High throughput direct end sequencing of BAC clones.
Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed. (+info)
Specific DNA recognition by F Factor TraY involves beta-sheet residues.
The F Factor TraY protein is a sequence-specific DNA-binding protein required for efficient conjugal transfer. Genetic and biochemical studies indicate that TraY has two functional roles in conjugation. TraY binds to the PY promoter to up-regulate transcription of tra genes. TraY also binds to the plasmid origin of transfer (oriT), serving as an accessory protein in the nicking of F Factor in preparation for transfer. TraY is thought to belong to the ribbon-helix-helix family of transcription factors. These proteins contact DNA using residues of an antiparallel beta-sheet. We engineered and characterized six TraY mutants each having a single potential beta-sheet DNA contact residue replaced with Ala. Most TraY mutants had significantly reduced affinity for the TraY oriT binding site while possessing near wild-type stability and nonspecific DNA recognition. These results indicate that TraY beta-sheet residues participate in DNA recognition, and support inclusion of TraY in the ribbon-helix-helix family. (+info)
Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.
In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number. (+info)
Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.
DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background. (+info)
A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.
Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors. (+info)
Comparison of proteins involved in pilus synthesis and mating pair stabilization from the related plasmids F and R100-1: insights into the mechanism of conjugation.
F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus. (+info)
Genomic subtraction identifies Salmonella typhimurium prophages, F-related plasmid sequences, and a novel fimbrial operon, stf, which are absent in Salmonella typhi.
Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57. (+info)
Analysis of F factor TraD membrane topology by use of gene fusions and trypsin-sensitive insertions.
This report describes a procedure for characterizing membrane protein topology which combines the analysis of reporter protein hybrids and trypsin-sensitive 31-amino-acid insertions generated by using transposons ISphoA/in and ISlacZ/in. Studies of the F factor TraD protein imply that the protein takes on a structure with two membrane-spanning sequences and amino and carboxyl termini facing the cytoplasm. It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins was ambiguous, based on the trypsin cleavage behavior of a 31-residue insertion. (+info)