Escherichia coli gene ydeA encodes a major facilitator pump which exports L-arabinose and isopropyl-beta-D-thiogalactopyranoside.
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Inactivation of the Escherichia coli gene ydeA, which encodes a member of the major facilitator superfamily, decreased the efflux of L-arabinose, thereby affecting the expression of AraC-regulated genes. In addition, overexpression of ydeA decreased the expression of genes regulated by isopropyl-beta-D-thiogalactopyranoside. (+info)
Hensin, the polarity reversal protein, is encoded by DMBT1, a gene frequently deleted in malignant gliomas.
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The band 3 anion exchanger is located in the apical membrane of a beta-intercalated clonal cell line, whereas the vacuolar H(+)-ATPase is present in the basolateral membrane. When these cells were seeded at confluent density, they converted to an alpha-phenotype, localizing each of these proteins to the opposite cell membrane domain. The reversal of polarity is induced by hensin, a 230-kDa extracellular matrix protein. Rabbit kidney hensin is a multidomain protein composed of eight SRCR ("scavenger receptor, cysteine rich"), two CUB ("C1r/C1s Uegf Bmp1"), and one ZP ("zona pellucida") domain. Other proteins known to have these domains include CRP-ductin, a cDNA expressed at high levels in mouse intestine (8 SRCR, 5 CUB, 1 ZP), ebnerin, a protein cloned from a rat taste bud library (4 SRCR, 3 CUB, 1 ZP), and DMBT1, a sequence in human chromosome 10q25-26 frequently deleted in malignant gliomas (9 SRCR, 2 CUB, 1 ZP). Rabbit and mouse hensin genomic clones contained a new SRCR that was not found in hensin cDNA but was homologous to the first SRCR domain in DMBT1. Furthermore, the 3'-untranslated regions and the signal peptide of hensin were homologous to those of DMBT1. Mouse genomic hensin was localized to chromosome 7 band F4, which is syntenic to human 10q25-26. These data suggest that hensin and DMBT1 are alternatively spliced forms of the same gene. The analysis of mouse hensin bacterial artificial chromosome (BAC) genomic clone by sequencing and Southern hybridization revealed that the gene also likely encodes CRP-ductin. A new antibody against the mouse SRCR1 domain recognized a protein in the mouse and rabbit brain but not in the immortalized cell line or kidney, whereas an antibody to SRCR6 and SRCR7 domains which are present in all the transcripts, recognized proteins in intestine, kidney, and brain from several species. The most likely interpretation of these data is that one gene produces at least three transcripts, namely, hensin, DMBT1, and CRP-ductin. Hensin may participate in determining the polarized phenotype of other epithelia and brain cells. (+info)
A 12-Mb complete coverage BAC contig map in human chromosome 16p13.1-p11.2.
(75/4994)
We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs. (+info)
Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes.
(76/4994)
ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd(+) bacterium as the result of replication errors or recombination-dependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with approximately 600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage. (+info)
Stress-induced membrane association of the Streptococcus mutans GTP-binding protein, an essential G protein, and investigation of its physiological role by utilizing an antisense RNA strategy.
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SGP (for Streptococcus GTP-binding protein) is a Streptococcus mutans essential GTPase which has significant sequence identity to the previously identified Escherichia coli Era protein and to numerous other prokaryotic GTPase proteins of unknown function. Recent studies in our laboratory have addressed the possible role of SGP in the stress response of the oral pathogen S. mutans. Here we report that during growth in the early stationary phase, and in response to elevated temperatures or acidic pH, the distribution of SGP between the cytoplasm and the membranes of S. mutans cells varies. Immunoblot analysis of soluble and membrane protein fractions collected from the mid-log and early stationary growth phases of bacterial populations grown at normal temperature (37 degrees C) and at the elevated temperature of 43 degrees C, or at acidic pH, demonstrated that the total amount of SGP increased with the age of the bacterial culture, elevated temperature, or acidic pH. Furthermore, it was established that a substantial amount of SGP is associated with the membrane fraction under stress conditions. In order to investigate the physiological role of SGP, we constructed an S. mutans strain capable of chromosomal sgp antisense RNA expression, which interferes with the normal information processing of the sgp gene. Utilizing this strain, we determined conditions whereby the streptococcal cells can be depleted of SGP, thus avoiding the problem of constructing a conditional lethal system. From the results of measurements of the nucleotide pools extracted from the antisense strain and its isogenic counterpart, we propose that one of the physiological roles of SGP is regulation and modulation of the GTP/GDP ratio under different growth conditions. Moreover, we observed that in SGP-depleted cells the levels of glucan-binding protein A (GbpA) substantially increased, suggesting that GbpA may have stress response-related physiological functions. Finally, the potential applications of the antisense RNA approach that we employed are discussed. (+info)
The gene locus yijP contributes to Escherichia coli K1 invasion of brain microvascular endothelial cells.
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Most cases of Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. A TnphoA mutant of E. coli K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the TnphoA insertion site was cloned from RS218, and the cloned DNA complemented the TnphoA mutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical yijP gene (also called f577) in the E. coli K-12 genome. Sequence analysis indicated that the E. coli K1 yijP gene likely encodes a 66. 6-kDa membrane protein. Deletion and complementation experiments indicated that the yijP gene was involved in E. coli K1 invasion of BMEC, i.e., the invasive ability of E. coli K1 was significantly reduced after yijP was deleted and was restored by complementation with a plasmid containing the yijP open reading frame. This is the first demonstration that the yijP gene locus plays a role in the pathogenesis of E. coli K1 meningitis. (+info)
Combined genetic and physical map of the complex genome of Agrobacterium tumefaciens.
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A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies. (+info)
Mutations affecting motifs of unknown function in the central domain of nitrogen regulatory protein C.
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The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of sigma54 holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated ntrC genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of glnA transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrCWT). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrCWT at low concentrations. Their elevated activation capacities remain to be explained. (+info)