A cross-hybridization method for DNA mapping with photobiotin-labeled probes

An efficient procedure combining the Southern Cross method described by H. Potter and D. Dressler (1986, Gene 48, 229-239) and the use of nonradioactive reporter molecules which allows fast restriction mapping of DNA molecules up to 40 kb is described. In this method, photobiotin-labeled fragments for one restriction enzyme cross-hybridize with at least five unlabeled digests transferred onto nylon membranes. Hybridization spots are revealed on recipient membranes at a homologous fragment cross, allowing direct restriction map construction. In comparison with 32P "cross-blot" hybridization, photobiotin labeling is safer, faster, and easier to dispose of.     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization.

Four restriction endonucleases, AseI (5'-ATTAAT), SpeI (5'-ACTAGT), DraI (5'-TTTAAA), and SnaBI (5'-TACGTA), generated DNA fragments of suitable size distributions for mapping the genome of Rhodobacter sphaeroides by transverse alternating field electrophoresis. AseI produced 17 fragments, ranging in size from 3 to 1,105 kilobases (kb), SpeI yielded 16 fragments (12 to 1,645 kb), DraI yielded at least 25 fragments (6 to 800 kb), and SnaBI generated 10 fragments (12 to 1,225 kb). A total genome size of approximately 4,400 +/- 112 kb was determined by summing the fragment lengths in each of the digests generated by using the different restriction endonucleases. The total genomic DNA consisted of chromosomal DNA (3,960 +/- 112 kb) and the five endogenous plasmids (approximately 450 kb total) whose cognate DNA fragments have been unambiguously identified. A number of genes have been physically mapped to the AseI-generated restriction endonuclease fragments of total genomic DNA by Southern hybridization analysis with either homologous or heterologous specific gene probes or, in the case of several auxotrophic and pigment-biosynthetic mutants apparently generated by Tn5, a Tn5-specific probe. Other genes have been mapped by a comparison with wild-type patterns of the electrophoretic banding patterns of the AseI-digested genomic DNA derived from mutants generated by the insertion of either kanamycin or spectinomycin-streptomycin resistance cartridges. The relative orientations, distance, and location of the pufBALMX, puhA, cycA, and pucBA operons have also been determined, as have been the relative orientations between prkB and hemT and between prkA and the fbc operon.     

Monitoring mammalian genome rearrangement with mid-repetitive sequences as probes

By utilization of mid-repetitive sequences, the intracisternal A particle (IAP) gene, as a probe, genome rearrangement involving IAP genes and their neighboring sequences in rodent cells can be monitored. This is based on electrophoretic separation of the twice digested restriction fragments of genomic DNA in a 2-D pattern. The first digestion was done in solution followed by electrophoresis of the restriction fragments in the first dimension. A second restriction enzyme digestion was carried out in situ in the gel followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. After Southern blotting, the DNA on the filter is hybridized with a probe that is a fragment located near the 5' end of the IAP gene, but does not overlap with the 5' long terminal repeat (LTR). The exposed X-ray film revealed about 370 distinct spots in the 2-D maps. In comparing the 2-D maps, genome rearrangement involving IAP was detected.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.     

Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(exo-) DNA polymerase and RFLP analysis

In this report we describe a simple and rapid protocol for reliable quantitation of mitochondrial DNA (mtDNA) mutations, which is basically a modification of the traditional polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP method has been of limited use for the accurate determination of ratios of mutant and wild type molecules, largely owing to the formation of heteroduplex molecules by PCR and incompleteness of restriction digestion. In order to overcome this problem, we have introduced a single-step primer extension reaction using Vent(R)(exo-) DNA polymerase and a fluorescence-labeled primer to the standard assay. The labeled homoduplex molecules are then digested with a restriction endonuclease, and the nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN analysis software. The amount of mutant mtDNA is readily estimated from fluorescence intensities of the wild-type and mutant mtDNA fragments corrected for incomplete digestion as monitored by a homologous control fragment. The accuracy of the improved protocol was determined by constructing standard curves obtained from defined mixtures of genomic DNA containing homoplasmic wild-type and mutant mtDNA. The expected values were obtained, with an observed correlation coefficient of 0.997 and a typical variability of +/-5% between repeated measurements. Further validation of the protocol is provided by the screening of five patients and unaffected subjects carrying the guanine to adenine transition at the nucleotide 3460 of the mitochondrial genome responsible for the mitochondrial disorder of Leber's hereditary optic neuropathy.     

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.     

Physical and genetic map of Streptococcus thermophilus A054.

The three restriction endonucleases SfiI, BssHII, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054. A total of 5, 8, and 24 fragments were produced with SfiI, BssHII, and SmaI, respectively. An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes. Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfiI, BssHII, and SmaI fragments. All restriction fragments were arranged in a unique circular chromosome. Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map. Among them, six rrn loci were precisely located. The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites. Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome. One was associated with the tandem rrnD and rrnE loci, and the other was mapped in the region of the lactose operon.     

Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome.

We describe a compilation of 79 known genes of Bradyrhizobium japonicum 110, 63 of which were placed on a correlated physical and genetic map of the chromosome. Genomic DNA was restricted with enzymes PacI, PmeI, and SwaI, which yielded two, five, and nine fragments, respectively. Linkage of some of the fragments was established by performing Southern blot hybridization experiments. For probes we used isolated, labelled fragments that were produced either by PmeI or by SwaI. Genes were mapped on individual restriction fragments by performing gene-directed mutagenesis. The principle of this method was to introduce recognition sites for all three restriction enzymes mentioned above into or very near the desired gene loci. Pulsed-field gel electrophoresis of restricted mutant DNA then resulted in an altered fragment pattern compared with wild-type DNA. This allowed us to identify overlapping fragments and to determine the exact position of any selected gene locus. The technique was limited only by the accuracy of the fragment size estimates. After linkage of all of the restriction fragments we concluded that the B. japonicum genome consists of a single, circular chromosome that is approximately 8,700 kb long. Genes directly concerned with nodulation and symbiotic nitrogen fixation are clustered in a chromosomal section that is about 380 kb long.     

A related moderately repetitive DNA family in the nematodes Ascaris lumbricoides and Panagrellus silusiae.

Digestion of genomic DNA from the nematodes Panagrellus silusiae and Ascaris lumbricoides with restriction endonuclease BamH1 releases a 0.7 kilobase (kb) fragment. The 0.7 kb fragment from both nematodes was cloned onto E. coli plasmid pUC19. Using representative clones as DNA hybridization probes, it was found that (i) the BamH1 fragments cross-hybridize; (ii) a ladder-effect with multiples of 0.7 kb was evident in both species after hybridization to genomic DNA and (iii) the genomic copy number of BamH1 elements is 150 and 195 for P. silusiae and A. lumbricoides respectively. DNA sequence analysis of the inserts, AL700-1 and PS700-1, revealed nucleotide blocks with over 85% similarity. No open reading frames are present in either DNA fragment. Neither fragment hybridizes to genomic DNA from Caenorhabditis elegans. Northern blot hybridization indicated that the 0.7 kb element is transcribed into poly(A)(-)-RNA in P. silusiae; but, is not transcribed in adult Ascaris muscle. Thus, P. silusiae and A. lumbricoides share a homologous, tandemly arrayed, moderately repetitive DNA family.     

Molecular analyses of a repetitive DNA sequence in wheat (Triticum aestivum L.).

A repetitive sequence designated WE35 was isolated from wheat genomic DNA. This sequence consists of a 320-bp repeat unit and represents approximately 0.002% of the total wheat DNA. It is unidirectionally distributed either continuously or discretely in the genome. Ladder-like banding patterns were observed in Southern blots when the wheat genomic DNA was restricted with endonuclease enzymes EcoRI, HincII, NciI, and NdeI, which is characteristic for tandemly organized sequences. Two DNA fragments in p451 were frequently associated with the WE35 repetitive unit in a majority of lambda wheat genomic clones. A 475-bp fragment homologous to the 5'-end long terminal repeat (LTR) of cereal retroelements was also found in some lambda wheat genomic clones containing the repetitive unit. Physical mapping by fluorescence in situ hybridization (FISH) indicated that one pair of wheat chromosomes could be specifically detected with the WE35 positive probe p551. WE35 can be considered a chromosome-specific repetitive sequence. This repetitive unit could be used as a molecular marker for genetic, phylogenetic, and evolutionary studies in the tribe Triticeae.     

Fine Structure Recombinational Analysis of Cloned Genes Using Yeast Transformation

We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, we show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, we determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast.     

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

A combined physical and genetic map of Pseudomonas aeruginosa PAO

A combined physical and genetic map of Pseudomonas aeruginosa PAO was constructed by pulsed-field gel electrophoresis and Southern hybridization using cosmid clones from a genomic library carrying known genes. A total of 37 SpeI restriction fragments have been mapped on the 5862 kb genome, and fragment contiguity demonstrated by hybridization with clones from a SpeI junction fragment library and fragments obtained by partial SpeI digestion, both derived from the P. aeruginosa PAO chromosome.     

Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996     

A 'Southern Cross' method for the analysis of genome organization and the localization of transcription units

A 'Southern Cross' hybridization method is described which permits the rapid restriction mapping of DNA molecules, up to 40 kb in size, for at least ten enzymes in a single operation. The procedure allows the full set of 32P-end-labelled fragments derived from one restriction enzyme digest to intersect and attempt to hybridize to the gel-separated fragments of as many as ten unlabelled digests immobilized on parallel sheets of filter paper. A two-dimensional array of hybridization spots is revealed on each recipient paper, indicating which radioactive and non-radioactive DNA fragments have sequences in common. A restriction map can then be directly and simply deduced from the matrix of hybridization spots in each cross-blot. The method affords advantages over other procedures for obtaining restriction maps in terms of the time required, the number of restriction enzymes that can be mapped, and the potential for eliminating ambiguity. It is also sufficiently sensitive to detect DNA rearrangements and restriction-site polymorphisms in moderately complex genomes. Furthermore, the procedure is applicable to other aspects of the study of genome organization: for example, the exon and intron areas of a segment of cloned genomic DNA can be identified by cross-hybridizing a set of radioactive restriction fragments from the genomic clone against immobilized RNA from a cell type of interest.