Finn Haugli

Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus

A synthetic oligodeoxynucleotide probe was used to clone the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. The sequence of a 2050-bp restriction fragment containing the gene was determined. Analysis of the gene-derived amino acid (aa) sequence showed that this exoenzyme is probably synthesized as a 283-aa precursor with a 24-aa signal peptide and a 14-aa propeptide. The mature, secreted enzyme comprises 245 aa residues. Sonicates of Escherichia coli HB101 carrying the gene on a multicopy plasmid showed phospholipase C activity. This activity was inhibited by Tris, a known inhibitor of the B. cereus enzyme and also by antiserum raised against pure B. cereus phospholipase C. We conclude therefore that the gene is expressed in E. coli. The cloning and sequencing described here complete the first step toward using in vitro mutagenesis for investigations of the structure-function relationships of B. cereus phospholipase C.

In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis

DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin‐ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron‐containing and intron‐less amoebae are mated. The mobility process is fast, being completed in 5–10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron‐encoded protein was expressed in Escherichia coli and found to be an endonuclease, I‐Dir I, that cleaves an intron‐less ribosomal DNA allele at the intron‐insertion site, and is probably involved in intron homing. The endonuclease I‐Dir I seems to be a rare example of a protein that is expressed from a ribozyme‐processed RNA polymerase I transcript in vivo.

Structure and evolution of myxomycete nuclear group I introns: a model for horizontal transfer by intron homing

SummaryWe have examined five nuclear group I introns, located at three different positions in the large subunit ribosomal RNA (LSU rRNA) gene of the two myxomycete species, Didymium iridis and Physarum polycephalum. Structural models of intron RNAs, including secondary and tertiary interactions, are proposed. This analysis revealed that the Physarum intron 2 contains an unusual core region that lacks the P8 segment, as well as several of the base-triples known to be conserved among group I introns. Structural and evolutionary comparisons suggest that the corresponding introns 1 and 2 were present in a common ancestor of Didymium and Physarum, and that the five introns in LSU rRNA genes of these myxomycetes were acquired in three different events. Evolutionary relationships, inferred from the sequence analysis of several different nuclear group I introns and the ribosomal RNA genes of the intron-harbouring organisms, strongly support horizontal transfer of introns in the course of evolution. We propose a model that may explain how myxomycetes in natural environments obtained their nuclear group I introns.

Rapid disappearance of one parental mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum

SummaryFive haploid amoebal strains of the myxomycete Physarum polycephalum, each with a distinct mitochondrial genotype, were crossed in all pairwise combinations. The mitochondrial genotype in the diploid plasmodia resulting from these isogamous matings were found to be transmitted uniparentally. This uniparental inheritance could be arranged in a dominant hierarchical order. Time-course analysis of the presence of mitochondrial genotypes in the zygotes and young developing plasmodial genotypes is virtually completed during the first two nuclear cycles in the zygote/differentiating plasmodium. To our knowledge this is the first report indicating an active mechanism involving the degradation of mitochondrial genomes in sexual crosses.

Extrachromosomal ribosomal DNA of Didymium iridis: sequence analysis of the large subunit ribosomal RNA gene and sub-telomeric region

SummaryThe ribosomal DNA of the myxomycete Didymium iridis is organized as extrachromosomal linear molecules of about 20 kb, containing only one transcription unit of the ribosomal RNA genes. We have determined the sequence of the large subunit ribosomal RNA (LSU rRNA) gene as well as the sub-telomeric and telomeric regions. The LSU rRNA gene was found to encode a 3857 nucleotide-long LSU rRNA, interrupted by a transcribed spacer and two group I introns. A complete secondary structure model of D. iridis LSU rRNA has been constructed. The compact sub-telomeric region of D. iridis rDNA was found to contain several directly repeated sequence elements that include the simple telomere motif TTAGGG. Based on pairwise comparisons of LSU rRNA sequences, the time of divergence between the two myxomycete genera Didymium and Physarum was estimated.

Group I introns in biotechnology: prospects of application of ribozymes and rare-cutting homing endonucleases

Abstract Group I introns are sporadically, but universally distributed among different genes and genomes. They are distinguished from other classes of introns by a characteristic RNA structure and splicing mechanism. Group I introns may encode two different gene products with relevance to biotechnology: group I ribozymes and rare-cutting homing endonucleases. General aspects on molecular and biological features of naturally occurring group I introns are briefly discussed in the first part of the chapter. The second part focuses on group I ribozymes and their potential as tools in molecular biology and therapy, including both gene therapy and chemotherapy. Finally, rare-cutting homing endonucleases are reviewed and considered as molecular tools in genome mapping and genome engineering.

DNA replication in physarum polycephalum: Characterization of replication products made in isolated nuclei

Abstract Nuclei isolated from synchronous S-phase plasmodia of the myxomycete Physarum polycephalum were competent in production of low molecular weight DNA replication intermediates. Furthermore, these nuclei showed some competence in joining these fragments into DNA of intermediate molecular weight. The DNA molecules made in vitro could be correlated with products made in vivo .

Enrichment and screening of heat-sensitive mutants of Physarum polycephalum

A new method for the isolation of temperature-sensitive mutants of Physarum polycephalum is described. It involves enrichment and prescreening of mutagenized amoebae followed by screening at both the plasmodial and amoebal stage. A total of 74 temperature-sensitive strains were recovered of which 26 were temperature-sensitive only as plasmodia, 35 only as amoebae and 13 in both stages. After a shift to the nonpermissive temperature, DNA and protein synthesis were followed in temperature-sensitive plasmodia to discover if the lesion affected functions of the nuclear cycle.

Toward a DNA Transformation System for Physarum polycephalum

Physarum polycephalum is an organism with unusually flexible possibilities for genetic analysis of a variety of functions. One of the foundations for making such a claim is that through the life cycle the following nuclear conditions can be achieved; haploid uninucleate; diploid homo- or heterozygous uninucleate; haploid multinucleate; diploid homo- or heterozygous multinucleate; haploid-haploid heterokaryons; diploid-diploid (homo- or heterozygous) heterokaryons; diploid-diploid heterokaryons; and heterochronous heterokaryons of all the ploidy types listed. In addition, the organism has natural cell cycle synchrony, interesting differentiation processes, and motility systems.