The Cryphonectria parasitica mitochondrial rns gene: Plasmid-like elements, introns and homing endonucleases

The mt-rns gene of Cryphonectria parasitica is 9872 bp long and includes two group I and two group II introns. An analysis of intronic protein-encoding sequences revealed that LAGLIDADG ORFs, which usually are associated with group I introns, were transferred at least twice into group II introns. A plasmid-like mitochondrial element (plME) that appears in high amounts in previously mutagen-induced mit1 and mit2 hypovirulent mutants of the Ep155 standard virulent strain of C. parasitica was found to be derived from a short region of the mt-rns gene, including the exon 1 and most of the first intron. The plME is a 4.2-kb circular, multimeric DNA and an autonomously-replicating mtDNA fragment. Although sexual transmission experiments indicate that the plME does not directly cause hypovirulence, its emergence is one manifestation of the many complex molecular and genetic events that appear to underlie this phenotype.     

Targeted gene disruption in Francisella tularensis by group II introns

Francisella tularensis is a highly infectious Gram-negative bacterium that is the causative agent of tularemia. Very little is known about the molecular mechanisms responsible for F. tularensis virulence, in part due to the paucity of genetic tools available for the study of F. tularensis. We have developed a gene knockout system for F. tularensis that utilizes retargeted mobile group II introns, or “targetrons”. These targetrons disrupt both single and duplicated target genes at high efficiency in three different F. tularensis subspecies. Here we describe in detail the targetron-based method for insertional mutagenesis of F. tularensis genes, which should facilitate a better understanding of F. tularensis pathogenesis. Group II introns can be adapted to inactivate genes in bacteria for which few genetic tools exist, thus providing a powerful tool to study the genetic basis of bacterial pathogenesis.     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa

Summary The complete nucleotide sequence of the gene coding for one of the carboxymethycellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most pseudomonas genes, the CMCase sequence does not have a high G+C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.Abbreviations CMCase carboxymethylcellulase - PAGE polyacrylamide gel electrophoresis - IPTG isopropyl--D-thiogalactoside - CAT chloramphenicol acetyl transferase     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Summary The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

Nuclear and mitochondrial revertants of a yeast mitochondrial tRNA mutant

Summary We isolated revertants capable of respiration from the respiratory deficient yeast mutant, FF1210-6C/ 170, which displays greatly decreased mitochondrial protein synthesis due to a single base substitution at the penultimate base of the tRNA^Asp gene on mitochondrial (mt) DNA. Three classical types of revertant were identified: (1) same-site revertants; (2) intragenic revertants which restore the base pairing in the acceptor stem of the mitochondrial tRNA^Asp; and (3) extragenic suppressors located in nuclear DNA. In addition a fourth type of revertant was identified in which the mutant tRNA^Asp is amplified due to the maintenance of both the original mutant mtDNA and a modified form of the mutant mtDNA in which only a small region around the tRNA^Asp gene is retained and amplified. The latter form resembles the mtDNA in vegetative petite (rho ^-) strains which normally segregates rapidly from the wild-type mtDNA. Each revertant type was characterized genetically and by both DNA sequence analysis of the mitochondrial tRNA^Asp gene and analysis of the quantity and size of RNA containing the tRNA^Asp sequence. These results indicate that the mitochondrial tRNA^Asp of the mutant retains a low level of activity and that the presence of the terminal base pair in tRNA^Asp is a determinant of both tRNA^Asp function and the maintenance of wild-type levels of tRNA^Asp.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.