Dissection of the Salmonella typhimurium genome by use of a Tn5 derivative carrying rare restriction sites

Wong, Kwok Wong Kwong Kwok; McClelland, Michael

doi:10.1128/jb.174.11.3807-3811.1992

Cited by 30 publications

(17 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We further characterized RSP435 by mapping its chromosomal location by means of Southern blots to Bln I and Xba I restriction fragments of the S. typhimurium genome separated by PFGE (17,18,26). The gene hybridized at high stringency to two genomic fragments.…”

Section: Methodsmentioning

confidence: 99%

“…*Constructed by generalized transduction with P22 HT12/4 int3 (16) grown from the donor strain TT3338 (gal: :TnlO). As a result, a Bln I fragment of about 200 kb, which corresponds to 18-21.5 min, was "dropped out" from the Bln I-A fragment (1600 kb) when the chromosome was digested with Bln I and resolved by pulsed-field gel electrophoresis (PFGE) (17,18 (20) into the Srf I site of the pCR-script SK(+) vector (Stratagene), mobilized into singlestranded phage, and sequenced using the Sequenase kit (United States Biochemical). PFGE.…”

Section: Methodsmentioning

confidence: 99%

“…PFGE. Genomic DNA from a late logarithmic phase S. typhimurium LT2 culture was prepared in InCert agarose (FMC) as described (17,18). The DNA was cleaved with 10 units of Bln I (Takara Biochemical, Berkeley, CA) or Xba I (Stratagene) for 4 hr in 1 x KGB (22), with potassium acetate replacing potassium glutamate.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.

Wong

McClelland

1994

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

101

View full text Add to dashboard Cite

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to identify conditionaily expressed genes in prokaryotes. Differential gene expression in SalmoneUa typhimurium LT2 in response to peroxide treatment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typhimurium infection. To identify genes inducible by hydrogen peroxide stress, total RNA from peroxide-treated and untreated bacterial cultures were RAP rmgerprinted with six different arbitrarily selected primers. A 435-base RAP product that was differentialy amplified by RAP using the reverse sequencing primer was cloned and sequenced. Northern blot analysis confirmed that the RNA corresponding to this clone, RSP435, was induced when bacteria were treated with hydrogen peroxide. The RNA was not induced in an oxyRI mutant that constitutively expresses a subset of hydrogen peroxide-inducible genes. Using pulsedfield gel electrophoresis and dot blot hybridization to an array of induced Mud-P22 integrations, the gene corresponding to RSP435 was mapped to two places, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in different parts of the genome. Identification, cloning, and mapping of the conditionaHly expressed RSP435 cDNA were performed entirely by physical means, demonstrating that the strategy should complement genetic methods for many prokaryotic or archaebacterial systems and should be applicable to organisms in which genetic methods are difficult to perform or have not yet been developed.Two similar methods for fingerprinting of eukaryotic RNA have been developed (1, 2) based on arbitrarily primed PCR (RAP) (3, 4). In principle, fingerprinting of RNA by RAP (1, 5) can be used on prokaryotic RNAs because it does not rely on poly(A)-tailed RNA that is rare in mRNAs isolated from bacteria (6). To demonstrate that the method can be used to identify differentially expressed genes of bacteria we searched for stress-induced RNAs in Salmonella typhimurium.Enterobacteria respond to various stimuli such as oxidative stress, extreme pH, anaerobiosis, heat shock, osmotic shock, and starvation, by changing the expression of groups of genes termed "stimulons" (7,29). Assorted genetic tools have been used to study these regulatory networks, including operon fusions (8), promoter probe plasmids (9, 10), and positively selected promoter probes (11). An example of the differential expression of sets of genes due to external stimuli is the response of S. typhimurium to oxidative stress. When bacteria invade the mammalian host, activated granulocytes may employ millimolar concentrations of peroxide and superoxide anion during phagocytosis in an attempt to kill the bacteria (12, 13). In response, the bacteria can induce stimu-The publication costs of this article were defrayed in part by page charge payment. This article must therefore...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.

Wong

McClelland

1994

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

101

View full text Add to dashboard Cite

show abstract

“…The fragments were arranged on a circular map by using cloned genes as probes, using transposon TnlO inserted at known sites to add a restriction site, and using "linking probes" derived from induction of lysogens for Mud-P22 (3,59). Further locations on the genome have been determined with TnS derivatives (58).…”

mentioning

confidence: 99%

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

1993

View full text Add to dashboard Cite

Endonuclease digestion of the 4,800-kb chromosome of SabloneUa lyphimurium LT2 yielded 24 XbaI fragments, 12 BIn! fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis.The 90-kb plasmid pSLT has oneXbaI site and one BinI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent TnlO insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and Bin1 sites in TnlO. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn1O insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the Ceu! digestion site, all four with the tRNA gene for glutamate have theXbaI and the BinI sites, but only four of the seven have the Bin! site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BinI sites outside the mrn genes show very little conservation.

show abstract

“…Strains were constructed via P22 HT12/4 int-3 transductions (6) as described previously (16). TnlO insertions (13), Tn5(pfm) insertions (15), and Mud-P22 strains (1) have been described previously.…”

Section: Methodsmentioning

confidence: 99%

High-resolution restriction map for a 240-kilobase region spanning 91 to 96 minutes on the Salmonella typhimurium LT2 chromosome

Wong

Rudd

et al. 1994

J Bacteriol

Self Cite

View full text Add to dashboard Cite

A hierarchical approach allows the completion of contiguous sets of overlapping clones for small regions of a genome, one at a time rather than tackling the whole genome at once. On the basis of the BlnI restriction map for Salmonella typhimurium LT2, we dissected the chromosome into 21 different fragments by using a TnS transposon carrying a BlnI site. Dissected chromosomal fragments were purified by pulsed-field gel electrophoresis and used as probes for sorting a lambda DASHII genomic library of 2,304 primary clones. A total of 129 clones identified as spanning the region from 91 min to 98 min were partly ordered on the basis of the intensity of hybridization with mitomycin-induced Mud-P22 phage DNAs from insertions with pac sites in opposite orientations at 93 min used as probes. Decreased signal intensity with the Mud-P22 probes corresponded to the increased distance of the clone from the site of Mud-P22 insertion and allowed the clones to be placed in two groups from 91 min to 93 min and from 93 min to 98 min and into four intensity categories within the two groups. A member of each category was used to generate a riboprobe from the T3 promoter flanking the insert. This probe identified overlapping clones among the 129 clones. This subchromosomal library was then screened again with riboprobes from nonoverlapping clones. After four cycles of this strategy, a minimal contiguous sequence of 19 partly overlapping clones was selected for restriction mapping. A detailed map of 378 sites for eight restriction enzymes is presented for a region of about 240 kb. Working clockwise, the following genes were placed on this physical map on the basis of their restriction maps: malFEK, lamB, malM, lexA, qor, dnaB, air, uvrA, proP, pmrB, pmrA, melA, meiB, phoN, amiB, mutL, and miaA.In 1987, a restriction map of the Escherichia coli genome was produced by using partial restriction digests of 3,400 bacteriophage lambda clones generated with the eight restriction enzymes BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, and PvuII (7). The genomic sequence of E. coli is going to be completed soon (2 enzymes used for E. coli was employed here. However, we took a different approach which allows the completion of contiguous sequences (contigs) for manageable regions of the genome, one at a time rather than tackling the whole genome at once, and which requires restriction mapping of only a small number of the lambda clones. MATERIALS AND METHODSPhage and bacterial strains. All bacterial strains were LT2 derivatives. Strains were constructed via P22 HT12/4 int-3 transductions (6) as described previously (16). TnlO insertions (13), Tn5(pfm) insertions (15), and Mud-P22 strains (1) have been described previously.Construction of pulsed-field gel electrophoresis strains. A set of random Tn5(pfm) insertions were generated and mapped as described previously (14

show abstract

Dissection of the Salmonella typhimurium genome by use of a Tn5 derivative carrying rare restriction sites

Cited by 30 publications

References 25 publications

Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.

Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

High-resolution restriction map for a 240-kilobase region spanning 91 to 96 minutes on the Salmonella typhimurium LT2 chromosome

Contact Info

Product

Resources

About