Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.     

Physical analysis and mapping of the Mycoplasma pneumoniae chromosome.

Field inversion gel electrophoresis was used for analysis of the chromosome of Mycoplasma pneumoniae. The restriction endonuclease SfiI (5'-GGCCNNNNNGGCC-3') generated 2 M. pneumoniae DNA fragments of approximately 437 and 357.5 kilobase pairs (kbp), whereas 13 restriction fragments ranging in size from 2.4 to 252.0 kbp resulted from digestion with ApaI (5'-GGGCCC-3'). Totaling the sizes of the individual restriction fragments from digestion with SfiI or ApaI yielded a genome size of 794.5 or 775.4 kbp, respectively. A physical map of the M. pneumoniae chromosome was constructed by using a combination of techniques that included analysis by sequential or partial restriction endonuclease digestions and use as hybridization probes of cloned M. pneumoniae DNA containing ApaI sites and hence overlapping adjacent ApaI fragments. Genetic loci for deoC, rrn, hmw3, and the P1 gene were identified by using cloned DNA to probe ApaI restriction fragment profiles.     

Construction of an EcoRI restriction map of Mycoplasma pneumoniae and localization of selected genes.

A restriction map of the genome of Mycoplasma pneumoniae, a small human pathogenic bacterium, was constructed by means of an ordered cosmid library which spans the complete bacterial chromosome. The positions of 143 endonuclease EcoRI restriction fragments were determined and aligned with the physical map. In addition, restriction sites for the rare-cutting enzymes XhoI (25 sites), ApaI (13 sites), NotI (2 sites), and SfiI (2 sites) were included. The resulting map consists of 185 restriction sites, has a mean resolution of 4.4 kbp, and predicts a genome size of 809 kbp. In addition, several genes were identified and mapped to their respective genomic EcoRI restriction fragments.     

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.     

ECD--a totally integrated database of Escherichia coli K12.

We have compiled the DNA sequence data for E. coli available from the GENBANK and EMBL data libraries and independently from the literature. Starting with this update of our Escherichia coli database (ECD release 20) we provide major changes compared to previous issues. This update not only represents another substantial increase in sequence information, it also allows now to find the exact physical location of each individual gene or regulatory region, even regarding discrepancies in nomenclature. In order to save space this printed version does not contain the database itself anymore, but we provide several examples. The complete database is publically available in electronic form together with a self explaining application program or as a flat file. The complete compilation including a full set of genetic map data and the E. coli protein index can be obtained in machine readable form from the EMBL data library as a part of the CD-ROM issue of the EMBL sequence database, released and updated every three months. After deletion of all detected overlaps a total of 2,878,364 individual bp is found to be determined till the end of June 1994. This corresponds to a total of 60.98% of the entire E. coli chromosome consisting of about 4,720 kbp. This number may actually be higher by 9161 bp derived from other strains of E. coli.     

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.     

Physical map of the linear chromosome of the bacterium Borrelia burgdorferi 212, a causative agent of Lyme disease, and localization of rRNA genes.

The spirochete Borrelia burgdorferi, which causes Lyme disease, and other members of the Borrelia genus are unique among characterized bacteria in having a linear chromosome. A restriction map of the chromosome of B. burgdorferi 212 was constructed by making extensive use of digestions in agarose blocks of restriction endonuclease fragments or chromosomal DNA that had been purified by pulsed-field gel electrophoresis. A total of 47 digestion sites for the enzymes SgrAI, SacII, MluI, BssHII, EagI, SmaI, NaeI, and ApaI were located. In most regions of the map, the gap between sites is 50 kbp or less, and 122 kbp is the largest distance between adjacent sites. The mapping data were consistent with previous conclusions that the B. burgdorferi chromosome is linear. The total size of the B. burgdorferi 212 chromosome was determined to be 946 kbp from the sums of the sizes of SacII, MluI, BssHII, and SmaI fragments, making it one of the smallest known bacterial chromosomes. The rRNA genes were found to be located near the center of the chromosome. One copy of the 16S rRNA gene (rrs) and two copies of the 23S rRNA gene (designated rrlA and rrlB), the latter pair in a tandem repeat, were detected. This particular complement of these two genes has not been reported for another bacterium.     

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.     

Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci.

Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD alpha and GAD beta, and these differed by only five amino acids. Both GAD alpha and GAD beta contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gadA and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.     

The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli

The DNA sequence of the Escherichia coli metK gene has been determined. Protein sequence data for purified S-adenosylmethionine synthetase have also been obtained and confirm that metK is the structural gene for S-adenosylmethionine synthetase in E. coli. The sequence of the amino-terminal 35 residues of purified S-adenosylmethionine synthetase localizes the beginning of the coding region of the DNA. The open reading frame extends 1152 bases and codes for a 384-residue protein of Mr = 41,941. The gene is transcribed clockwise on the E. coli chromosome. The DNA region 5' to the coding region was found to contain symmetrical sequences suggestive of operator structures and homologous to sequences upstream from other met genes sharing the same regulatory mechanism.     

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.     

Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions

We report yeast/Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZ gene of E. coli. The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII. The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene. All the restriction sites have been phased to three different reading frames. Two series of vectors have been constructed. The first series (YEp) has two origins of replication (ori), i.e., of the yeast 2 mu circle and of the ColE1 plasmid of E. coli, and can therefore replicate autonomously in both organisms. These shuttle vectors also have the ApR gene of E. coli and either the yeast LEU2 or URA3 genes to allow for selection of both E. coli and yeast transformants. The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2 mu ori. The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA. None of the vectors express beta-galactosidase (beta Gal) in yeast or E. coli in the absence of inserted yeast promoter sequences. The 5'-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors. In-frame gene fusions can be detected by beta Gal activity when either yeast or E. coli clones are plated on media containing XGal indicator. Quantitative determinations of promoter activity were made by colorimetric assay of beta Gal activity in whole cells. Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.     

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.     

Amplification and characterization of a beta-globin gene synthesized in vitro.

Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.     

Compilation of DNA sequences of Escherichia coli K12 (ECD and ECDC; update 1995).

We have compiled the DNA sequence data for Escherichia coli available from the GenBank and EMBL data libraries and independently from the literature. Unlike the previous updates of our E.coli databases, we provide the most recent version preferentially via the World Wide Web System (use URL: http://susi.bio.unigiessen.de/usr/local/www++ +/html/ecdc.html). Our database includes an assembled set of contiguous sequences. Each of these contigs compiles all available sequence information, including those derived from a variety of elder sequences. The organization of the database allows one to find the exact physical location of each individual gene or regulatory region, even regarding discrepancies in nomenclature. The WWW program allows access into the original EMBL and SWISSPROT datafiles. A FASTA and BLAST search may be performed online. Besides the WWW format a flat file version may be obtained via ftp. The complete compilation, including a full set of genetic map data and the E.coli protein index, can be obtained in machine readable form from the EMBL data library as a part of the CD-ROM issue of the EMBL sequence database, released and updated every three months. After deletion of all detected overlaps a total of 3 333 878 individual bp was determined by the end of September 1995. This corresponds to a total of 71.71% of the entire E.coli chromosome consisting of about 4720 kbp. About 94 kbp (2%) are available additionally, but have not yet been definitely mapped.