Plasma cell-regulated polyadenylation at the Ig gamma 2b secretion-specific poly(A) site.

We found that the sequences downstream of the Ig gamma 2b secretory-specific (sec) poly(A) site play an important role in the preferential production of sec Ig mRNA during plasma B cell development. The Ig gamma 2b mRNA production in a deletion mutant (delta-Kpn) lacking the Ig sec poly(A) site and downstream consensus element (dsc) has been previously shown to default to the use of the downstream membrane-specific (mb) poly(A) site. In this study restoration of the Ig sec poly(A) site and dsc to the delta-Kpn gene causes a significant increase in the use of the sec poly(A) site vs mb poly(A) site in stable transfectants of plasma but not memory B cell tumors, indicating plasma cell-specific recognition of the Ig sec dsc. Restoration of the poly(A) cleavage site alone to delta-Kpn did not restore regulation. Substitution of an SV40 downstream poly(A) element for the Ig dsc in the delta-Kpn gene also does not restore regulation. The data further indicate that although the Ig dsc is clearly very important in the plasma cell-regulated expression, the difference in the processing ratios of the restored vs the intact Ig gamma 2b gene in plasma cells suggests that there are other yet to be defined sequences that may also play a role in the intact gene. Insertion of a 130-nucleotide segment of the gene containing the Ig sec poly(A) site and dsc into a heterologous, guanosyl phosphotransferase gene resulted in plasma cell-regulated polyadenylation of the sec poly(A) site. Neither the mb nor the SV40 early poly(A) sites and their respective dscs, in similar gpt chimeras, were regulated. Therefore the region downstream of the Ig sec poly(A) site plays an essential role in regulating polyadenylation at the sec poly(A) site in plasma cells but not memory cells. A model involving a plasma cell-specific recognition factor for the Ig sec dsc is presented.     

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.     

The in vivo use of alternate 3'-splice sites in group I introns.

Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used.     

Cell-specific expression of the carrot EP2 lipid transfer protein gene.

A cDNA corresponding to a 10-kD protein, designated extracellular protein 2 (EP2), that is secreted by embryogenic cell cultures of carrot was obtained by expression screening. The derived protein sequence and antisera against heterologous plant lipid transfer proteins identified the EP2 protein as a lipid transfer protein. Protein gel blot analysis showed that the EP2 protein is present in cell walls and conditioned medium of cell cultures. RNA gel blot analysis revealed that the EP2 gene is expressed in embryogenic cell cultures, the shoot apex of seedlings, developing flowers, and maturing seeds. In situ hybridization showed expression of the EP2 gene in protoderm cells of somatic and zygotic embryos and transient expression in epidermis cells of leaf primordia and all flower organs. In the shoot apical meristem, expression is found in the tunica and lateral zone. In maturing seeds, the EP2 gene is expressed in the outer epidermis of the integument, the seed coat, and the pericarp epidermis, as well as transiently in between both mericarps. Based on the extracellular location of the EP2 protein and the expression pattern of the encoding gene, we propose a role for plant lipid transfer proteins in the transport of cutin monomers through the extracellular matrix to sites of cutin synthesis.     

Decreased expression of Fc gamma RIII mRNA in leukemic granulocytes.

Morphologically mature granulocytes from patients with chronic myeloid leukemia show significant impairment in their ability to internalize aggregated IgG, a ligand that is rapidly phagocytosed by normal human granulocytes. With a view to understand the molecular basis of this defect, normal and leukemic granulocytes were examined for the steady-state levels of mRNA for Fc gamma RIII, a membrane-associated receptor that initially binds and traps the IgG-opsonized antigens. Northern blot analyses revealed that the level of the specific mRNA in CML granulocytes was between 0.08 and 0.69 times that seen in the normal granulocytes. This could be one of the contributory factors for the observed endocytic defect in the leukemic granulocytes.     

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.     

Duplication of the human immunoglobulin heavy chain gamma 2 gene.

The five C gamma genes in the human immunoglobulin heavy chain region show nonrandom association and segregation as haplotypes. From the study of genetic variation in C gamma genes of 58 healthy Caucasian volunteers, we have identified a haplotype that involves a duplication of C gamma 2. This haplotype contains both the 13.5-kilobase (kb) and 25-kb BamHI fragment alleles of C gamma 2. In addition, the patterns and relative intensity of BamHI fragments containing C gamma genes were those expected for genomic DNA containing three copies of C gamma 2 for every two copies of the four other C gamma genes. A new EcoRI polymorphism in C gamma 4 was useful in defining the haplotype containing the duplication. Alleles of the C gamma genes in the duplication haplotype, including Gm markers of C gamma 1 and C gamma 3 and DNA polymorphisms of C psi gamma, C gamma 2, and C gamma 4, were consistent with its origin from an unequal crossover between the two common C gamma haplotypes, H1 and H2. This recombinant haplotype, which has been designated H2;1(gamma 2 dup) to reflect its origin, occurred with a frequency of .043 in a random sample of 116 chromosomes.