Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKLO2 consists of lysis genes S, R, and Rz from bacteriophage A under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKLO2 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. cakoacetcus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. cakoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage 4X174 and causes lysis ofE. coli and P. stutzeri bacteria by activating cellular autolysins.Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKLO1-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.The microbial world is characterized by the collective ability of its members to rapidly colonize and thrive in a vast range of habitats. Some of these environments are characterized by such extreme physical and/or chemical conditions that all other forms of life are excluded. The metabolic opportunism of microbes results, on the one hand, from the exceptional physiological versatility and biochemical diversity of the microbial world as a whole and, on the other, from the ability of individual populations, when under appropriate selection pressure, to rapidly evolve new metabolic potential as a result of the acquisition of new genetic information through mutation and efficient gene transfer mechanisms. Although gene transfer mechanisms such as conjugation, transduction, and transformation have been extensively studied in the laboratory and participating cellular components have been identified and characterized to a considerable extent (for reviews, see, e.g., references 4 and 14), only limited information is available on natural gene transfer in the environment (17). Since genetic flux in microbial communities is a critical component of t...
In a search for genes involved in X-linked mental retardation we have analyzed the expression pattern and genomic structure of human MAGED2. This gene is a member of a new defined MAGE-D cluster in Xp11.2, a hot spot for X-linked mental retardation. Rat and mouse orthologues have been isolated. In contrast to the genes of the MAGE-A, MAGE- B and MAGE-C clusters, MAGED2 is expressed ubiquitously. High expression was detected in specific brain regions and in the interstitium of testes. Five SNPs in the coding region of human MAGED2 were characterized and their allele frequencies determined in a German and Turkish population.
The Wieacker-Wolff syndrome (WWS, MIM* 314580), first described clinically in 1985, is an X-linked recessive disorder. In earlier studies, linkage between the WWS gene and DXYS1 at Xq21.2 and DXS1 at Xq11 as well as AR at Xq12 was reported. Here we report on a linkage analysis using highly polymorphic, short terminal repeat markers located in the segment from Xp21 to Xq24. No recombination between the WWS locus and ALAS2 or with AR (z = 4.890 at θ = 0.0) was found. Therefore, the WWS locus was assigned to a segment of approximately 8 cM between PFC (Xp11.3-Xp 11.23) and DXS339 (Xq11.2-Xq13).
Cotransformation frequencies of 16, 39, 51, and 60% were observed when donor alleles were separated by distances of 9.2, 7.4, 6.3, and 5.1 kb, respectively, in donor Acinetobacter calcoaceticus DNA. A different and unexpected pattern was observed when the distance between recipient alleles was reduced from 9.2 to 5.1 kb. Ligation of unlinked chromosomal DNA fragments allowed them to be linked genetically through natural transformation.Natural transformation (12) has been useful in the analysis of catabolic pathways in Acinetobacter calcoaceticus (1, 3-8, 10, 14, 15, 18), but, as yet, there has been little evidence correlating cotransformation frequencies with the physical distance between alleles (11). An opportunity to investigate such correlations was presented by elucidation of the pca-qui-pob gene cluster. The pca genes encode enzymes required for growth with protocatechuate (15), and this metabolite can be produced by either the action of qui gene products on quinate (7,8) or the pobA-encoded metabolism of p-hydroxybenzoate (3). Null pca mutations prevent growth with both p-hydroxybenzoate and protocatechuate (9), whereas null pob mutations allow growth with protocatechuate (1). Cotransformation frequencies can be determined by transforming recipient strains, blocked in both pca and pob, with DNA containing the wildtype alleles. Wild-type pca DNA can be selected by demanding growth with protocatechuate, and cotransformation of wild-type pob DNA can be assessed as the frequency of the selected transformants that grow with p-hydroxybenzoate. Engineered deletions removing segments of qui DNA do not impede the growth of cells with either protocatechuate or p-hydroxybenzoate (7,8). Therefore, we were able to examine how variations in distance caused by the deletion of DNA between pca and pob influenced their cotransformation frequencies.Organization of wild-type and mutant genes in the investigated strains is presented in Fig. 1. In the wild-type background of strain ADP4021, pcaH19 and pobR5 are separated by about 9.2 kb, and transformation with donor DNA in which the wild-type alleles were identically separated yielded a cotransformation frequency of 16% (Fig. 2). The frequency increased to 39, 51, and 60% when the donor alleles were separated by 7.4, 6.3, and 5.1 kb, respectively (Fig. 2). This information may provide a rough guide to the correlation between cotransformation frequencies and the linear distances between transformed alleles in a wild-type background, but the results must be regarded with caution because a different pattern was observed when DNA from the same donors was provided to recipient strain ADP699, in which pcaH19 and pobR5 are separated by only 5.1 kb. As shown in Fig. 2, the cotransformation of markers in ADP699 was essentially invariant at 35% when the separation of donor alleles ranged between 5.1 and 7.4 kb (Fig. 2). The most remarkable finding was the difference in the cotransformations of ADP4021 and ADP699 when donor DNA provided alleles separated by 5.1 kb. In this comparison, ...
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