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M S Neuberger

Publications and source records attributed to M S Neuberger.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1987

Regulation of membrane IgM expression in secretory B cells: translational and post-translational events.

IgM secreting cells express little or no membrane IgM. This is not always due to absence of the relevant mRNA. To investigate the synthesis and processing of membrane (micron) and secreted (microseconds) polypeptides in secretory B cells, myeloma cells were transfected either with a plasmid containing an intact mu gene or with one only capable of directing micron (not microseconds) mRNA synthesis. Although myeloma transfectants could make abundant levels of micron mRNA, they did not express IgM on the cell surface. In the myeloma host, micron mRNA is translated some 5-fold less efficiently than microseconds mRNA. However, this translational control does not totally preclude micron synthesis, indicating post-translational regulatory events. No difference between micron and microseconds chains could be detected in their rate of assembly with light chains or in their stability, although both types of heavy chain were degraded more rapidly when synthesized in the absence of light chain, or when the hydrophobic nature of the leader sequence was destroyed by site-directed mutagenesis. However, whereas intracellular microseconds chains in IgM-secreting plasmacytoma were found to be concentrated in the Golgi, the micron chains were mainly located in the endoplasmic reticulum. Retention in the endoplasmic reticulum is also observed for both micron and microseconds when synthesized in the absence of light chain. We propose that it is the expansion of the endoplasmic reticulum that accompanies B cell to plasma cell differentiation which is in part responsible for the down-regulation of surface IgM expression. Such a mechanism may also affect the expression of other surface proteins.

Animals↗
PubMed1987

Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.

Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.

Animals↗
PubMed1987

Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain.

Plasmids were constructed in which expression of genes encoding the heavy and light chains of a hapten-specific IgM antibody is under control of a heat shock promoter. Glioma, phaeochromocytoma and other non-lymphoid cell lines transfected with the plasmids were able to process and secrete immunoglobulin following heat induction. The glioma transfectants were studied in detail and were shown to secrete polymeric IgM in a yield similar to that obtained with a plasmacytoma. However, the glioma IgM was not associated with J chain and was largely composed of pentamers and hexamers. Thus, neither J chain nor other lymphoid-specific proteins are required for assembly and secretion of polymeric IgM although the absence of J chain may encourage hexamer formation.

Animals↗
PubMed1986

Production of antibody-tagged enzymes by myeloma cells: application to DNA polymerase I Klenow fragment.

Myeloma DNA expression systems can be used for the synthesis and secretion of antibody/enzyme recombinant molecules. Here we describe the construction of a myeloma cell-line that secretes a hapten-specific antibody/enzyme hybrid molecule, in which the antibody Fc portion has been replaced by the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). This Fab-PolIk hybrid molecule is secreted in good yield from the myeloma transfectants, can be purified to homogeneity in a single step on hapten-Sepharose columns, and exhibits PolIk activity as judged by its use in dideoxy nucleotide sequencing. Thus Fab-PolIk can be used for the same purposes as conventional PolIk but has the advantage that it is easily purified to homogeneity in a one-step purification from culture medium.

Animals↗
PubMed1985

Chromosome translocation activates heterogeneously initiated, bipolar transcription of a mouse c-myc gene.

In many mouse plasmacytomas, the active c-myc gene has been truncated by chromosome translocation with the resultant severance of the protein-coding sequence from the normal promoter. Transcripts of such truncated c-myc genes were analyzed by Northern blotting, nuclease S1 mapping, primer extension assays and cDNA cloning. We conclude that transcription originates from multiple initiation sites on both c-myc coding and non-coding strands with the two-sets of transcripts derived from adjacent but essentially non-overlapping regions located greater than 1 kb from the translocation junction. In X63Ag8, where c-myc is translocated to the immunoglobulin C gamma 2b gene, the c-myc non-coding strand transcripts include the translocation junction and then splice directly into the gamma 2b CH1 exon. We propose that chromosome translocation activates a cryptic promoter in the first intron and that the heterogeneously initiated, bipolar transcription reflects the absence of a suitably placed TATA box element.

Animals↗
PubMed1985

Transcription cell type specificity is conferred by an immunoglobulin VH gene promoter that includes a functional consensus sequence.

Immunoglobulin heavy chain gene transcription was studied using DNA transfection. The enhancer element identified in the mouse heavy chain locus was active in pre-B and plasmacytoma cell lines, but no activity was detected in two T cell lymphomas. However, even in the absence of the enhancer, cell type specificity of immunoglobulin gene transcription was still retained. We have used gene fusions to show that transcription cell type specificity is conferred by a VH gene promoter. Deletion analysis of this VH promoter indicates that a conserved octamer found 5' of the TATA box in immunoglobulin V genes is a functional part of the tissue-specific promoter upstream element.

Animals↗
PubMed1983

Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells.

A plasmid including a mouse immunoglobulin mu gene was transfected into the IgG-secreting human lymphoid line HMy2 and mouse B- and pre-B-cell lines WEHI 231 and 18-81; stably transfected cells were selected. Transfected HMy2 cells synthesized mouse immunoglobulin mu chains as a major secreted protein but the WEHI 231 and 18-81 transfectants transcribed the introduced mu gene at lower levels. In HMy2 transfectants, most of the transcription of the introduced heavy chain gene initiated 40 and 62 bp upstream of the beginning of the VH exon translation start, although a small proportion of transcripts initiating further upstream was detected. WEHI 231 and 18-81 transfectants gave a much higher proportion of upstream initiation. Transient expression of the VH exon was monitored following transfection of mouse myeloma with the VH gene DNA in various plasmid constructs. VH transcription was only observed if the plasmids contained a segment derived from the large VH-CH intron of the immunoglobulin heavy chain locus. This segment, located between JH and switch regions, functioned both downstream of the VH exon and upstream in either orientation. The existence of a transcription enhancer element in this region is therefore proposed.

Animals↗
PubMed1981

Purification and properties of D-ribulokinase and D-xylulokinase from Klebsiella aerogenes.

The D-ribulokinase and D-xylulokinase of Klebsiella aerogenes were purified to homogeneity from Escherichia coli K12 construct strains that synthesized these enzymes constitutively. The D-ribulokinase, which is encoded in the ribitol operon, is active as a dimer of 60 000 subunit mol.wt., whereas the D-xylulokinase, which is encoded in the D-arabitol operon, is active as a dimer of 54 000 subunit mol.wt. The amino acid compositions and N-terminal sequences of both pentulokinases are reported. The Kapp. values of the enzymes for their D-pentulose substrates were determined, and the D-ribulokinase was shown to have a low-affinity side-specificity for ribitol and D-arabitol. These results are discussed in the context of the evolution of the Klebsiella aerogenes pentitol operons.

Amino Acids↗
PubMed1981

Sequence of a secondary phage lambda attachment site located between the pentitol operons of Klebsiella aerogenes.

We have determined the nucleotide sequence of a secondary phage lambda attachment site (att) located between the structural genes of the ribitol and D-arabitol catabolic operons of Klebsiella aerogenes. The core region of this secondary attachment site (sequence: GGTTTTTTCGATTAT) shows considerable homology with the 15-base-pair core region common to both the phage att and the primary bacterial att of Escherichia coli K12 (sequence: GCTTTTTTACTAA); however, there is no such clear homology between the sequences flanking the cores of the primary att and this secondary att. Integration of phage lambda into the K. aerogenes secondary att occurred by recombination between the core region of the phage att and an oligo(T.A) stretch located within the K. aerogenes secondary att.

Attachment Sites, Microbiological↗
PubMed1981

Activation of mouse complement by monoclonal mouse antibodies.

The ability of monoclonal mouse IgM, IgD and IgG anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) antibodies to activate mouse complement was studied using a hemolytic assay. Efficient hemolysis was obtained with IgG2a, IgG2b and IgG3 antibodies but no lysis was observed using a monoclonal IgD. Of several IgG1 antibodies tested, three gave no detectable hemolysis, although weak but significant hemolysis was obtained with two other IgG1. IgM was found to be powerfully hemolytic in that it was effective at lower concentrations than were IgG. However, using complement from mouse strains carrying the H-2k haplotype it was found that, under conditions of complement limitation and saturating antibody, a fixed amount of complement could lyse about 3 to 4 times as many IgG-coated sheep red cells as IgM-coated red cells. This discrimination between IgM and IgG as regards the efficiency of complement utilization is controlled by a gene in the S region of H-2 and is not apparent with complement from mice carrying the b, d or s allele at that locus.

Animals↗
PubMed1981

Switch from hapten-specific immunoglobulin M to immunoglobulin D secretion in a hybrid mouse cell line.

From a hybrid mouse cell line (B1-8) that secreted an IgM, lambda 1 anti-(4-hydroxy-3-nitrophenyl)acetyl antibody but that had no detectable surface IgM, selection for a variant with lambda 1 chains on the surface resulted in the isolation of a line that had switched from mu to delta expression. The surface and secreted Igs of this line were typed as IgD with two monoclonal antibodies, and the parental IgM and variant IgD molecules carried the same variable regions as judged by hapten-binding and idiotypic analysis. The surface and secreted delta chains of the IgD variant have apparent molecular weights of 64,000 and 61,000, respectively. However, the unglycosylated secreted delta polypeptide chain has a molecular weight of only 44,000. The secreted IgD exists predominantly in the delta 2 lambda A2 form, does not contain J protein, is relatively stable in serum, and does not fix complement.

Animals↗
PubMed1981

Structure of an experimentally evolved gene duplication encoding ribitol dehydrogenase in a mutant of Klebsiella aerogenes.

We have previously described a system of experimental evolution in which many of the mutants of Klebsiella aerogenes selected for faster growth on xylitol ('evolvants') synthesized elevated levels of ribitol dehydrogenase and have presented genetic evidence implicating gene duplication in the enzyme superproduction in some of the evolvants. Here we describe a physical approach to the screening for gene duplications and subsequent structure determination. Nick-translated, cloned ribitol operon (rbt) DNA was used as a hybridization probe to identify fragments containing rbt operon sequences in restriction digests of total bacterial DNA. Whilst several of the evolvants probably harbour duplications spanning the entire rbt operon, one of the spontaneously arising evolvants (strain A3) was shown to harbour a small (5.8 kilobase pairs) direct DNA repeat which encodes the dehydrogenase (but not the kinase) of the closely linked D-arabitol operon as well as the dehydrogenase (but not the kinase) of the rbt operon. The hybridization data suggest that there are 4 to 5 copies of the repeat arranged contiguously on the chromosome. The genetic instability of strain A3, the rbt fragment hybridization pattern of an A3 segregant and the activities of the pentitol catabolic enzymes in A3 are all consistent with the proposed gene duplication structure.

Base Sequence↗
PubMed1980

Minicircular ColE1-related DNA in strains of Klebsiella aerogenes selected for fast growth on xylitol.

We have previously described a large family of mutants of Klebsiella aerogene which were selected by continuous on xylitol and which superproduce ribitol dehydrogenase. One of these strains was found to harbour a high copy number 2.1 x 10(6) dalton plasmid. This plasmid is a deletion derivative of a low copy number 3.5 x 10(6) dalton plasmid present in the ancestral strain of K. aerogenes. However, since these plasmids do not contain the genes required for pentitol catabolism and some enzyme-superproducing strains have lost all DNA homologous to the plasmids, they are not implicated in the fast growth on xylitol. The plasmids contain regions of homology with the Escherichia coli plasmid ColE1.

Alcohol Oxidoreductases↗
PubMed1979

Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.

Amino Acids↗