Search PubMed⌕ Search

Biomedical subjects

S L Morrison

Publications and source records attributed to S L Morrison.

At least 145 records · Page 8Linked to original sources
PubMed1986

Membrane IgM, IgD, and IgG act as signal transmission molecules in a series of B lymphomas.

Increases in intracellular free calcium concentration ((Ca2+)i) were observed in response to anti-immunoglobulin (Ig) antibodies in each of six B cell tumors or B cell hybridomas bearing mu or delta chains on their cell surface. The BAL17 cell line, bearing mu and delta chains on its surface, behaved similarly to mature B cells in the following respects. Anti-IgM and anti-IgD antibodies caused increases in (Ca2+)i and inositol phospholipid metabolism; the initial increases in (Ca2+)i were derived partly from an intracellular Ca2+ pool; lipopolysaccharide, phorbol myristate acetate (PMA), B cell stimulatory factor-1, and antibodies to class I and class II major histocompatibility molecules and to the Fc gamma receptor failed to cause increases in (Ca2+)i or in inositol phospholipid metabolism; and increases in (Ca2+)i and inositol phospholipid metabolism in response to anti-Ig were inhibited by pretreatment with PMA. Furthermore A20, an IgG2a bearing lymphoma, showed increases in (Ca2+)i in response to anti-IgG2a, and a lymphoma cell line (6G8-2E10) expressing membrane IgG2b as a result of DNA-mediated transfer of the gamma 2b H chain gene, showed increases in (Ca2+)i in response to anti-IgG2b. These results indicate that Ig-bearing lymphomas display early events in B cell activation after receptor cross-linkage and can be used for detailed studies of the activation process.

Animals↗
PubMed1986

Sequences 3' of immunoglobulin heavy chain genes influence their expression.

The function of sequences 3' of Ig genes in controlling their expression has been investigated by analyzing mutants of Ig-producing cells and by gene transfection experiments. A mutant of an IgA producing myeloma was isolated whose steady-state level of heavy chain mRNA and protein was decreased. Analysis of the mutant showed it had deleted at least 4 kb of DNA immediately 3' of the alpha gene and introduced at least 5 kb of non-Ig sequence in its place. Examination of nuclear RNA showed no accumulation of aberrant transcripts or altered processing patterns. Instead, the transcription rate of heavy chain in the mutant was approximately 1/7 of that in its parent. This mutant suggests that sequences 3' of Ig genes facilitate their transcription; alternatively, the non-Ig sequences may act to depress transcription. A complete gamma 2b heavy chain gene containing both the secreted and membrane exons was transfected into lymphoid cells. The ratio of membrane/secreted Ig mRNA produced by the transfectants was found to reflect the phenotype of the recipient cell; myelomas made mostly secreted mRNA while lymphomas made only a slight excess of secreted mRNA. When a heavy chain was used with a deletion of sequences within the IVS between the secreted and membrane exons which left the AATAA poly A addition signal intact, but removed the site of cleavage and polyadenylation, the processing ratio was altered so that predominantly membrane Ig was produced in transfected myeloma cells. The alteration in processing could be a result of the deletion of the normal site for poly A addition; alternatively it could be a result of the deletion of another sequence which is recognized by processing enzymes. A 13 bp sequence (GTCCTGGTTCTTT), was found to be highly conserved both in position and sequence in human and mouse gamma chain genes. When a gene with a deletion which left the poly A addition site and the conserved sequence intact was used for transfection, the processing pattern was found to be identical to that of a wild type heavy chain gene.

Animals↗
PubMed1986

Sequences near the 3' secretion-specific polyadenylation site influence levels of secretion-specific and membrane-specific IgG2b mRNA in myeloma cells.

The expressed immunoglobulin gamma 2b (IgG2b) heavy-chain gene of 4T001 was cloned into the shuttle vector pSV2-gpt and transfected into myeloma J558L and lymphoma A20.2J. Northern blots indicated that the transfected gamma 2b gene was processed in a manner similar to the endogenous heavy chain in both lymphoma and myeloma cells. To identify sequences important for immunoglobulin mRNA processing, we constructed deletions around the secretion-specific polyadenylation site and introduced the deleted genes into J558L cells. The BAL deletion lacked 670 base pairs of intervening sequence between secreted and membrane regions; the Kpn deletion lacked 830 base pairs in this region. J558L cells transfected with either the entire gamma 2b gene or the delta BAL vector produced predominantly secretion-specific gamma 2b mRNA and protein. J558L cells transfected with the delta Kpn vector produced approximately equimolar amounts of secretion-specific and membrane-specific gamma 2b mRNA. Both 55,000-dalton secreted and 62,000-dalton putative surface IgG2b proteins were detected in the delta Kpn transfectants. We conclude that sequences absent in the Kpn deletion but present in the BAL deletion exert an important role in the production of secretion-specific mRNA. The Kpn deletion removes the normal site of cleavage and poly(A) addition, and it is possible that it is the absence of this site which changes the processing pattern. Alternatively, it is possible that sequences absent in the Kpn deletion but present in the BAL deletion function in regulating the production of predominantly secretion-specific mRNA in myeloma cells. The possible role of a highly conserved sequence found in this region is discussed.

Animals↗
PubMed1986

Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products.

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.

Alleles↗
PubMed1985

Transfectomas provide novel chimeric antibodies.

Methods have been developed to transfect immunoglobulin genes into lymphoid cells. The transfected genes are faithfully expressed, and assembly can occur both between the transfected and endogenous chains and between two transfected chains. Gene transfection can be used to reconstitute immunoglobulin molecules and to produce novel immunoglobulin molecules. These novel molecules can represent unique combinations of heavy and light chains; alternatively, by means of recombinant DNA technology, genes can be assembled in vitro, transfected, and expressed. The end products of such manipulations include chimeric molecules with variable regions joined to different isotypic constant regions; this is possible both within and between species. It is also possible to synthesize altered immunoglobulin molecules, as well as molecules having immunoglobulin sequences fused with nonimmunoglobulin sequences (for example, enzyme sequences).

Animals↗
PubMed1985

C57BL/6 x BALB/c hybridomas produce IgA which assembles into molecules with covalent bonds between heavy chains (H) and light chains (L), and into molecules lacking covalent bonds between H and L.

Examination of the gel electrophoresis patterns of 14C-biosynthetically labeled immunoglobulin from C57BL/6 X BALB/c IgA hybridomas reveals that each of the monoclonal cell populations produces two different forms of IgA: molecules with heavy chains (H) and light chains (L) joined by disulfide bonds, as well as molecules with H and L being noncovalently associated. The possible origin of this was explored: Southern blot analysis of the hybridoma DNA indicated that only one alpha gene is expressed by each cell line; hybridoma cells labeled in the presence of the N-glycosylation inhibitor tunicamycin exhibit both forms; and electrophoresis of biosynthetically labeled spleen cell IgA from C57BL/6, BALB/c and (C57BL/6 X BALB/c) F1 mice shows that BALB/c mice produce only the noncovalently associated form, while C57BL/6 and (C57BL/6 X BALB/c) F1 mice produce both. Possible mechanisms by which two types of IgA may be assembled by the same hybridoma cell are discussed.

Animals↗
PubMed1985

Alternative splicing patterns in an aberrantly rearranged immunoglobulin kappa-light-chain gene.

Using a 15-nucleotide primer specific for the immunoglobulin kappa-chain gene, we synthesized cDNA from the mRNA of an anti-alpha(1----6)dextran hybridoma. The hybridoma had been produced using MPC-11 as the parental myeloma. Hybridization and sequence analysis of one clone showed that it was derived from a 1.2-kilobase (kb) kappa-chain mRNA that lacked a joining minigene segment (J). The mRNA had the leader region correctly spliced to the variable region (V) but, in the absence of a J, V kappa was flanked by 62 nucleotides (3202-3263) from the intervening sequence (between J5 and the kappa-chain constant region gene C kappa) before being spliced to C kappa. This mRNA originated from the kappa-chain-fragment gene of MPC-11 but differed from the previously described 0.8-kb kappa-chain-fragment mRNA [Choi, E., Kuehl, W.M. & Wall, R. (1980) Nature (London) 286, 776-779; Seidman, J.G. & Leder, P. (1980) Nature (London) 286, 779-783] in which the leader sequence is spliced directly to C kappa. This 1.2-kb mRNA was present as a polyadenylylated species in total cellular RNA but could not be detected in cytoplasmic RNA. Thus, it either failed to be transported out of the nucleus or was rapidly degraded in the cytoplasm. These studies show that transcripts of the kappa-chain-fragment gene are processed by two distinct splicing pathways to yield either a 0.8-kb mRNA with the leader region spliced directly to C kappa or a 1.2-kb mRNA with leader, V, 62 nucleotides of the intervening sequence, and C kappa.

Animals↗
PubMed1985

Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice.

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.

Amino Acid Sequence↗
PubMed1985

A human-mouse chimeric immunoglobulin gene with a human variable region is expressed in mouse myeloma cells.

We have constructed and obtained expression of a chimeric human-mouse immunoglobulin gene after transfection into mouse myeloma cells. A human VDJH gene segment was joined to a mouse C kappa gene in the plasmid vector pSV2-gpt, and the construct was transfected into J558L cells by protoplast fusion. Analyses of six transformants by RIA and SDS-PAGE indicated that the chimeric protein was synthesized in large amounts in five. A kappa-specific transcript was observed by Northern blot analysis. Four out of five clones were stable producers of this chimeric chain over a period of 10 mo.

Animals↗
PubMed1984

Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.

We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.

Animals↗
PubMed1984

Loss of a consensus splice signal in a mutant immunoglobulin gene eliminates the CH1 domain exon from the mRNA.

A series of mouse myeloma mutants, derived from a cell line of the murine MPC-11 tumor (gamma 2b, kappa), resemble human heavy-chain disease in their loss of an internal domain (exon). In these mutants, most of the gamma 2b CH1 exon was present in the nuclear RNA but was removed during splicing to form the mature cytoplasmic RNA. Amino acid sequence studies of one mutant (10.1) are consistent with the loss of the complete CH1 domain. A second mutant cell line (I17) derived from 10.1 and containing the same CH1 alteration was shown by S1 nuclease protection experiments to have an additional mRNA deletion spanning the CH2-CH3 domain boundary. This second deletion was shown to result from a genomic alteration that provided a marker for the isolation of the expressed H-chain allele. To determine the basis of the CH1 splicing defect, the 117 genome-expressed gamma 2b constant region DNA was cloned. Sequence studies showed a deletion of 99 nucleotides around the 3' end of the CH1 domain, which removed the splice site and flanking DNA, apparently causing the aberrant splicing of the RNA transcript. The sequence deleted in the mutant is flanked by short repeats of the octameric sequence CCAGCCAG in the wild-type gene. In the mutant, one copy of the repeat, in addition to the sequences between the repeats, has been lost.

Amino Acid Sequence↗
PubMed1983

A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene.

We have studied the DNA sequences required for high level expression of a cloned heavy chain immunoglobulin gene stably introduced into mouse myeloma cells by DNA transfection. We found that DNA sequences derived from the germ line JH-C mu region are required for accurate and efficient transcription from a functionally rearranged VH promoter. Similar to viral transcriptional enhancer elements, these cellular sequences stimulate transcription from either the homologous VH gene segment promoter or a heterologous SV40 promoter. They are active when placed on the 5' or 3' side of the rearranged VH gene segment and they function when their orientation is reversed. However, unlike viral enhancers, the Ig gene enhancer appears to act in a tissue-specific manner, since it is active in mouse B cells but not in mouse fibroblasts. The nucleotide sequence of the Ig enhancer region contains repeating elements that closely resemble sequence the possible role of tissue-specific transcription in cell differentiation and malignant transformation.

Animals↗
PubMed1983

Immunoglobulin gene expression in transformed lymphoid cells.

Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt. The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months. Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line. The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis. Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule. No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.

Animals↗
PubMed1983

Ultraviolet-Stimulated KHCO(3) Efflux from Rose Cells: Regulation of Cytoplasmic pH.

Suspension-cultured cells of Rosa damascena that have been irradiated with ultraviolet light (254 nanometers, 2.1 x 10(4) joules per square meter) rapidly lose K(+) and HCO(3) (-) ions to the medium. If the HCO(3) (-) is derived from respiratory CO(2) inside the cell, then loss of HCO(3) (-) should be accompanied by an acidification of the cytoplasm. Estimates of the pH of control and ultraviolet-irradiated cells by (31)P-nuclear magnetic resonance spectroscopy indicated that, following irradiation, the pH of both cytoplasm and vacuole dropped by 0.2 to 0.3 units. This change was not as great as was predicted from the observed HCO(3) (-) loss. Analysis of nitrogenous compounds in the cell suggested that reduction of nitrate and synthesis of gamma-aminobutyric acid absorbed some of the protons formed by the synthesis and dissociation of bicarbonate.

Journal Article↗
PubMed1982

Immunochemical characterization of binding sites of hybridoma antibodies specific for alpha (1 leads to 6) linked dextran.

The combining sites of seven BALB/c IgM, four BALB/c IgA and one C57BL/6 IgA hybridoma antibodies specific for alpha (1 leads to 6) linked dextran were probed by precipitin and precipitin inhibition assays. The 12 antibodies are able to bind to linear determinants in the interior of the dextran molecule; some have sites complementary to six alpha (1 leads to 6) linked glucose residues and other have sites complementary to seven alpha (1 leads to 6) linked glucose residues. From the analysis of the precipitins and precipitin inhibitions, it is concluded that no two hybridoma proteins have identical binding sites.

Animals↗