tissue specific (active in all B cell lines tested and not in a T cell line), and independent of NF kappa B, similar to the mouse lambda enhancers recently reported. The human lambda enhancer is active in both mouse and human B cell lines; interestingly, the mouse lambda enhancers are active in mouse lines but not in a human B cell line. DNA sequence comparison of the mouse and human lambda enhancers indicates a higher degree of homology (average of 72.5%) within the 111-bp enhancer core region identified here than for the remaining flanking sequence compared (average of 42%). This discovery of an enhancer in the human lambda locus (HuE lambda), which is clearly distinct from that of the H and L chain loci, will help to determine the mechanism for the ordered expression and rearrangement of these gene complexes in B cell ontogeny. The presence of only one enhancer in the human C lambda complex 3' of all the C genes suggests that the evolutionary duplication of the L locus differs from that seen in mouse; in mouse the duplication unit was JCJC-enhancer, whereas the human JC lambda s duplicated without the enhancer.

Animals↗
PubMed1991

A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci.

A transgene, pHRD, is highly methylated in 12 independent mouse lines when in a C57BL/6 strain background, but becomes progressively less methylated when bred into a DBA/2 background. Transgenes inherited from the mother are generally more methylated; however, this parental effect disappears following continued breeding into the nonmethylating strain. Mapping experiments using BXD recombinant inbred mice as well as other inbred strains indicate that a single strain-specific modifier (Ssm-1) linked to, but distinct from, Fv-1 is responsible for the strain effect. In addition to the methylated and unmethylated transgenic phenotypes, certain mice exhibit a partial methylation pattern that is a consequence of an unusual cellular mosaicism. The pHRD transgene, containing target sequences for the V(D)J recombinase, undergoes site-specific recombination only in lymphoid tissues. This V-J joining is restricted primarily to unmethylated transgene copies.

Animals↗
PubMed1991

Precursors of both conventional and Ly-1 B cells can escape feedback inhibition of Ig gene rearrangement.

Experiments with transgenic mice carrying rearranged Ig transgenes have shown that membrane bound Ig molecules cause feedback inhibition of endogenous Ig gene rearrangement. However, this inhibition is never complete. It has been postulated that escape from feedback may be a property of the Ly-1 B cell subset, whereas rearrangement of endogenous Ig genes may be completely inhibited in conventional B cells. This possibility was investigated in transgenic mice carrying a lambda transgene under the control of the H chain enhancer. It was found that kappa producing B cells in these lambda transgenic mice were for the most part, although not exclusively, of the conventional B cell phenotype. Examination of peritoneal exudate cells revealed that a large proportion of Ly-1 B cells also express kappa. Adoptive transfer of bone marrow from adult lambda transgenic mice, a source of conventional B cell precursors, resulted in the production of relatively high levels of serum kappa 2 to 3 mo after transfer into recipient SCID mice. A high proportion of donor B cells in the spleen produced endogenous kappa protein with or without co-production of lambda. It is concluded that precursors of both conventional and Ly-1 B cells can escape feedback inhibition of L chain gene rearrangement.

Animals↗
PubMed1991

Factors affecting the rearrangement efficiency of an Ig test gene.

A rearrangement test gene, pHRD, containing the mouse IgH enhancer and the metallothionein promoter, has previously been shown to rearrange efficiently after transfection into a pre-B cell line. Experiments are now reported that assess the requirements of the DNA substrate as well as of the transfected cells for efficient rearrangement. It was found that deletion of the metallothionein promoter or substitution of the IgH enhancer by the kappa enhancer did not affect rearrangement. However, deletion of the Ig enhancer reduced the efficiency. Transfection of pHRD into stable hybrids of pre-B cells and myeloma cells resulted in a high frequency of rearrangement only if certain myeloma chromosomes were lost. Furthermore, pHRD introduced into rearrangement incompetent myeloma cells upon subsequent cell fusion with pre-B cells was rearranged only very rarely and then apparently only immediately after cell fusion. Stable pre-B cell x myeloma hybrids that retained the critical myeloma chromosomes were found to have lost VDJ recombinase activity and transcripts of the RAG-1, RAG-2 and TdT genes. It is concluded that transcription, i.e., the copying of the DNA by polymerase, is probably not required for rearrangement, but that the rearrangement substrate must be in an "open" chromatin state, such as may be provided by transcriptional factors. Furthermore, the absence of rearrangement in myeloma cells is apparently due to the continued action of an inhibitor of rearrangement.

Animals↗
PubMed1991

Mutation pattern of immunoglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication.

We have previously demonstrated that B lymphocyte specific somatic mutations are introduced into the variable regions of immunoglobulin kappa transgenes in two independent transgenic mouse lines. The frequency, distribution and nature of these mutations strongly suggest that they arose as a result of the process of somatic hypermutation, which is responsible, in part, for affinity maturation during an immune response. Unexpectedly, in these multiple copy transgenic lines, many of the transgene copies showed no evidence of somatic mutation. This paradox was addressed by determining the sequence of each transgene copy in several B cell hybridomas derived from a mouse line carrying three copies of the kappa transgene. It was found that the somatic hypermutation process in different B cells from the same mouse preferentially targets one, but not the same, transgene copy. We present a model, based on the pattern of this targeting, which links somatic hypermutation to the orientation of the Ig gene relative to the direction of DNA replication.

Animals↗
PubMed1990

Analysis of somatic mutations in kappa transgenes.

We have examined the nature and localization of somatic mutations in three kappa transgenes cloned from IgG-secreting hybridomas. All of the mutations identified were single base substitutions. Mutations were localized to the variable (V) region and its flanking sequences. In every case, the nuclear matrix association region, kappa enhancer, and C gene were spared. These data indicate that the rearranged kappa gene contains the necessary sequences for targeting of the mutation process, and suggest that the observed localization of mutations to the V region reflects the inherent specificity of this mutation process.

Animals↗
PubMed1990

Differential splicing of thymosin beta 4 mRNA.

A cDNA clone was isolated from a mouse pre-B cell line, the sequence of which has a very high homology with rat and human thymosin beta 4 genes. However, the mouse clone has an insertion of 98 bp relative to the published rat and human sequences upstream of the coding region. By isolation of a second set of clones from a different cDNA library and by cloning a PCR amplified region of mouse genomic DNA it was confirmed that the insertion is not a cloning artifact. Furthermore, it was shown by RNase protection assays with RNA from the pre-B cell line that two sizes of thymosin beta 4 mRNA exist, a long form containing the 98 nucleotide insertion, and a short form that corresponds to the known rat and human mRNA. The short form is about 50 times more abundant than the long form. Analysis of genomic DNA by sequencing and Southern blotting revealed that both forms are encoded by a single gene in the mouse. The two forms of mRNA arise by differential RNA splicing; the long mRNA contains three separate exons, whereas the short mRNA is missing exon 2. The long mRNA is present in two different pre-B cell lines, spleen and thymus, but could not be detected in brain, liver, and kidney. It is possible that the longer mRNA, which encodes a hydrophobic NH2-extension of six additional amino acids, plays a role in lymphocyte function or development. In contrast to the mouse which has a single thymosin beta 4 gene, rat and human have multiple homologs. Most or all of these also contain sequences that cross-hybridize with the newly discovered exon 2. A polymorphic thymosin beta 4 gene has been found in human DNA.

Amino Acid Sequence↗
PubMed1990

A novel enhancer in the immunoglobulin lambda locus is duplicated and functionally independent of NF kappa B.

As a first step toward defining the elements necessary for lambda immunoglobulin gene regulation, DNase I hypersensitive sites were mapped in the mouse lambda locus. A hypersensitive site found 15.5 kb downstream of C lambda 4 was present in all the B-cell but not in the T-cell lines tested. This site coincided with a strong B-cell-specific transcriptional enhancer (E lambda 2-4). This novel enhancer is active in myeloma cells, regardless of the status of endogenous lambda genes, but is inactive in a T-cell line and in fibroblasts. The enhancer E lambda 2-4 functions in the absence of the transcription factor NF kappa B, which is necessary for kappa enhancer function. No evidence could be found for NF kappa B binding by this element. Rearrangement of V lambda 2 to JC lambda 3 or JC lambda genes deletes E lambda 2-4; however, a second strong enhancer was found 35 kb downstream of C lambda 1, which cannot be eliminated by lambda gene rearrangements. The second lambda enhancer (E lambda 3-1) is 90% homologous to the E lambda 2-4 sequence in the region determined to comprise the active enhancer and likewise lacks the consensus binding site for NF kappa B. The data support a model for the independent activation of kappa and lambda gene expression based on locus-specific regulation at the enhancer level.

Animals↗
PubMed1990

Delay of early B-lymphocyte development by gamma 2b immunoglobulin transgene: effect on differentiation-specific molecules.

Mice transgenic for gamma 2b Ig heavy chain were examined for alterations in B-cell differentiation and endogenous Ig gene rearrangement and expression. Fresh bone marrow from these mice was markedly reduced in BP-1+ cells and there were small reductions in B220+ and sIg+ cells. A-MuLV (Abelson murine leukemia virus) transformants from these bone marrow cells showed little alteration in Ig gene rearrangement and expression when compared to controls, however. Isolation of the B-lymphoid compartment from these mice in vitro using LBMC (lymphoid bone marrow cultures) enabled more detailed characterization of the effects of the transgene. LBMC derived from gamma 2b transgenic mice had similar growth kinetics, but a 4-5-week delay in the expression of endogenous mu Ig in comparison to control cultures. Nucleic acids derived from these early cultures prior to endogenous mu Ig expression showed reduced Ig JH rearrangements, some sterile mu transcription, low levels of BP-1 expression, and virtually undetectable TdT (terminal deoxynucleotidyl transferase) expression. Thus, this gamma 2b transgene appears able to affect early B-lymphocyte development.

Animals↗
PubMed1989

Alteration of the B cell surface phenotype, immune response to phosphocholine and the B cell repertoire in M167 mu plus kappa transgenic mice.

M167, mu plus kappa, transgenic mice have been analyzed for the expression of the transgene product as a cell surface, Ag-specific receptor and for their ability to respond to Ag. The vast majority of B cells in these H + L transgenics (97 to 99%) express large amounts of the transgene product on their surface and are capable of binding phosphocholine. A total of 4 to 30% of the B cells also express endogenous IgM and IgD H chain products. After immunization with phosphocholine (PC)-conjugated keyhole limpet hemocyanin, more than 1000 micrograms/ml of anti-PC antibody bearing the transgene IgMa allotype marker are produced. Surprisingly, significant amounts of anti-PC antibodies that express the endogenous, IgMb allotype, are also produced; however, these antibodies lack the T15-idiotype which dominates the anti-PC response in their nontransgenic littermate controls. The B cells producing these endogenous anti-PC antibodies also fail to switch to IgG anti-PC synthesis, whereas B cells producing anti-keyhole limpet hemocyanin antibodies readily undergo class switching. These last two observations may be due to the fact that the endogenous anti-PC antibody actually results from mixed mu a + mu b molecules in which the transgene encoded H and L chains are most likely responsible for the binding of PC. Thus, a switch of the endogenous isotype from mu b to IgG would result in a loss of specificity for PC in the IgG molecules produced using the endogenous VH-gene product(s), and mu a + gamma b hybrid molecules are not likely to be formed. This hypothesis is supported by the fact that the majority of (mu a + mu b) hybridomas have the mu b-allotype joined with a VH region other than the VH1 gene which is required for PC-binding and T15 idiotype expression.

Animals↗
PubMed1989

Inhibition of immunoglobulin gene rearrangement by the expression of a lambda 2 transgene.

The rearrangement of Ig genes is known to be regulated by the production of H and kappa L chains. To determine whether lambda L chains have a similar effect, transgenic mice were produced with a lambda 2 gene. It was necessary to include the H chain enhancer, since a lambda gene without the added enhancer did not result in transgene expression. The lambda 2 transgene with the H enhancer was expressed in lymphoid cells only. The majority of the B cells of newborn transgenic mice produced lambda, whereas kappa + cells were reduced. Concomitantly, serum levels of kappa and kappa mRNA were diminished. By 2 wk after birth the proportion of kappa-expressing cells was dramatically increased. Adults had reduced proportions of B cells that produced lambda only, but the levels of lambda were still higher than in normal littermates. Also, kappa + cells were still lower than in normal mice. Analysis of hybridomas revealed that reduction of kappa gene rearrangement was the basis for the decreased frequency of kappa + cells. Furthermore, many cells also contained an unrearranged H chain allele. It was concluded that feedback inhibition by the lambda 2 together with endogenous H protein may have inhibited recombinase activity in early pre-B cells, leading to inhibition of both H chain and kappa gene rearrangement. Thus, lambda 2 can replace kappa in a feedback complex. The levels of serum lambda 1 and, to a lesser degree, of spleen lambda 1 mRNA were reduced in the lambda 2 transgenic mice. However, the proportion of hybridomas with endogenous lambda gene rearrangement was at least as high as in normal mice. It was therefore concluded that the suppression of functional lambda 1 may be a consequence of decreased selection of endogenous lambda-producing cells because of the excess of transgenic lambda. The escape of kappa-producing cells from feedback inhibition may be the result of several mechanisms that operate to varying degrees, among them: (a) kappa rearrangement during a period in which the recombinase is still active after appearance of a lambda 2/mu stop signal; (b) a B cell lineage that is not feedback inhibited at the pre-B cell stage; (c) subthreshold levels of transgenic lambda 2 in some pre-B cells; and (d) loss of the lambda 2 transgenes in rare pre-B cells.

Animals↗