System to study horizontal gene exchange among microorganisms without cultivation of recipients

Ribosomal RNA genes are characterized by highly conserved sequences and are present in multiple copies in most prokaryotic chromosomes. In principle, therefore, they might serve as sites for homologous recombination between unrelated microorganisms. Plasmids containing 23S ribosomal gene sequences, from different bacteria, which had been interrupted by insertion of a kanamycin-resistance gene, were used to transform Acinetobacter sp. DSM587 (former name: Acinetobacter calcoaceticus BD413-ivl10). In all cases, homologies between the 23S rRNA genes of phylogenetically distant bacteria and Acinetobac-ter sp. DSM587 were sufficient for replacement recombination events. The integration events, resulting in inactivation of any one of the seven rrn operons of Acinetobacter sp. DSM587, had no observable influence on cell growth. These results suggest the possibility of rRNA genes serving as natural vehicles for horizontal gene transfer. They also provide the basis of a novel strategy to analyse gene transfer without selection or cultivation of recipient cells. Because of the highly conserved structure of bacterial rrn operons, recombination events subsequent to gene transfer can be readily identified by polymerase chain reaction amplification of the recombinant sequence using a universal forward primer for the 16S rRNA gene and a reverse primer specific for the integrated marker gene.     

Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs.

Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals.     

Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences

Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences.     

Evolution of mercuric reductase (merA) gene: a case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA is key gene in mer operon and has been used for this study. Phylogenetic analysis of aligned merA sequences shows broad similarities to the established 16S rRNA phylogeny. But there is no separation of bacterial merA from archael merA which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.     

DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer

Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160 232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy—in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.     

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.     

Crop improvement through modification of the plant's own genome

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.     

Evolution of mercuric reductase (<Emphasis Type="Italic">merA</Emphasis>) gene: A case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA gene is key gene in mer operon and has been used for this swtudy. Phylogenetic analysis of aligned merA gene sequences shows broad similarities to the established 16S rRNA gene phylogeny. But there is no separation of bacterial merA gene from archael merA gene which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA gene and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

Nucleotide sequence and evolution of the 18S ribosomal RNA gene in maize mitochondria.

The nucleotide sequence of the gene coding for the 18S ribosomal RNA of maize mitochondria has been determined and a model for the secondary structure is proposed. Dot matrix analysis has been used to compare the extent and distribution of sequence similarities of the entire maize mitochondrial 18S rRNA sequence with that of 15 other small subunit rRNA sequences. The mitochondrial gene shows great similarity to the eubacterial sequences and to the maize chloroplast, and less similarity to mitochondrial rRNA genes in animals and fungi. We propose that this similarity is due to a slow rate of nucleotide divergence in plant mtDNA compared to the mtDNA of animals. Sequence comparisons indicate that the evolution of the maize mitochondrial 18S, chloroplast 16S and nuclear 17S ribosomal genes have been essentially independent, in spite of evidence for DNA transfer between organelles and the nucleus.     

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

New gene transfer methods.

The intentional introduction of recombinant DNA molecules into a living organism can be achieved in many ways. Viruses have been making a living by practicing gene transfer for millennia. Recently, man has gotten into the act. The paradigm employed is fairly straightforward. First, a way must be found to move genetic information across biological membrane barriers. Then, presumably, DNA repair mechanisms do the rest. The array of methods available to move DNA into the nucleus provides the flexibility necessary to transfer genes into cells as physically diverse as sperm and eggs. Some of the more promising alternative strategies such as sperm-mediated gene transfer, restriction enzyme-mediated integration, metaphase II transgenesis, and a new twist on retrovirus-mediated gene transfer will be discussed, among other methods.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Stable genetic transformation of garlic plants using particle bombardment

The improvement of garlic plants (Allium sativum L.) via biotechnological approaches is currently limited by the lack of an applicable direct gene transfer system. In this paper, we present the development of a genetic transformation system using particle bombardment for gene delivery and immature clove-derived callus as the gene target. Plasmid DNA (pBI221.23), containing the selectable "hpt" gene for hygromycin resistance and the reporter "gus" gene, was delivered into callus tissue that had been previously treated with aurintricarboxylic acid as an endogenous nuclease inhibitor. The transformed calli were selected using hygromycin B, regenerated, and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical beta-glucuronidase assay. The results indicated that biolistic transformation can lead to the transfer, expression and stable integration of a DNA fragment into garlic chromosomal DNA. The relative simplicity of this system is a good recommendation for its future use in the production of genetically modified garlic plants.