Physical map of the seven ribosomal RNA genes of Escherichia coli.

Escherichia coli DNA was digested with restriction endonucleases BamHI, PstI, EcoRI, SalI, HindIII, XhoI, BglII, SmaI, HpaI and with selected double and triple combinations of the same enzymes. The digests were electrophoresed and hybridized with 32P-labelled ribosomal RNA by using the Southern blotting technique. The resulting bands could be arranged into seven groups, and it was possible to construct a unique physical map of the seven rRNA genes (operons) of the bacterial chromosome. Mapping information obtained on several transducing phages and recombinant plasmids carrying rRNA genes, and mapping data published in the literature helped to determine the final map. The results suggest that phage lambda daroE152 carries a "hybrid" rRNA gene which was probably formed by recombination between two different chromosomal rRNA genes.     

Study on rRNA genes in Mycobacterium smegmatis

The number of rRNA genes in Mycobacterium smegmatis was examined by hybridization of BamHI and SalI digests of chromosomal DNA with 3'-end-labeled 5S, 16S, 23S rRNA and tRNA. Each RNA probe gave two hybridization bands. The PstI fragments of 6.6 kilobases were cloned to pBR322. The cloned DNA was characterized by restriction endonuclease mapping, DNA-RNA hybridization, and the R-loop technique.     

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.     

Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21.

The number of rRNA genes in Streptomyces lividans was examined by Southern hybridization. Randomly labeled 23 and 16S rRNAs were hybridized with BamHI, BglII, PstI, SalI, or XhoI digests of S. lividans TK21 DNA. BamHi, BglII, SalI and XhoI digests yielded six radioactive bands each for the 23 and 16S rRNAs, whereas PstI digests gave one band for the 23S rRNA and one high-intensity band and six low-density bands for the 16S rRNA. The 7.4-kilobase-pair BamHI fragment containing one of the rRNA gene clusters was cloned into plasmid pBR322. The hybrid plasmid, pSLTK1, was characterized by physical mapping, Southern hybridization, and electron microscopic analysis of the R loops formed between pSLTK1 and the 23 and 16S rRNAs. There were at least six rRNA genes in S. lividans TK21. The 16 and 23S rRNA genes were estimated to be about 1.40 and 3.17 kilobase pairs, respectively. The genes for the rRNAs were aligned in the sequence 16S-23S-5S. tRNA genes were not found in the spacer region or in the context of the rRNA genes. The G + C content of the spacer region was calculated to be approximately 58%, in contrast to 73% for the chromosome as a whole.     

Chromosomal Organization of Rrna Operons in Bacillus Subtilis

Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion.     

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.     

A specialized host-vector system for the in Vivo cloning of the trp operon of wild-type and mutant strains of Salmonella typhimurium by generalized transduction

Using in vitro methods, a 14.2-kb EcoRI fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant delta trpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a SalI fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-SalI upstream flanking sequence and the 3.2-kb SalI-EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecA-dependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the differentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cellular viability and of plasmid stability.     

Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.     

Mechanism of pBR322 transduction mediated by cytosine-substituting T4 bacteriophage

Summary A cytosine-substitution type mutant of bacteriophage T4 (T4dC phage) has been shown to mediate the transfer of plasmid pBR322. The transduction frequency was around 10^-2 per singly infected cell at low multiplicity of infection. The transductants contained either a monomer or multimers of pBR322. The transducing capacity of T4dC phage was resistant to methylmethanesulfonate treatment. The results of Southern blotting experiments have indicated that the pBR322 DNA exists as head-to-tail concatemers in the transducing particles. The mechanism of transfer of pBR322 mediated by T4dC phages is discussed     

Physical map of Campylobacter jejuni TGH9011 and localization of 10 genetic markers by use of pulsed-field gel electrophoresis.

The physical map of Campylobacter jejuni TGH9011 (ATCC 43430) was constructed by mapping the three restriction enzyme sites SacII (CCGCGG), SalI (GTCGAC), and SmaI (CCCGGG) on the genome of C. jejuni by using pulsed-field gel electrophoresis and Southern hybridization. A total of 25 restriction enzyme sites were mapped onto the C. jejuni chromosome. The size of the genome was reevaluated and was shown to be 1,812.5 kb. Ten C. jejuni genetic markers that have been isolated in our laboratory were mapped to specific restriction enzyme fragments. Furthermore, we have accurately mapped one of the three rRNA operons (rrnA) and have demonstrated a separation of the 16S and 23S rRNA-encoding sequences in one of the rRNA operons.     

Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli.

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

Analysis of operons encoding 23S rRNA of Clostridium botulinum type A.

Southern hybridization analysis of Clostridium botulinum type A chromosomal DNA indicated the presence of six copies of the 23S rRNA gene. Fragments of DNA encoding 23S rRNA were amplified by polymerase chain reaction and cloned in Escherichia coli. Three clones examined by restriction enzyme and sequence analysis were found to be derived from different operons. Sequence determination of the entire insert of two clones revealed nine nucleotide changes in the genes coding for 23S rRNA (99.7% sequence identity) between operons encoded on the same chromosome, showing microheterogeneity in the rRNA operons of this organism.     

Stability of the hybrid plasmid pIM138 and its curing by some eliminating agents

Hybrid plasmid pIM138 was constructed by insertion of a chromosomal fragment with the threonme operon fromEscherichia coli into the pBR322 vector. Molar mass of pIM138 was 2.8 Mg/mol. Heteroduplexes between pBR322 vector and pIM138 hybrid DNA molecules were prepared. The hybrid plasmid shows a high stability against the curing effect of rifampicin and clorobiocm inE. coli SK1590thr host.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.