A physical map of the genome of Ureaplasma urealyticum 960T with ribosomal RNA loci.

A physical map is presented for the 900 kilobase pair genome of Ureaplasma urealyticum 960T, locating 29 sites for 6 restriction endonucleases. The large restriction fragments were separated and sized by pulsed-field agarose gel electrophoresis (PFGE). Their locations on the map were determined by probing Southern blots of digests with individual fragments isolated from other digests and by correlating the products of double digestions and partial digestions. An end-labelling technique was used to detect small fragments not readily observed by PFGE. Two loci for rRNA genes have been determined by probing with cloned DNA.     

Preparation and FIGE separation of infrequent restriction fragments from Mycoplasma mycoides DNA

Use of procedures for obtaining satisfactory preparation and digestion of intact DNA of Mycoplasma mycoides subsp. mycoides Y in agarose blocks is reported. The use of inverted field agarose gel electrophoresis (FIGE) for separation of the small number of fragments derived from the genome by several restriction endonuclease digestions is shown. An effect that fragments containing replication forks remain in the well during FIGE, distorting the representative yield of restriction fragments on the gels, is overcome by incubating cells with chloramphenicol for 1 1/2 h before harvest to allow rounds of replication to go to completion without new initiations of DNA synthesis.     

Genomic maps of some strains within the Mycoplasma mycoides cluster

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.     

Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of M_r = 126 × 10⁶. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis.The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 10⁶ molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.     

Physical mapping of the Mycoplasma capricolum genome

A physical map of Mycoplasma capricolum ATCC 27343 genome was constructed, based on estimation of the restriction fragment sizes by pulse-field electrophoresis. The linkage order of restriction fragments was determined by two-dimensional electrophoresis of partial and complete single digests and complete double digests and by Southern hybridization analysis. The genome size was established at 1155.5 kb, and 26 cleavage sites for 7 endonucleases were assigned to the map.     

Isolation of a second cryptic plasmid from Mycoplasma mycoides subsp. mycoides

Plasmids have rarely been detected in organisms constituting the genus Mycoplasma. Recently, the isolation of a cryptic plasmid from Mycoplasma mycoides subsp. mycoides has been described, and we report here the isolation of a second cryptic plasmid from this species. Restriction map and Southern blot analyses show that the second plasmid is distinct from the previously described plasmid, although a limited region of homology was detected. The availability of mycoplasmal cryptic plasmids may lead to the development of cloning vectors that replicate in these organisms.     

Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome

The restriction enzyme DraI cleaves the Caulobacter crescentus genome into at least 35 fragments which have been resolved in agarose gels using pulsed-field-gradient gel electrophoresis (PFGE). When digests were performed using DNA from strains containing Tn5 insertion mutations, altered band migrations were observed. Using PFGE with the appropriate pulse times, size differences as small as 2% could be resolved in large fragments. Using this approach, we have constructed a partial physical map of the genome which correlates well with the C. crescentus genetic map and have shown the size of the genome to be approx. 3800 kb. Using hybridization with cloned genes, we have determined the map locations of five previously unmapped genes. In addition, we have shown that PFGE can be used to rapidly determine the map locations of new insertion mutations or the sizes of deletion mutations.     

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.

A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Enzymes of Purine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides were proposed previously from studies of its usage of radioactive purines and pyrimidines. To interpret more fully the pattern of purine usage, we have assayed cell-free extracts of this organism for several enzymes associated with the salvage synthesis of purine nucleotides. M. mycoides possessed phosphoribosyltransferases for adenine, guanine, and hypoxanthine, purine nucleoside phosphorylase, GMP reductase, GMP kinase, adenylosuccinate synthetase, and adenylosuccinate lyase. Purine nucleoside kinase and adenosine deaminase were not detected. Examination of kinetic properties and regulation of some of the above enzymes revealed differences between M. mycoides and Escherichia coli. Most notable of these were the greater susceptibility of the enzymes from M. mycoides to inhibition by nucleotides and the more widespread involvement of GMP as an inhibitor. Observations on enzyme activities in vitro allow an adequate explanation of the capacity of guanine to provide M. mycoides with its full requirement for purine nucleotides.     

Studies of lactate dehydrogenase of Mycoplasma mycoides var. mycoides.

Polyacrylamide gel electrophoresis and isoelectric focusing techniques have been used to compare NAD-dependent L(plus) lactate dehydrogenases (LDH) from ten different strains of Mycoplasma mycoides var. mycoides. The enzymes were not distinguished from one another, or from normal bovine LDH 1 by these methods. The kinetic behaviour of LDH form M. mycoides (T1 vaccine strain) suggested that the enzyme could readily reduce pyruvate or oxidize lactate in a manner which, in vertebrates, requires two different isoenzymes.     

The impact of two-dimensional pulsed-field gel electrophoresis techniques for the consistent and complete mapping of bacterial genomes: refined physical map of Pseudomonas aeruginosa PAO.

The SpeI/DpnI map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was refined by two-dimensional (2D) pulsed-field gel electrophoresis techniques (PFGE) which allow the complete and consistent physical mapping of any bacterial genome of interest. Single restriction digests were repetitively separated by PFGE employing different pulse times and ramps in order to detect all bands with optimum resolution. Fragment order was evaluated from the pattern of 2D PFGE gels: 1. Partial-complete digestion. A partial restriction digest was separated in the first dimension, redigested to completion, and subsequently perpendicularly resolved in the second dimension. 2D-gel comparisons of the ethidium bromide stain of all fragments and of the autoradiogram of end-labeled partial digestion fragments was nearly sufficient for the construction of the macrorestriction map. 2. Reciprocal gels. A complete restriction digest with enzyme A was run in the first dimension, redigested with enzyme B, and separated in the second orthogonal direction. The order of restriction digests was reverse on the second gel. In case of two rare-cutters, fragments were visualized by ethidium bromide staining or hybridization with genomic DNA. If a frequent and a rare cutter were employed, linking fragments were identified by end-labeling of the first digest. 3. A few small fragments were isolated by preparative PFGE and used as a probe for Southern analysis.--38 SpeI and 15 DpnI fragments were positioned on the map. The zero point was relocated to the 'origin of replication'. The anonymous mapping techniques described herein are unbiased by repetitive DNA, unclonable genomic regions, unfavourable location of restriction sites, or cloning artifacts as frequently encountered in other top-down or bottom-up approaches.     

Chromosome map of the thermophilic archaebacterium Thermococcus celer.

A physical map for the chromosome of the thermophilic archaebacterium Thermococcus celer Vu13 has been constructed. Thirty-four restriction endonucleases were tested for their ability to generate large restriction fragments from the chromosome of T. celer. Of these, the enzymes NheI, SpeI, and XbaI yielded the fewest fragments when analyzed by pulsed-field electrophoresis. NheI and SpeI each gave 5 fragments, while XbaI gave 12. The size of the T. celer chromosome was determined from the sum of the apparent sizes of restriction fragments derived from single and double digests by using these enzymes and was found to be 1,890 +/- 27 kilobase pairs. Partial and complete digests allowed the order of all but three small (less than 15 kilobase pairs) fragments to be deduced. These three fragments were assigned positions by using hybridization probes derived from these restriction fragments. The positions of the other fragments were confirmed by using hybridization probes derived in the same manner. The positions of the 5S, 16S, and 23S rRNA genes as well as the 7S RNA gene were located on this map by using cloned portions of these genes as hybridization probes. The 5S rRNA gene was localized 48 to 196 kilobases from the 5' end of the 16S gene. The 7S RNA gene was localized 190 to 504 kilobases from the 3' end of the 23S gene. These analyses demonstrated that the chromosome of T. celer is a single, circular DNA molecule. This is the first such demonstration of the structure of an archaebacterial chromosome.     

Isolation of Mycoplasma mycoides, mycoides (LC variant), from two naturally aborted caprine fetuses

Described in this study are 2 cases of caprine abortion associated with the LC type of Mycoplasma mycoides , subsp. mycoides . This mycoplasma and Mycoplasma mycoides , subsp. capri had been previously reported in adult goats in this herd. The abortion took place in the latter part of gestation. Samples from heart blood, lung and pleural exudate were collected for the isolation of mycoplasmas and other bacterials in both fetuses. Two strains of Mycoplasma mycoides , subsp. mycoides (LC variant) were isolated. The only gross lesion of the internal organs in the aborted fetuses was congestion of the lungs. Microscopic lesions were encountered in the lungs, and these were characterized by patchy to diffuse pneumonia. Exfoliated cells, many alveolar macrophages, scattered neutrophils and lymphocytes were seen in the lumen of the terminal airways and alveolar spaces. This report appears to be the first isolation of Mycoplasma mycoides , subsp. mycoides (LC variant) from aborted caprine fetuses.     

Physical maps of the genomes of three Bacillus cereus strains.

NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B. cereus type strain ATCC 14579 have been established and compared with the previously established map of B. cereus ATCC 10987. Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains. The sizes of the genomes varied between 5.4 and 6.3 Mb. The maps were constructed by hybridization of 42 random probes, prepared from B. cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis. Nine probes were specific for ATCC 10987 only. Probes for five B. subtilis and five B. cereus genes were also used. The NotI restriction fragment patterns of the four strains were strikingly different.     

Location of sites of transposon Tn916 insertion in the Mycoplasma mycoides genome.

The genome of Mycoplasma mycoides subsp. mycoides GC1176-2 was cleaved into six large fragments by the endonuclease KpnI which also cleaved the transposon Tn916 once. This has allowed genomic mapping of insertion sites for 50 transformants of GC1176-2 containing Tn916. Almost all of the mapped sites were clearly separate. The transformants provide a bank of genomes each with a KpnI site at a different position to facilitate mapping of gene loci.     

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.     

Relationships between strains of Mycoplasma mycoides subspp. mycoides and capri studied by two-dimensional gel electrophoresis of cell proteins.

Strains of Mycoplasma mycoides subsp. mycoides have been divided into small colony (SC) and large colony (LC) types (Cottew & Yeats, 1978). The protein patterns of representative strains of these two types and of M. mycoides subsp. capri were compared by a high resolution, two-dimensional gel electrophoretic method. The results suggest that the LC strains are more closely related to M. mycoides subsp. capri than to the SC strains of M. mycoides subsp. mycoides.     

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.