Biomedical subjects
Publications and source records attributed to M J Shulman.
We have identified the mutation in an IgM hybridoma mutant line which produces truncated mu protein. DNA sequencing has revealed that two base pairs are deleted in the Cmu3 domain, resulting in an early termination of protein synthesis by forming an in-frame termination codon (UGA). The mutant mu chain is incorporated into monomeric IgM which is secreted efficiently.
A nonfunctional immunoglobulin kappa-chain gene previously shown to contain intracisternal A-particle (IAP) sequences within one of its introns was further characterized by DNA and RNA blot analysis. The results of these experiments indicate that the joining of the IAP sequences to the kappa-chain gene was a consequence of an insertion event which presumably involved a complete IAP gene. It is postulated that the insertion of the IAP gene has altered the secondary or tertiary structure of the kappa-chain pre-mRNA, resulting in the utilization of a "cryptic" 5' splice site. The detection of numerous IAPs in isogenous wild-type hybridoma cells by electron microscopy suggests that transposition of IAP genes might proceed via the reverse transcription of IAP-associated RNA. These findings raise the possibility that transposition of IAP genes might also occur in other IAP-positive mouse cells. In particular, insertional mutagenesis by IAP genes in IAP-producing tumor cells could play a role in tumor cell heterogeneity or tumor progression. We have also investigated the possibility that IAP-related sequences might be present in the human genome. Using nonstringent hybridization conditions, multiple discrete restriction fragments could be detected in human DNA with several mouse IAP-specific probes.
Each of two severely defective mouse kappa-chain genes has acquired a different intracisternal A particle (IAP) element within one of its introns. One IAP element generated 6-base-pair direct repeats upon insertion. In contrast, the other IAP element was not flanked by direct repeats and was missing a single nucleotide from its 3' terminus. Sequence analysis of the latter IAP element demonstrated that its long terminal repeats were not identical. Nevertheless, the long terminal repeats were organized like proviral long terminal repeats, and this IAP element did contain two regions that were analogous to retroviral priming sites for RNA-directed DNA synthesis. The region that corresponded to a retroviral tRNA primer binding site was complementary to the 3' ends of all mammalian phenylalanine tRNAs. These findings are discussed in the context of the presumed mode of transposition of IAP elements involving the reverse transcription of IAP RNA.
Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in kappa chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing mu heavy chain fragments and provide information on the requirements of IgM assembly and mu gene expression. We also describe a transfer system for the mu and kappa genes which will be useful in analyzing the structural basis of IgM function.
Mutants of an IgM producing hybridoma cell line were isolated which produce mu heavy chain fragments. Two such mutants were found to have internal deletions in the mu gene and the nucleotide sequence of the deletion endpoints was determined. No evidence was found for a role of the heavy chain switch region in the formation of these deletions. The implications of these mutants in defining the requirements of immunoglobulin gene expression are discussed.
The rearranged immunoglobulin heavy (mu) and light (kappa) chain genes cloned from the Sp6 hybridoma cell line producing immunoglobulin M specific for the hapten 2,4, 6-trinitrophenyl were inserted into the transfer vector pSV2-neo and introduced into various plasmacytoma and hybridoma cell lines. The transfer of the mu and kappa genes resulted in the production of pentameric, hapten-specific, functional IgM.
Eight mutant hybridoma lines are described, which synthesize short immunoglobulin mu chains. Four internal deletions were mapped by Southern blot analysis. They are shown to remove DNA from either part or all of the first, and first and second, constant mu exons. The sizes of the deletions range between 0.6 and 5 kb, leaving an equal or unequal number of splice signals. Shorter mu RNA of one size was found irrespective of whether an exon was completely or only partially deleted. These results preclude exclusive 3' (constant region) to 5' (variable region) directional splicing of the mu RNA. No important signals seem to reside in the deleted DNA stretches affecting the transcription or the correct RNA splicing of the remaining exons. The internal mu protein deletions revealed unusual covalent light chain attachment demonstrating functional homology between the first (normally used) and fourth mu constant domain. The other mu protein deletions (10, 11, and 12 kd) involved neither gross DNA nor RNA lesions and are considered to be due to premature chain termination. Since secretion is found in most of the mutant IgM-producing lines, no single one of the four mu constant domains (including the C-terminal one which contains the so-called secretory piece) is necessary for secretion.
The kappa light chain genes from two mutant hybridoma cell lines defective in kappa light chain synthesis were isolated and compared to the wild-type kappa light chain gene. In each case, the mutant kappa light chain genes were found to contain repetitive DNA elements in their intervening sequences that were not present in the intervening sequences of the wild-type kappa light chain gene. These elements were found to be related to the genes of intracisternal A particles. These results suggest that the decreased production of kappa light chain in the mutant cell lines is due to the presence of the intracisternal A particle-related genes.
Using a hybridoma cell line which secretes hapten-specific immunoglobulin M (IgM), we have isolated a variety of mutants which produce abnormal immunoglobulin. Immunoglobulin was tested for the size and composition of the component heavy and light chains and for variable and constant region related functional and serological activities. Some mutants secrete IgM which seems to be defective in hapten binding; others make IgM which appears not to activate complement. Many of the mutants secrete monomeric as opposed to pentameric IgM. In some cases, the defect apparently correlates with structural alterations in the mu heavy chain: partial deletion, polypeptide addition, and abnormal glycosylation have been observed. These mutant cell lines provide a means of identifying the structural basis of IgM function and of studying the biochemistry of IgM synthesis and processing.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
We have investigated immunoglobulin production by cloned hybrid cells. gamma2b heavy chains, but not gamma1 or gamma21, exist in two forms, differing in molecular weight, galactose labeling and charge. Hybrid molecules containing gamma2b and gamma1 heavy chains were secreted by hybridoma cells synthesizing Ig molecules of both subclasses. However, cells synthesizing both IgM and IgG chains did not secrete mu-gamma hybrid molecules. We have surveyed many new hybridomas derived from different mouse strains: 10% of those secreting IgM produce a mu chain which is about 5000 daltons larger than normal mu. Two IgM-secreting lines were studied in detail and were found to bear mu but not delta in their membrane. Intra- and extracellular mu differ in molecular size; pentameric IgM-containing molecules of both sizes were observed in the membrane fraction.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.