PubMed Health⌕ Search

Biomedical subjects

M S Neuberger

Publications and source records attributed to M S Neuberger.

At least 19 recordsLinked to original sources
PubMed1991

The B-cell antigen receptor of the five immunoglobulin classes.

Several proteins associate with surface IgM to form the antigen receptor. We show that just two, the alpha and beta associated chains, are sufficient to reconstitute an IgM surface receptor in fibroblasts. Contrary to expectation, a common alpha chain associates with all five immunoglobulin classes. We propose that B-cell antigen receptors consist of a common alpha/beta heterodimer associated with each immunoglobulin class. But the classes differ both in the glycosylation of their associated alpha chain and in their dependence on alpha/beta for surface transport.

Amino Acid Sequence↗
PubMed1991

The mouse IgH 3'-enhancer.

A lymphoid-specific transcription enhancer element has recently been identified at the far 3' end of the rat immunoglobulin heavy chain (IgH) locus. Sequence analysis presented here reveals that this enhancer is flanked by a 350-bp invert repeat, giving a structure reminiscent of a transposable element. We therefore screened for the equivalent enhancer in the mouse to determine whether its presence was conserved during evolution. A mouse homologue was indeed identified and is located 16 kb downstream of the C alpha 1 exon. It is also flanked by invert repeats and these are not repeated throughout the genome. The mouse and rat enhancers retain high sequence homology. As regard activity, the IgH 3'-enhancer is lymphoid specific. However, this activity was detected in two plasmacytoma lines tested but not in two B cell lymphomas nor in HeLa cells suggesting that the enhancer may only play a stage-specific role during lymphocyte differentiation. As regards function within the IgH locus, we found that inclusion of the mouse IgH 3'-enhancer (in addition to the intron-enhancer) on mu gene expression plasmids effected a small increase in mu mRNA levels in stable plasmacytoma transfectants.

Animals↗
PubMed1991

Somatic hypermutation of immunoglobulin kappa may depend on sequences 3' of C kappa and occurs on passenger transgenes.

We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism.

Amino Acid Sequence↗
PubMed1990

The importance of the 3'-enhancer region in immunoglobulin kappa gene expression.

The first enhancers to be identified in the immunoglobulin gene loci are located in the J-C intron. However, deletion of the immunoglobulin kappa intron-enhancer has little effect on the transcription of kappa transgenes. Here we ask whether the second kappa enhancer which we recently identified at the 3'-end of the locus plays a role in kappa gene expression. We show that its omission leads to 20-40 fold lower expression of kappa transgenes and to poor allelic exclusion. Transfection experiments show that activity of the 3'-enhancer, like that of the kappa-intron enhancer, can be induced in a pre-B cell line by incubation with bacterial lipopolysaccharide. Whereas induction of the kappa-intron enhancer is due to induction of NF-kappa B activity, deletion mapping of the 3'-enhancer localises its activity to a 50 nucleotide region that lacks an NF-kappa B site; indeed the 3'-enhancer allows kappa expression in a cell line which lacks NF-kappa B. Thus, both the 3'- and intron-enhancers can be induced at the same stage of differentiation but by distinct pathways. Furthermore, unlike the intron-enhancer, the 3'-enhancer plays a critical role in the transcription of rearranged immunoglobulin kappa genes.

Animals↗
PubMed1990

Lymphoid-specific transcriptional activation by components of the IgH enhancer: studies on the E2/E3 and octanucleotide elements.

The IgH enhancer is a strong lymphoid-specific activator and is composed of multiple factor-binding motifs. One of these, the octamer, is common to enhancer and promoter, binds ubiquitous and lymphoid-specific factors and is able to act as a lymphoid-specific transcriptional activator. However, it is also found as an essential component of promoters active in non-lymphoid cells. From analysis of the activities of synthetic promoters, we suggest that recruitment of the lymphoid-specific octamer-binding protein next to the TATA is sufficient to create a functional lymphoid-specific promoter whereas the ubiquitous octamer binding protein is not active in single copy but can act in concert with other promoter binding factors. However, the activity of the IgH enhancer is not dependent on the octamer and we identify the E2/E3 elements as also being sufficient to confer lymphoid-specificity on a linked gene. Activity of the E2/E3 region results from the synergistic activity of the two motifs, E2 alone being able to confer a low level of activity which is dramatically increased by the adjacent E3. Thus, in the case of both the E2/E3 and the octamer motifs, interactions between adjacent elements can play a critical role in determining the tissue specificity of activity.

Base Sequence↗
PubMed1990

A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus.

The expression of immunoglobulin heavy-chain (IgH) genes is generally thought to be regulated by the combination of the VH promoter with the enhancer element which is located in the JH-CH intron. This is probably an oversimplification: there are cell lines that transcribe IgH genes despite the deletion of the intron-enhancer. These findings could imply that other enhancer element(s) exist in the IgH locus. Here we show that a strong B-cell-specific enhancer is indeed located at the 3'-end of the rat IgH locus, 25 kilobases downstream of C alpha. This enhancer should be retained downstream of all rearranged IgH genes, regardless of the VH or CH segment used. Taken together with analogous findings for the mouse kappa locus, the results prompt a re-evaluation of the mechanism of regulation of immunoglobulin gene transcription. Furthermore, unlike the intron-enhancer, the IgH 3' enhancer would become linked to a c-myc that rearranges into an IgH switch region. The IgH 3' enhancer could therefore play a part in the activation of the translocated c-myc genes in rat immunocytomas, mouse plasmacytomas and Burkitt lymphomas.

Animals↗
PubMed1990

The sequence of the mu transmembrane segment determines the tissue specificity of the transport of immunoglobulin M to the cell surface.

Membrane IgM is expressed on the surface of B lymphocytes. It is not transported to the surface of transfected plasmacytoma or COS cells. Here, we show that mutation of four hydrophilic amino acids in the microm transmembrane is sufficient to overcome the intracellular retention of membrane IgM in non-B cells. This suggests that the B cell-specific IgM-associated proteins that have been postulated to assist the transport of membrane IgM to the cell surface (3) act either by forming a hydrophobic sheath that surrounds the microm transmembrane segment or by displacing an interaction with this segment that would otherwise cause retention. Experiments with a CD8/mu hybrid H chain indicate that the proteins that assist the transport of membrane IgM to the B cell surface at most need the mu CH4 and transmembrane/cytoplasmic portion for interaction.

Amino Acid Sequence↗
PubMed1990

Expression and targeting of intracellular antibodies in mammalian cells.

Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm and led us to ask whether antibodies could be further targeted to the nucleus. Mutations were therefore made in which the leader sequence of the light chain was replaced by the nuclear localization signal of the SV40 large T antigen. Transfectants in which the heavy chain lacking the hydrophobic leader was expressed together with a light chain carrying the nuclear localization signal were selected and a nuclear distribution of the assembled antibody was found. Thus, it should prove possible to target a specific antibody to the cell nucleus with the aim of interfering with the function of a nuclear antigen.

Amino Acid Sequence↗
PubMed1990

Construction, function and immunogenicity of recombinant monoclonal antibodies.

Much of the interest in chimaeric antibodies stems from their presumed lack of immunogenicity in humans. We have tested this assumption using mouse models and have concluded that whilst chimaerisation can certainly lead to a reduction in the total anti-antibody response, a substantial anti-variable region response can nevertheless remain. As it therefore seems likely that determinants within the variable region lead to this anti-antibody response, we have initiated experiments to test whether it might be possible to use transgenic mice that carry human immunoglobulin gene segments in their germline configuration as a means of making a repertoire of entirely human antibodies.

Animals↗
PubMed1989

The immunogenicity of chimeric antibodies.

Mice were immunized with model xenogeneic (both the VH frameworks and the CH domains of human origin), chimeric (just VH frameworks human), or self antibodies, and the antiantibody responses were dissected. Only the self antibody did not elicit a response. A strong response was elicited by the most xenogeneic antibody with approximately 90% against the C and approximately 10% against the V. The anti-V response was not attenuated in the chimeric antibody, demonstrating that foreign VH frameworks can be sufficient to lead to a strong antiantibody response. The magnitude of this xenogeneic anti-VH response was similar to that of the allotypic response elicited by immunizing mice of the Igha allotype with an Ighb antibody. Thus, although chimerization can diminish antiantibody responses, attention should be paid both to V region immunogenicity and to polymorphism.

Animals↗
PubMed1989

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice.

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.

Animals↗
PubMed1989

The immunoglobulin kappa locus contains a second, stronger B-cell-specific enhancer which is located downstream of the constant region.

The description of cell lines capable of transcribing immunoglobulin heavy or light chain genes in the apparent absence of an active enhancer has led us to look for novel enhancers in the immunoglobulin gene loci. Here we show that there is a second B-cell-specific enhancer in the mouse kappa locus and that this is located 9 kb downstream of C kappa. This enhancer is some 7-fold stronger than the kappa-intron enhancer and shows striking sequence homologies to the lymphotropic papovavirus, IgH and kappa-intron enhancers. The location of the kappa 3' enhancer between C kappa and the RS element means that it is deleted in some B cells that express lambda light chains.

Animals↗
PubMed1989

A repertoire of monoclonal antibodies with human heavy chains from transgenic mice.

The introduction of human immunoglobulin gene segments in their unrearranged configuration into the germ line of mice might allow the production of a repertoire of human antibodies. Such transgenic mice could be used for the production of human monoclonal antibodies against human antigens. To test the feasibility of this approach, mice were created that carry a human heavy-chain minilocus comprising unrearranged immunoglobulin variable, diversity, and joining elements linked to a human mu-chain gene. The gene segments of this minilocus are rearranged in a large proportion of cells in thymus and spleen but not in nonlymphoid tissue. Some 4% of the B lymphocytes synthesize human mu chains resulting in a serum titer of about 50 micrograms of transgenic IgM antibody per ml. Hybridomas were established from the transgenic mice that stably secreted several micrograms of antibodies containing human mu heavy chains per milliliter.

Animals↗
PubMed1989

Cellular selection leads to age-dependent and reversible down-regulation of transgenic immunoglobulin light chain genes.

Analysis of immunoglobulin expression in mice transgenic for either a kappa light chain (driven by the kappa enhancer) or lambda light chain (driven by the IgH enhancer) revealed that the transgenic light chains are expressed by the majority of B lymphocytes in the neonatal mice. However, the proportion of B cells that express the transgenes at a detectable level decreases rapidly with age, with a concomitant increase in cells expressing rearrangements of one of the endogenous light chain loci. This appears to be the result of cellular selection. The down-regulation of transgene expression is not due to an irreversible mechanism as incubation of adult splenic lymphocytes with bacterial lipopolysaccharide leads to a rapid increase in the expression of the transgenic light chain on the B cell surface. In mice carrying the lambda transgene (but not in mice carrying the kappa transgene) the change with age in the pattern of transgene expression is accompanied by a shift towards B cells that do not express surface IgD. This shift towards IgM+/IgDlow B cells is also observed in mice transgenic for a chloramphenicol acetyltransferase gene linked to the IgH enhancer. This suggests that the down-regulation of IgD may either be due to the expression of a transgene that impairs B cell development or, alternatively, could be associated with the molecular events responsible for the down-regulation of IgH enhancer activity. The results also draw attention to the contribution of cellular selection in determining the pattern of expression of immunoglobulin transgenes and emphasize the importance of in vivo analysis of neonatal as well as adult transgenic mice.

Aging↗
PubMed1988

The intron requirement for immunoglobulin gene expression is dependent upon the promoter.

Transfection assays were used to assess the need for an intron in order to obtain expression of cytoplasmic immunoglobulin mu mRNA. An intron is required when transcription is driven by an immunoglobulin promoter/enhancer combination, although this requirement is not specific for a particular intron. However, this need for an intron is dependent upon the promoter used. Whilst an intron is required in the case of immunoglobulin or beta-globin promoters, it is not in the case of cytomegalovirus or heat-shock promoters. The data point to a connection between the promoter and RNA processing or export.

Enhancer Elements, Genetic↗
PubMed1988

The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes.

The increase in abundance of immunoglobulin heavy-chain (IgH) mRNA that accompanies development of a B cell into a plasma cell is mainly due to post-transcriptional events. By constructing mu genes, whose expression is under inducible control, we determined the half-life of pulsed mu transcripts to be approximately 20 hr in plasmacytoma hosts and approximately 3 hr in B-cell lymphomas. Interestingly, a mu gene with a mutated signal sequence that is found on free (rather than membrane-bound) polysomes decays in the plasmacytoma host with a shortened half-life of 3 hr. Thus, a change in the turnover rate of IgH mRNA plays an important role in the cell-type specificity of immunoglobulin gene expression; this change may be a consequence of the fact that IgH mRNA is located in the membrane-bound polysome fraction.

Animals↗