Optional introns in mitochondrial DNA of Podospora anserina are the primary source of observed size polymorphisms.

The significant differences in mitochondrial genome size among seven races (B, E, M, T, U, W, and Y) of Podospora anserina have been found to be primarily due to the presence and/or absence of introns, including four introns not previously known to be optional. Information from physical mapping of races M and T, and sequence data from races A and s, was used to identify regions likely to contain insertions or deletions, which were then characterized using PCR and sequence analysis. Newly confirmed optional introns are the first intron of the large ribosomal RNA (LSUr1), the single intron of NADH dehydrogenase subunit 3 (ND3i1), the single intron in ATPase subunit 6 (ATPase6), and the fifth intron of cytochrome oxidase subunit I (COIi5). We have also found that race M exists in two forms as determined by mitochondrial DNA. These results bring to nine (including races A and s) the number of races characterized by mitochondrial intron content with a total of six known optional introns and one optional insertion. Eight of the nine races contain a distinct set of introns, providing a more reliable means for identification and comparison. The identification of optional mitochondrial introns in P. anserina may have evolutionary implications regarding the transfer and/or mobility of these introns.

Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro.

An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene. The stpA gene, which was localized to 60.24 min on the E. coli chromosome, encodes a 15.3-kDa protein. Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios. Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates. These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements. Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed. Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure.

The P2 element of the td intron is dispensable despite its normal role in splicing.

The P2 region of group I introns has been proposed to be involved in the correct positioning of the P1 5'-splice site duplex in the catalytic core (Michel, F., and Westhof, E. (1990) J. Mol. Biol. 216, 585-610). The behavior of delta P2 deletion mutants of the td intron is consistent with this hypothesis. The delta P2 mutants are capable of site-specific hydrolysis, indicating that the conformation of the ribozyme is not grossly altered, but they are incapable of transesterification reactions at the splice sites, as would be predicted if P1 is not appropriately aligned within the catalytic core. Nevertheless, the function of the P2 element can be bypassed in specific pseudorevertants isolated by genetic selection from the delta P2 mutants. These results, together with phylogenetic data, support the existence of alternate strategies to create a functional P1-core interaction.

A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion.

A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene. Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo. Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts. Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts. This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E. coli were attributable to the presence of the overlapping transcript. Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement. This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.

Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron. Analysis of the P6-L6a region.

Non-directed mutagenesis and phylogenetic comparison suggest that certain elements of the bacteriophage T4 td group Ia intron are dispensable to self-splicing. The L6-P6a-L6a region was identified as a potential non-essential element, and was removed by sequential deletions extending from the L6a loop toward the P6 pairing. Assays for splicing indicate that as long as the P6 pairing is maintained, the 1016 nucleotide td intron can be reduced to less than 250 nucleotides while maintaining function in vivo and in vitro. The P6 pairing appears to be essential for splicing while P6a is not. In addition, a spontaneous pseudorevertant of a splicing-defective deletion was isolated and shown to result from a single nucleotide change in the predicted L6a loop. This genetic suppressor mimics the ability of Mg2+ to reverse the phenotype of the deletion, suggesting that function is restored by structural stabilization of P6. The tolerance of this region to deletion prompted us to split the ribozyme core in L6a, to generate precursors that might function in trans. Indeed, the two half-molecules do associate to form a bimolecular complex that yields accurately ligated exons both in vitro and in vivo. The biological implications of these results, as well as the usefulness of trans-splicing for generating unprocessed precursors in vitro are discussed.