Localized gene-specific induction of accessibility to V(D)J recombination induced by E2A and early B cell factor in nonlymphoid cells.

Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VkappaI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vkappa locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the D(H), Vkappa, and Vlambda loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.

Related alphaN- and epsilonN-methyltransferases methylate the large and small subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is methylated at the ax amino position of the N-terminal methionyl residue of the processed and assembled form of the small subunit (SS), and is also methylated in some species at the epsilon-amino group of lysine-14 in the large subunit (LS). The gene (rbcMT-S) and cDNAs for the SS alphaN-methyltransferase (SSMT) from spinach (Spinach oleracea) have been cloned, sequenced, and expressed. The gene is closely related to a previously characterized LS methyltransferase (Rubisco LSMT) cDNA from pea (Rubisco LSMT) and a Rubisco LSMT gene from tobacco. Sequence analysis of the cDNA and transcript mapping experiments demonstrate that the rbcMT-S pre-mRNAs experience alternative 3' splice site selection, such that mRNAs for a long form with a four amino acid insertion and a short form are expressed at approximately equal abundance. The coding sequence of spinach SSMT includes a putative targeting presequence with sequence identity at a plastid processing site. A N-terminal truncated form of spinach SSMT was expressed and purified from E. coli cells. Both long and short forms of the cDNAs were shown to catalyze methylation of the a amine of the N-terminal methionine of the SS of Rubisco.

Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase gene in tobacco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) epsilon N-methyltransferase (Rubisco LSMT) catalyzes the posttranslational methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco in many higher plant species including tobacco. The tobacco Rubisco LSMT gene (rbcMT-T) and its cDNA were isolated, sequenced, and characterized. The gene contains 6 exons and spans about 6 kb. Primer extension analysis indicated one transcription start site located 93 nt upstream of the translation initiation site. Sequence analysis of the 5'-flanking region suggests several potential binding sites for transcription factors, including 7 GT-1 elements and an HSP-70.5 element. Gene dosage analysis by Southern hybridization demonstrated that the tobacco rbcMT-T gene is present as a single copy in the tobacco haploid genome. The full-length cDNA for tobacco rbcMT-T is 1974 nt in length excluding the 3' poly(A)15 tail, and encodes a 491 amino acid polypeptide with a molecular mass of ca. 56kDa. The deduced amino acid sequence of tobacco Rubisco LSMT has 64.5% identity and 75.3% similarity with the sequence of pea Rubisco LSMT, and both proteins contain several copies of a conserved imperfect leucine-rich repeat motif.