Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.     

Mutations in genes downstream of the rpoN gene (encoding σ54) of Klebsiella pneumoniae affect expression from σ54-dependent promoters

Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from sigma 54-dependent promoters. We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified.     

PCR-based gene disruption and recombinatory marker excision to produce modified industrial Saccharomyces cerevisiae without added sequences

The dominant selectable Kanr marker, which confers geneticin resistance in yeast, is extensively used for PCR based disruption of genes in functional analysis studies in laboratory strains of Saccharomyces cerevisiae. We have developed a gene disruption cassette, which incorporates the Kanr marker, and direct repeat sequences designed from the target gene to enable the deletion of the gene without the introduction of added DNA sequences. We report on the disruption of the HO gene as a test case, using the hodr-Kanr-hodr cassette. The cassette was shown to integrate at the HO locus and the Kanr marker excised by recombination between the two direct repeat sequences. The disruption/excision event resulted in the removal of one direct repeat and the coding sequence of the gene, and hence in this case loss of HO function, with the introduction of no foreign or additional sequences, including the Kanr marker. Having been derived from the target site, the remaining direct repeat sequence is native sequence in its native location. This design template has the potential to be adapted to other genes, and as such will be of advantage in instances such as the optimization of strains by recombinant DNA technology where the retention of minimal or no foreign sequences is desired.     

Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains.

The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.     

New integrative method to generate Bacillus subtilis recombinant strains free of selection markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP beta-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P(lysA) promoter with that of the P(blaP) beta-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.     

Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.     

Bacillus sporulation gene spo0H codes for sigma 30 (sigma H).

The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively. The two spo0H gene products are 91% identical to one another and about 25% identical to most of the procaryotic sigma factors. The predicted proteins have a conserved 14-amino-acid sequence at their amino terminal end, typical of sigma factors. Antibodies raised against the spo0H gene product of B. licheniformis specifically react with RNA polymerase sigma factor protein, sigma 30, purified from B. subtilis. We conclude that the spo0H genes of B. licheniformis and B. subtilis code for sigma 30, now known as sigma H.     

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.     

The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes: characterization of reovirus core protein sigma 2.

The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes were determined to gain insight into the structure and function of the S2 translation product, virion core protein sigma 2. The S2 sequences of the type 1 Lang, type 2 Jones, and type 3 Dearing strains are 1,331 nucleotides in length and contain a single large open reading frame that could encode a protein of 418 amino acids, corresponding to sigma 2. The deduced sigma 2 amino acid sequences of these strains are very conserved, being identical at 94% of the sequence positions. Predictions of sigma 2 secondary structure and hydrophobicity suggest that the protein has a two-domain structure. A larger domain is suggested to be formed from the amino-terminal three-fourths of sigma 2 sequence, which is separated from a smaller carboxy-terminal domain by a turn-rich hinge region. The carboxy-terminal domain includes sequences that are more hydrophilic than those in the rest of the protein and contains sequences which are predicted to form an alpha-helix. A region of striking similarity was found between amino acids 354 and 374 of sigma 2 and amino acids 1008 and 1031 of the beta subunit of the Escherichia coli DNA-dependent RNA polymerase. We suggest that the regions with similar sequence in sigma 2 and the beta subunit form amphipathic alpha-helices which may play a related role in the function of each protein. We have also performed experiments to further characterize the double-stranded RNA-binding activity of sigma 2 and found that the capacity to bind double-stranded RNA is a property of the sigma 2 protein of prototype strains and of the S2 mutant tsC447.     

Gene cassettes from the insert region of integrons are excised as covalently closed circles

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.     

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool.