Human ureaplasmas show diverse genome sizes by pulsed-field electrophoresis.

Contour clamped homogeneous field (CHEF) agarose gel electrophoresis (AGE), ramped to give linear separation of DNA molecules of 600-1600 kilobase pairs (kbp), was used to determine mobilities for full-sized genomic DNA of the serotype standard strains of the human genital mollicutes, Ureaplasma urealyticum relative to yeast chromosomal DNA markers. Indicated genome sizes (in kbp) were 760 for the four biotype 1 strains and 840-1140 for eleven biotype 2 strains. Other estimates were: 720 for Mycoplasma hominis, 1070 for Mycoplasma hyopneumoniae, 890 for Mycoplasma flocculare, 1180 and 1350 for Mycoplasma mycoides subsp. mycoides Y and GC1176-2, respectively, and 1650 and 1580 for Acholeplasma laidlawii B and PG 8, respectively. These data supplement previous evidence from CHEF AGE that the genomes of the Mycoplasmataceae are diverse in size with some larger than previously estimated from DNA renaturation kinetics.     

Reiterated DNA sequences defining genomic diversity within the species Mycoplasma hyorhinis

Genomic restriction fragments isolated from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae were shown by DNA hybridization and nucleotide sequence analyses to contain sequences common to these two species, as well as another porcine-derived mycoplasma, Mycoplasma flocculare. Intraspecies hybridization experiments using these fragments as probes indicated that the sequence is highly redundant in several strains of M. hyorhinis, but that there is diversity in the sizes of restriction fragments detected among these strains. In contrast, repetition of the sequence was limited in M. hyopneumoniae and M. flocculare, and no homologies to this repeated element were apparent in mycoplasma species isolated from animal hosts other than the swine. The reiterated sequence may reflect intraspecies genomic diversification in M. hyorhinis and its selective presence in otherwise unrelated species raises the possibility that it has been horizontally transmitted between these organisms.     

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 the physical maps of Mycoplasma hyopneumoniae and Mycoplasma flocculare and the location of rRNA genes on these maps.

The genomes of Mycoplasma flocculare and Mycoplasma hyopneumoniae, two mycoplasmas of the porcine respiratory system, were studied. Based upon antigenic cross-reactivity and DNA-DNA hybridization, these species have given indication of a close genetic relationship. By using field-inversion gel electrophoresis and employing the restriction digest fragments obtained from the gels as the probes, physical maps of the genomes of the two species were constructed. Mycoplasma hyopneumoniae is similar to M. flocculare in having a single set of rRNA genes and the 5S-rRNA gene is separated from the 16S and 23S rRNA genes. Based upon the location of the rRNA genes on the physical maps in both species, the distance between the 5S and the 16S and 23S rRNA genes is at least 150 kbp. Thus, there is further evidence for the close relationship between these organisms.     

Pulse-field electrophoresis indicates full-length Mycoplasma chromosomes range widely in size.

Full-size linear chromosomes were prepared from mycoplasmas by using gamma-irradiation to introduce one (on average) double-strand break in their circular chromosomes. Chromosome sizes were estimated by pulsed-field gel electrophoresis (PFGE) from the mobilities of these full-length molecules relative to DNA size references. Sizes estimated for Ureaplasma urealyticum T960 and 16 Mycoplasma species ranged from 684 kbp (M. hominis) to 1315 kbp (M. iowae). Using this sample, we found no correlation between the mobility of the full-size linear chromosomes and their G + C content. Sizes for A. laidlawii and A. hippikon were within the range expected from renaturation kinetics. PFGE size estimates are in good agreement with sizes determined by other methods, including electron microscopy, an ordered clone library, and summation of restriction fragments. Our estimates also agree with those from renaturation kinetics for both the largest and some of the smallest chromosomes, but in the intermediate size range, renaturation kinetics consistently provides lower values than PFGE or electron microscopy. Our PFGE estimates show that mycoplasma chromosomes span a continual range of sizes, with several intermediate values falling between the previously recognized large and small chromosome size clusters.     

Plasmodium falciparum: mapping genes to nine parasite chromosomes

Using pulsed field gel electrophoresis with pulse time of 120 sec, eight chromosomal DNA molecules from clone 7G8 of the Plasmodium falciparum Brazilian isolate IMTM22 were resolved. A ninth chromosomal molecule which did not enter the gel was identified at the slot by hybridization to two DNA probes and by restriction enzyme analysis. Thirteen parasite DNA sequences were mapped to the nine chromosomes, with at least one sequence mapped to each chromosome. The restriction enzyme NotI appeared to produce only one cut in the entire IMTM22 genome.     

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 Myxococcus xanthus chromosome.

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.     

A physical and genetic map of the Mycoplasma hyopneumoniae strain J genome

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.     

Size and physical map of the Campylobacter jejuni chromosome.

The chromosome of Campylobacter jejuni is circular and approximately 1700 kb in circumference. The size of the genome was determined by field inversion gel electrophoresis of restriction endonuclease fragments using lambda DNA concatamers and yeast chromosomes to calibrate the size of the fragments. In view of the low (32-35%) G + C content of the campylobacter genome, enzymes that recognizes GC-rich sequences were used. Of the enzymes tested BssHII (G/C(G)CGC), NciI (CC/CGCG) and SalI (G/TCGAC) appeared to be usable. Hybridization of labeled fragments with two or more fragments from digests with a different restriction enzyme gave the information to order the fragments on the C jejuni chromosome. The localization on the genome of the flagellin and ribosomal gene clusters was determined.     

Preparation of high molecular weight plant DNA and its use for artificial chromosome construction

Summary The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analyzed. Conditions have been established for the obtainment of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension. Restriction fragments were obtained by partial and total digestion with different endonucleases, and separated by pulsed-field gel electrophoresis. Ligation of partially EcoRI-digested DNA (range 30–300 kbp) followed by transformation of yeast spheroplasts gave rise to YACs with an average size of 60 kbp. The introduction of a DNA size-selection step before ligation led to production of YACs in the range of 100–200 kbp. Clones of up to 460 kbp were obtained by blunt-end ligation of pre-selected unrestricted DNA.Abbreviations 2,4-D 2,4-dichloro phenoxyacetic acid - 6BAP 6-benzylaninopurine - BFP bovine serum albumin 0.1%, Ficoll 400 0.1%; polyvinylpyrrolidone 0.1% - CHEF clumped homogeneous electric field - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - HMW high molecular weight - km kanamycin - LMP agarose low melting point agarose - MS Murashige and Skoog mediun - npt neomycin phosphotransferase - PEG polyethyleneglycol - PFGE pulsed-field gel electrophoresis - RFLP restriction fragrant lenght polymorphism - SDS sodium dodecyl sulphate - SSC sodium chloride 150 mM, sodiun citrate 15 nM, pH 7 - TAE TRIS-Acetate pH 8 40 mM, EDTA 2 mM - TE TRIS-HCl pH 8 10 mM, EDTA 1 mM - YAC yeast artificial chromosome     

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.     

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.     

Physical map of Mycoplasma gallisepticum.

Physical chromosomal maps of two Mycoplasma gallisepticum strains, R and ATCC 19610, were constructed by using field inversion gel electrophoresis. To assist in the ordering of chromosomal fragments and the construction of the chromosomal maps, the gram-positive transposon Tn4001 was modified to serve as a mobile restriction site. The total sizes of the M. gallisepticum R and ATCC 19610 genomes were estimated to be 1,037 and 998 kb, respectively. The restriction enzyme locations for EagI and SmaI were determined along with several transposon insertion sites. The two strain maps were similar except for three small deletions and one additional EagI site in strain ATCC 19610.     

Distinct mouse DNA sequences enable establishment and persistence of plasmid DNA polymers in mouse cells.

Distinct elements isolated from mouse genomic DNA confer on plasmid DNA the ability to persist at high copy numbers in mouse L fibroblasts (1). Field inversion gel electrophoresis demonstrated that - in contrast to our previous assumption - the persisting plasmid DNA does not exist extrachromosomally but as clusters of tandem repeats integrated into genomic DNA. Digestion with restriction endonucleases that do not cut within the plasmid DNA results in fragments of 50-300 kb in length indicating reiteration of 10-50 plasmid DNA molecules. Restriction with several enzymes that cut once or twice within the plasmid sequences lead to fragment(s) indicative for head-to-tail tandem repeats. In situ hybridization revealed signals for a long homogeneously staining region (HSR) in one or two chromosomes per cell nucleus. Possibilities how these elements could act in the establishment and/or maintenance of the head-to-tail polymers of plasmid DNA in mouse cells are discussed.     

Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy.

The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments.     

Comparison of genomes in Streptococcus thermophilus strains of different origins

According to DNA hybridization data, thermophilic streptococci used in Russia as starters in the dairy industry are divided into 6 different genomovars, with a degree of DNA homology not exceeding 20-50%. The analysis of genomes from these genomovars using SmaI restriction endonuclease and pulsed-field gel electrophoresis revealed a wide variability of the genome size. In some strains, the genome size considerably exceeded 2000 kbp. Most of the strains studied contained plasmids about 120 kbp in size.     

Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.     

Isolation of a repeated DNA sequence from Bordetella pertussis

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.     

Identification of a plant-derived mollicute as a strain of an avian pathogen, Mycoplasma iowae, and its implications for mollicute taxonomy.

Strain PPAV, a filamentous but nonhelical mollicute, was isolated from aborted apple seeds in France in late 1979. This organism grew well in SP-4 broth, fermented glucose, and required sterol for growth, and most of its properties suggested that it belonged to the genus Mycoplasma. However, it was serologically distinct; in addition, unlike other Mycoplasma species, genome measurements consistently yielded values of about 1,000 MDa (ca. 1,500 kbp), and the organism had a growth temperature optimum of 43 degrees C. A comparison of strain PPAV 16S rRNA sequences with those of other mollicutes revealed a high degree of sequence similarity to a strain of Mycoplasma iowae, which is commonly encountered in poultry. This relationship was confirmed by performing a restriction endonuclease pattern analysis and DNA-DNA hybridization tests. The genome size of type strain 695 of M. iowae was determined to be about 1,000 MDa (1,500 kbp) by renaturation kinetics, a value which is much higher than any other value known in the genus. Additional measurements by pulsed-field gel electrophoresis yielded values of 1,300 kbp for both strain PPAV and M. iowae. Subsequent phenotypic comparisons supported this relationship. Serologic tests with strain PPAV and other strains of M. iowae confirmed the findings of other investigators that this species is serologically heterogeneous. The high optimum temperature for growth of strain PPAV was also shared by a number of M. iowae isolates. Genome size is an inappropriate character for taxonomic assignment to the family Mycoplasmataceae because strain PPAV and other established species in this family are now known to have genomes ranging in size from 1,000 to 1,400 kbp.