Storage of monokaryotic strains of Podospora anserina

Maintenance of Podospora anserina strains for experimental purposes is very time consuming (see Esser 1969 Neurospora Newsl. 15:27-31) and methods have been published that address this issue by freezing ascospores at -80 oC (Begel and Belcour 1991 Fungal Genet. Newsl. 38:67). Although the latter approach does reduce the amount of time required for yearly sexual crosses and ascospore isolation, it does not resolve the problem of the time required to rapidly generate monokaryotic hyphae, that are needed as a source for inoculum for many types of experiments

Substrate structural requirements of Schizosaccharomyces pombe RNase P

AbstractRNase P from Schizosaccharomyces pombe has been purified over 2000-fold. The apparent Km for two S. pombe tRNA precursors derived from the supS1 and sup3-e tRNASer genes is 20 nM; the apparent Vmax is 2.5 nM/min (supS1) and 1.1 nM/min (sup3-e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action

Group II intron mobility occurs by target DNA-primed reverse transcription

AbstractMobile group II introns encode reverse transcriptases and insert site specifically into intronless alleles (homing). Here, in vitro experiments show that homing of the yeast mtDNA group II intron a12 occurs by reverse transcription at a double-strand break in the recipient DNA. A site-specific endonuclease cleaves the antisense strand of recipient DNA at position +10 of exon 3 and the sense strand at the intron insertion site. Reverse transcription of al2-containing pre-mRNA is primed by the antisense strand cleaved in exon 3 and results in cotransfer of the intron and flanking exon sequences. Remarkably, the DNA endonuclease that initiates homing requires both the a12 reverse transcriptase protein and a12 RNA. Parallels in their reverse transcription mechanisms raise the possibility that mobile group II introns were ancestors of nuclear non-long terminal repeat retrotransposons and telomerases

A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro

Reverse transcriptases (RTs) are RNA-dependent DNA polymerases that usually function in the replication of selfish DNAs such as retrotransposons and retroviruses. Here, we have biochemically characterized a RT-related protein, AbiK, which is required for abortive phage infection in the Grampositive bacterium Lactococcus lactis. In vitro, AbiK does not exhibit the properties expected for an RT, but polymerizes long DNAs of ‘random’ sequence, analogous to a terminal transferase. Moreover, the polymerized DNAs appear to be covalently attached to the AbiK protein, presumably because an amino acid serves as a primer. Mutagenesis experiments indicate that the polymerase activity resides in the RT motifs and is essential for phage resistance in vivo. These results establish a novel biochemical property and a non-replicative biological role for a polymerase

A diversity of uncharacterized reverse transcriptases in bacteria

Retroelements are usually considered to be eukaryotic elements because of the large number and variety in eukaryotic genomes. By comparison, reverse transcriptases (RTs) are rare in bacteria, with only three characterized classes: retrons, group II introns and diversity-generating retroelements (DGRs). Here, we present the results of a bioinformatic survey that aims to define the landscape of RTs across eubacterial, archaeal and phage genomes. We identify and categorize 1021 RTs, of which the majority are group II introns (73%). Surprisingly, a plethora of novel RTs are found that do not belong to characterized classes. The RTs have 11 domain architectures and are classified into 20 groupings based on sequence similarity, phylogenetic analyses and open reading frame domain structures. Interestingly, group II introns are the only bacterial RTs to exhibit clear evidence for independent mobility, while five other groups have putative functions in defense against phage infection or promotion of phage infection. These examples suggest that additional beneficial functions will be discovered among uncharacterized RTs. The study lays the groundwork for experimental characterization of these highly diverse sequences and has implications for the evolution of retroelements

Self-splicing of a group IIC intron: 5′ exon recognition and alternative 5′ splicing events implicate the stem–loop motif of a transcriptional terminator

Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interestingly, additional unexpected products were formed that were highly dependent on ionic conditions. These products were determined to represent alternative splicing events at the 5′ junction and cleavages throughout the RNA transcript. The alternative splicing and cleavage events occurred at cryptic splice sites containing stem–loop and IBS1 motifs, suggesting that the 5′ exon is recognized by both elements. These results provide the first example of a group II intron that uses 5′ splice sites nonadjacent to the ribozyme structure. Furthermore, the data suggest that IIC introns differ from IIA and IIB introns with respect to 5′ exon definition, and that the terminator stem–loop substitutes in part for the missing IBS2–EBS2 (intron and exon binding sites 2) interaction

Target Site Recognition by a Diversity-Generating Retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification

ORF-less and reverse-transcriptase-encoding group II introns in archaebacteria, with a pattern of homing into related group II intron ORFs

Although group II intron retroelements are prevalent in eubacteria, they have not been identified in archaebacteria in the first 10 genomes sequenced. However, the recently sequenced archael genome of Methanosarcina acetivorans contains 21 group II introns, including 7 introns that do not encode reverse transcriptase ORFs. To our knowledge, these are the first retroelements identified in archaebacteria, and the first ORF-less group II introns in bacteria. Furthermore, the insertion pattern of the introns is highly unusual. The introns appear to insert site-specifically into ORFs of other group II introns, forming nested clusters of up to four introns, but there are no flanking exons that could encode a functional protein after the introns have been spliced out

Evolution of group II introns

Present in the genomes of bacteria and eukaryotic organelles, group II introns are an ancient class of ribozymes and retroelements that are believed to have been the ancestors of nuclear pre-mRNA introns. Despite long-standing speculation, there is limited understanding about the actual pathway by which group II introns evolved into eukaryotic introns. In this review, we focus on the evolution of group II introns themselves. We describe the different forms of group II introns known to exist in nature and then address how these forms may have evolved to give rise to spliceosomal introns and other genetic elements. Finally, we summarize the structural and biochemical parallels between group II introns and the spliceosome, including recent data that strongly support their hypothesized evolutionary relationship.Ye