The lateral mobility of some membrane proteins is determined by their ectodomains.
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Animals↗
Biomedical subjects
A Bothwell
Publications and source records attributed to A Bothwell.
Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.
In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.
Animals↗
Isolation and expression of an IFN-responsive Ly-6C chromosomal gene.
The Ly-6 locus controls the expression of genes whose products in lymphoid cells are involved in the process of Ag-independent T cell activation. The Ly-6 locus contains multiple tightly linked genes which have been mapped to a specific region on murine chromosome 15. The present approach to further define the Ly-6 Ag is based on the transfection of cloned genes and identification of the expressed products by using mAb. Screening of Ly-6 related chromosomal clones revealed one that contains a gene that is closely related to yet distinct from that of the previously characterized Ly-6E.1 protein. Transfection of this chromosomal clone into COS cells shows that it contains the gene encoding Ly-6C.1 determinants. The expression of the transfected Ly-6C.1 gene is enhanced in COS cells following treatment with mouse IFN. Characterization of the DNA sequence of the Ly-6C.1 gene has established that it consists of four exons, the first of which is untranslated. Several possible regulatory elements have been identified in the putative promoter region of this gene (5' to the first exon), including a 28-base sequence closely resembling the consensus IFN-responsive sequence found in the promoter regions of other IFN-responsive genes.
Animals↗
The T-cell independent antigen, NP-ficoll, primes for a high affinity IgM anti-NP response.
In a number of different strains of inbred mice, immunization with a hapten coupled to a protein carrier results in production of homogeneous serum antibodies. At the genetic level this corresponds to the use of a very limited set of variable region genes in the actively secreting B-cells. In contrast, immunization with the same hapten coupled to a T-cell independent (TI) carrier produces a heterogeneous antibody response. Here we show that successive immunizations of C57BL/6 mice, first with the hapten NP coupled to ficoll, a TI carrier, and then one month later with a subliminal dose of the same hapten coupled to a protein carrier, generate a novel set of hybridomas. These hybridomas produce antibodies which are of the IgM isotope and which lack somatic mutation. Some of these antibodies have a much higher affinity for NP than do antibodies which use the prototypical gene combination (VH186.2-lamda 1) of the strain specific response in C57BL/6 mice.
Animals↗
A limited number of B cell lineages generates the heterogeneity of a secondary immune response.
We have studied the cellular basis for heterogeneity in the secondary immune response by creating a large set of B cell hybridomas from a single C57BL/6 mouse immunized with the hapten (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin, and searching among the cells for all possible sets of clonally related lines. Among 28 independent cell lines from a single animal we find that 21 of them fall into seven small families of from two to five members; only seven lines have no matches among the set. A statistical analysis determines that the number of genetically distinct B cell precursors whose progeny were isolated during this secondary response is 18, with 95% confidence limits of 14 and 26. The distribution of family sizes implies that each activated clone has proliferated to approximately the same extent. Complete sequencing of the variable region of immunoglobulin heavy chain mRNA from 19 of the cell lines reveals many shared somatic mutations among related lines, implying a pronounced founder effect has occurred to eliminate most of the progeny of each precursor B cell. This may be the result either of a high frequency of debilitating somatic mutation or from selection against cells still expressing the idiotype of primary anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies.
Animals↗
cDNA characterization of an Ly-6.2 gene expressed in BW5147 tumor cells.
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Amino Acid Sequence↗
Murine Ly-6 multigene family is located on chromosome 15.
Murine Ly-6-encoded molecules play an important role in the antigen-independent activation of lymphocytes. We have described the cloning of a cDNA encoding the protein component of an Ly-6 molecule. Hybridization studies indicated that this cDNA identified multiple DNA fragments on Southern blots. The banding pattern exhibits a restriction fragment length polymorphism from mice bearing either the Ly-6a or the Ly-6b allele. We have employed three independent chromosomal mapping techniques, somatic cell hybrids, in situ hybridization, and strain distribution pattern analysis of the restriction fragment length polymorphism of DNA from recombinant inbred lines, to ascertain the chromosomal origins of these bands. We report that all members of the Ly-6 multigene family are tightly linked on chromosome 15 and have been regionalized by in situ hybridization analysis to band 15E on the distal portion of this chromosome. Linkage analysis has indicated that the Ly-6 genes are located within 1 map unit of Env-54 (a retroviral envelope restriction fragment length polymorphism probe), 3 map units from ins-1, (insulin-related gene), and 4 map units from the protooncogene c-sis. The possible involvement of the Ly-6 lymphocyte activation and differentiation antigen genes in chromosome 15-related lymphoid malignancies is discussed.
Animals↗
Isolation of a murine Ly-6 cDNA reveals a new multigene family.
The Ly-6 alloantigens have been shown to play a critical role in T lymphocyte activation. To isolate a Ly-6 cDNA, synthetic oligonucleotides, based on the partial amino acid sequence of purified Ly-6E.1 protein, were used to probe a cDNA library. The synthetic oligonucleotides or the isolated cDNA detected a 1.1-kb RNA species. Sequence analysis of the cDNA clone revealed that the Ly-6E.1 protein consists of a 26-amino acid leader followed by a 108-residue, cysteine-rich, core protein with no N-linked glycosylation sites. Southern blot analysis of genomic DNAs revealed multiple bands indicating a family of related genes. Using recombinant inbred and Ly-6 congenic strains of mice, restriction fragment length polymorphisms were demonstrable, and correlated with the Ly-6 allotype of the DNA donors. This probe will enable further molecular genetic analysis of the role of Ly-6-linked proteins in the process of T lymphocyte activation. Isolation of Ly-6 genomic clones may promote a further understanding of the complex tissue-specific expression patterns characteristic of Ly-6-linked genes.
Amino Acid Sequence↗
Allotypic differences in murine mu genes.
We report the complete DNA sequence of a c-DNA clone of the heavy chain mu b allele of the C57BL/6 mouse. Comparisons have been made with the nucleotide sequences of the germ line BALB/c mu a and the plasmacytoma TEPC-183 mu a alleles reported elsewhere over the entire length of the coding and the 3' untranslated region. In contrast to the extensive differences between the gamma 2a a and b alleles we have reported earlier we see a very high degree of homology between the mu alleles. Only one of the nucleotide differences between C57BL/6 mu b and BALB/c mu a leads to an amino acid substitution. This single amino acid exchange must form the allotypic determinant of the mu b allele. A comparison of four different DNA sequences indicates that they are all distinct IgM alleles.
Amino Acid Sequence↗
Molecular characterization of antibodies bearing Id-460. I. The structure of two highly homologous VH genes used to produce idiotype positive immunoglobulins.
The heavy chain immunoglobulin genes encoding a variety of antibodies specific for DNP or Pasteurella pneumotropica and bearing the dominant idiotype of MOPC 460, Id-460, were cloned and sequenced. The VH genes encoding the M460 and D35 (DNP binding) antibodies were found to be homologous but not identical to the VH gene encoding the LB8 (P. pneumotropica binding) monoclonal antibody. Two of at least eight genes in the VH460 cross-hybridizing gene family can encode Id-460 positive antibodies. The VH460 gene family overlaps with the gene family described by VH36-60 and more completely describes this germ-line VH gene family. We have previously demonstrated a genetic requirement for VK1 expression in order to observe the expression of Id-460 in anti-DNP antibody responses. Southern blot analysis of these monoclonal antibody-producing cells demonstrates, as with the cross-hybridizing VH, that two cross-hybridizing VK1 genes (VK1A and VK1C) can be used to encode Id-460-positive immunoglobulins. We demonstrate that immunoglobulin idiotype determinant expression can be the result of the expression of nonidentical but highly homologous genes in the VH460 cross-hybridizing VH gene family and also in the VK1 cross-hybridizing VL family.
Animals↗
Mutational analysis of the immunoglobulin heavy chain promoter region.
Complete immunoglobulin heavy chain (IgH) genes (gamma and mu) containing the intronic IgH enhancer and mutations in the upstream promoter region were constructed in vitro and introduced into murine J558L myeloma cells by protoplast fusion. S1-nuclease mapping experiments demonstrated that IgH gene expression was extremely sensitive to mutation in an upstream region containing the octanucleotide sequence ATGCAAAT. Significant IgH mRNA levels were detected in RNA from cells transfected with IgH gene constructs in which all upstream sequences on the 5' proximal side of this element were deleted. Similar results were obtained using the precise inverse of the IgH octamer, which is found in the upstream promoter region of immunoglobulin light chain genes. Deletion of the IgH octamer, or point mutation of adenine to guanine at position 6, resulted in the loss of correctly initiated IgH mRNA. A DNA binding factor from J558L nuclear extracts was identified that appeared to recognize the octamer on the basis of differential binding to homologous restriction fragments containing the various mutations and that bound preferentially with octamer DNA fragments derived from functional relative to nonfunctional IgH constructs. Collectively, these data suggest that the octamer element contains residues that are critical to accurate immunoglobulin gene transcription and that may serve as part of a recognition locus for nuclear factors important to B-cell-specific immunoglobulin expression.
Animals↗
Molecular characterization of antibodies bearing Id-460. II. Molecular basis for Id-460 expression.
Id-460+ immunoglobulins can be induced in vivo by immunization with dinitrophenyl (DNP) or P. pneumotropica and form two nonoverlapping groups of antibodies with respect to antigen binding specificity. In this study, using Id-460+ antibodies of differing antigen binding specificities, we compared on the molecular genetic level the five gene segment combinations (VH, DH, JH, VL, and JL) that encode the variable regions of these idiotype-positive immunoglobulins. The Id-460 determinant appears to be a conformational or combinatorial determinant encoded by VH460 and VK1 crosshybridizing genes. DH, JH, and JK gene segments appear to have no measurable effect upon expression of Id-460. Finally, antigen binding specificity does not appear to simply localize to any particular gene segment but may in part be the result of somatic mutation and/or VDJH junctional sequences, whose length correlates roughly with antigen binding specificity.
Amino Acid Sequence↗
The T-cell-independent immune response to the hapten NP uses a large repertoire of heavy chain genes.
Hybridomas generated from C57BL/6 mice immunized with the hapten NP coupled to ficoll, a T-cell-independent carrier, produce monoclonal antibodies that use a large repertoire of VH regions and light chains. This contrasts with the homogeneity of the strain-specific response to NP observed with T-cell-dependent carriers, where most of the antibodies use a single VH region, V186.2, in combination with the lambda-1 light chain. There is no evidence for somatic mutation in any of the sequenced regions of the antibodies generated by NP-ficoll. Thus T cell participation is required for the homogeneity of the strain-specific hapten response, and probably for somatic mutation as well.
Animals↗
Physical linkage of the constant region genes for immunoglobulins lambda I and lambda III.
During differentiation from a stem cell to an antibody-secreting cell, the immunoglobulin genes within a B cell undergo a rearrangement that juxtaposes a variable region gene to a constant region gene. To analyze the genetic organization of an immunoglobulin gene family in nonrearranged, germ-line DNA, we have constructed a recombinant DNA library from randomly cleaved mouse kidney DNA fragments. From this library, we have isolated three overlapping recombinant clones containing the constant region gene for lambda I light chains (C lambda I). These clones spanned 24.9 kilobases of mouse DNA and contained no variable region sequences. Hybridization of these clones with lambda II cDNA demonstrated the presence of an additional constant region gene and a joining region 3.2 kilobases 5' of C lambda I. This gene was tentatively identified as C lambda III by the absence of an Ava I endonuclease site, which is present within C lambda II. The C lambda III amino acid sequence has recently been reported [Azuma, T., Steiner, L. A. & Eisen, H. N. (1981) Proc. Natl. Acad. Sci. USA 78, 569-573] and is very closely related to the C lambda II amino acid sequence.
Animals↗
Multiple expression of Ig lambda-chain encoding RNA species in murine plasmacytoma cells.
The murine plasmacytoma MOPC 315, which synthesizes a complete IgA molecule containing lambda 2 light chains, is shown to synthesize a polypeptide related to the lambda 1 light chain. The abnormal lambda 1 polypeptide (m.w. approximately equal to 18,000) is encoded by a shorter polyA+ mRNA than the lambda 1 mRNAs of lambda 1-producing plasmacytomas. Polypeptides containing lambda 1-specific antigenic determinants have been found to be synthesized both in vitro by translations of RNA and in intact cells. HOPC-1, a lambda 1-producing plasmacytoma, was found to synthesize, in addition to the normal lambda 1-encoding mRNA, a lambda 2 RNA of normal length. A lambda 2-producing hybridoma does not appear to synthesize RNA encoding lambda 1 sequences. The results presented here show that more than 1 light chain gene may be transcribed within a single plasmacytoma cell. Although more than 1 light chain gene may be transcribed, the product of only 1 gene appears to be utilized for the synthesis of complete immunoglobulin molecules. This suggests novel mechanisms for the control of immunoglobulin expression.
Animals↗
Characterization of a fused protein specified by the adenovirus type 2-simian virus 40 hybrid Ad2+ND1 dp2.
The adenovirus type 2-simian virus 40 (SV40) hybrid virus Ad2+ND1 dp2 (E. Lukanidin, manuscript in preparation) specified two proteins (molecular weights, 24,000 and 23,000) that are, in part, products of an insertion of SV40 early DNA sequences. This was demonstrated by translation in vitro from viral mRNA that had been selected by hybridization to SV40 DNA. These two phosphorylated, nonvirion proteins were produced late in infection in amounts similar to adenovirus 2 structural proteins and were closely related to each other in tryptic peptide composition. The portion of SV40 DNA (map units 0.17 to 0.22 on the SV40 genome) coding for these proteins was joined to sequences coding for the amino-terminal part of the adenovirus type 2 structural protein IV (fiber). The Ad2+ND1 dp2 23,000- and 24,000-molecular-weight proteins were hybrid polypeptides, with about two-thirds of their tryptic peptides contributed by the fiber protein and the remainder contributed by SV40 T-antigen. They shared with T-antigen (molecular weight, 96,000) a carboxy-terminal proline-rich tryptic peptide. Together, the tryptic peptide composition of these proteins and the known SV40 DNA sequences suggested the reading frame for the translation of T-antigen. The carboxy terminus for T-anigen would then be located on the SV40 genome map next to the TAA terminator triplet at position 0.175, 910 bases away from the cleavage site of the restriction endonuclease EcoRI. Seven host range mutants from Ad2+ND1 dp2 were isolated that had lost the capacity to propagate on monkey cells. They did not induce detectable levels of the hybrid proteins. Three of these mutants had lost the SV40 DNA insertion that codes in part for these proteins. Thus, in analogy to the Ad2+ND1 30,000-molecular-weight protein, the presence of these proteins correlates with the presence of the helper function for adenovirus replication on monkey cells.
Adenoviruses, Human↗