Heterologous Regions Research Articles

Genomic organization of the related Bacillus subtilis bacteriophages SPP1, 41c, rho 15, and SF6.

The genomes of the related virulent Bacillus subtilis bacteriophages SPP1, 41c, rho 15, and SF6 are partially circularly permuted and terminally redundant. Heteroduplex molecules were produced with various combinations of these DNAs. Their electron-microscopic analyses showed a consistent pattern of homologous and heterologous regions of DNA. Restriction maps of the phage DNAs were established. A comparison of these maps showed a pattern of conserved and variable DNAs compatible with the electron-microscopic analyses. In all phage genomes, regions specifying early and late functions were conserved. In each phage genome, such regions were separated by short segments of heterologous DNA characteristic for each phage.

The nucleotide sequences of the heterologous region between the genomes of Bacillus phages M2 and Nf that indicate the two phages are originally identical

It has been suggested that the heterologous population of Bacillus phage M2 is derived from an original clone, which is identical with phage Nf, by the deletion on a particular region of the genome. We have determined the nucleotide sequence of this region of M2 subclones and Nf genomes. The results clearly indicate that the homologous recombination through the short direct repeated sequence is the main cause of the varied deletions found in the genomes of M2 subclones.

Evidence that Bacillus phage M2 is derived from Nf by deletion in a particular region of the genome.

It has been shown that the restriction maps of Bacillus phages M2 and Nf are identical to each other except in the region 32 to 46% from the left ends of the genomes. Furthermore, in this region, genomes of the original stock of phage M2 were heterogeneous in length. We have found that M2 has the same origin as Nf and that the heterogeniety in the region is caused by deletions of various sizes. These results were obtained by electoron microscopic observations of heteroduplex molecules between the genomes of Nf and subclones of M2, and by fine physical mapping of the heterologous regions. Finally, it has been shown that these deletions affected phage morphology but not multiplication.

Recombination efficiency is a quadratic function of the length of homology during plasmid transformation of Bacillus subtilis protoplasts and Escherichia coli competent cells.

Recombination efficiency between transforming and resident plasmids was 100 times higher in Bacillus subtilis protoplasts than in Escherichia coli competent cells. In both systems it varied with the square of the length of the sequences common to two plasmids within the studied range 0.96-8.8 kb in B. subtilis, 0.72-3.9 kb in E. coli. The observed exponential dependence may be characteristic of recombination occurring within a relatively short interval bordered by relatively long heterologous regions.

Polarity in adenovirus recombination

The distributions of the crossovers necessary to generate ts + genomes have been examined in a collection of clonally unrelated ts + recombinants from a set of ts×ts adenovirus crosses. In a cross between two parents that are grossly heterologous between map units 80.2 and 91.5, the distribution of crossovers was significantly skewed toward the left-hand end of the genome, with a declining frequency proceeding rightward. This gradient of recombination was modified by the removal of the right-hand heterology and by the presence of another region of heterology between map units 3.67 and 10.11. In a cross where the ts markers were flanked by both heterologies, no gradient was observed and ts + recombinants were characterized by a higher rate of supernumerary crossovers. In a cross designed so that one is marker was internal to two heterologies, crossovers were found disproportionately between the second is marker and the nearby heterology. In addition, ts + recombinants formed by crossing over internal to the heterologies again were accompanied by a high frequency of supernumerary crossovers. Finally, ts + recombinant frequencies in crosses identical except for the presence of either one or two flanking heterologies were markedly lower in the latter case. These data, taken together, suggest that a major pathway of adenovirus recombination initiates at, or near, the molecular termini and is perhaps driven by the displaced single strands produced during DNA replication. Internal initiation, on the other hand, may employ these single strands to form genetic “patches.”

Fine scale interspersion of conserved sequences in the pea and corn chloroplast genomes

A comparison is made of the chloroplast genomes of two divergent higher plants, pea and corn. Reassociation kinetics analysis shows that only 33–34% of the chloroplast DNA (ct DNA) sequences are conserved in these two plants, which is equal to about 43 kilobases (kb). The restriction enzyme patterns produced by Eco RI, Bam HI, and Sal I are different for each ct DNA, as expected from the low level of homology. The total length of cross-reacting Eco RI fragments, assessed by blot hybridization methods, exceeds the reassociation kinetics estimate by at least 20 kb. An electron microscopic analysis of ct DNA heteroduplexes shows that the conserved regions are surprisingly short, and consequently, they are interspersed with divergent DNA. Fifty percent of the conserved regions are less than 550 bases; 10 sites are less than 150 bases. The median length of a heterologous region is 250 bases. The heteroduplexes fall into 4 classes, established by the position and size of the conserved and divergent regions, totaling 61 kb. One class has been identified as the ribosomal gene region: the corn Eco RI fragment, Eco RI A, which codes for the 16S and 23S cistrons (Bedbrook and Bogorad 1976), was reassociated with total pea ct DNA, and the products analyzed by electron microscopy. Only one pattern of heteroduplexes was observed. A stretch of almost completely conserved DNA, equivalent to 6.9 kb, extends from the 16S gene through the 23S gene, and therefore, includes the transcribed spacer separating these two cistrons. Heterologous regions occur immediately outside of the 5′ and 3′ ends of the 16S and 23S genes, respectively. A set of the 16S and 23S genes contribute about 4%, and the spacer 1.6%, to the level of sequence homology in each genome.

Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical.

The molecular relationships of the IncP1 plasmids RP1, RP4, R68 and RK2 were tested by electron microscopic examination of heteroduplexes. In several hybridization experiments molecules were detected which had a 7.8% portion of incomplete reannealing. This 'heterologous region' could be explained by the typical renaturation behaviour of the transposon Tn1. The identity of the Tn1 transposon present in RP1 and RP4 was proved by heteroduplex experiments with lambda phage DNA containing this transposon. These results indicated that the plasmids RP1 and RP4 are identical. Additional heteroduplex experiments between plasmids R68.45 and RP8 and between R68.45 and RK2 were performed. R68.45, a derivative of R68, has a small DNA insertion and RP8 can be regarded as a large insertion mutant of RP4; both insertions were used as single-stranded hybridization markers. From the hybrid molecules formed, it was deduced that R68 and RK2 are identical with RP1 and RP4 as far as molecular structure is revealed by the technique used.

Evolution of Drosophila mitochondrial DNAs Analysis of heteroduplex molecules

We have mapped the single block of non-homologous sequences and measured the extent and distribution of base-pair substitutions within the homologous sequences in Drosophila melanogaster: Drosophila virilis heteroduplex mitochondrial DNAs (mtDNAs). Of the 4.8 kilobases long, unusually (A + T)-rich region in D. melanogaster mtDNA, only 0.5 kilobases can react with related, but not identical sequences in D. virilis mtDNA, while the rest (4.3 kilobases in the long arm of a heteroduplex loop) is replaced by a shorter, non-homologous region (1.0 kilobases in the short arm of the loop). No additional heterologous regions are evident. Homologous sequences have accumulated on the average 15.5% base-pair changes. Regionally, these substitutions are relatively uniformly distributed (14.5–16.5%) except for a single, more conserved region (10–13%), which presumably represents the ribosomal cistrons. The lack of general sequence stability suggests that the invariant topographic organization of the nucleotide sequence, previously recognized among Drosophila mtDNAs, is under more stringent selection than the sequence per se.

Hotspots for generalized recombination in the Escherichia coli chromosome

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λ bio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA + function, the site-specific stimulation of recombination required recB + function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology. The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 10 3 bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.

Complex mitochondrial DNA in Drosophila.

The larval mtDNA isolated from D. virilis, D. simulans and D. melanogaster exists in complex molecular forms in addition to the simple monomeric circular form. The frequency of circular dimers and oligomers is highly elevated in apparently normal larval tissues. These complex forms of mtDNA are separable on agarose gels. Hind III restriction endonuclease and electron microscopic analyses used in the present study have revealed that circular dimers are simply the circular concatemers of two monomeric circles which are arranged in a head-to-tail structure with no detectable heterologous regions such as insertions or deletions. The electrophoretic patterns of Hind III digested mtDNAs of D. simulans and D. melanogaster (sibling species) are identical and distinguishable from that of distantly related species, D. virilis.

λimmλ·434: A phage with a hybrid immunity region

The genomes of coliphages λ and λimm434 are identical with the exception of the immunity region, which codes for the respective repressors and corresponding operators. Although previous genetic studies failed to detect any recombinations that might have occurred within the immunity regions of these two phages, electron-micrographic heteroduplex mapping demonstrated a short interval of homology between λ and λimm434 DNA located in this otherwise heterologous region. We report the isolation of a recombinant which resulted from a cross-over event between λ and λimm434 within the immunity region. This phage, denoted λimmλ·434, carries the leftward promoter-operator as well as gene rex of λ and the rightward promoter-operator as well as gene tof ( cro) of phage 434. Since the recombination occurred within the cI gene, phage λimmλ·434 must carry a hybrid repressor gene.

Abnormalities in synaptonemal complexes in pollen mother cells of a lily hybrid

Two abnormalities, one in lateral elements of synaptonemal complexes the other involving whole complexes, have been studied with the electron microscope in pollen mother cells of the lily hybrid, Lilium aureliensis × L. henryi, which with the light microscope showed almost complete bivalent formation at metaphase. Brief water treatment of pollen mother cells prior to fixation, revealed that the aberrant configurations in lateral elements arose by breakage and subsequent folding of severed fragments up to about 0.8 μm long. The abnormality ocurred at recognisable heterologous regions, apparently immediately after pairing. The folded fragments were eliminated from the chromosomes at some time during pachytene. Pseudo pairing was observed after synapsis between either more than one pair of homologues or one pair bent back on themselves, so as to produce polycomplexes. Seemingly, central elements could develop between lateral elements on their outer face under these conditions.

Homology and structural relationships between the dimeric and monomeric circular forms of mitochondrial DNA from human leukemic leukocytes.

Abstract Mitochondrial DNA occurs in multiple circular forms in human leukemic leukocytes. As part of an inquiry into the structural relationships between the forms, the 5- and 10-micron circular duplexes were separated and characterized by sedimentation procedures. The buoyant densities in neutral cesium chloride were indistinguishable, indicating that over-all base compositions of the two species are the same. The buoyant densities of the corresponding heavy and light strands in the two species were also indistinguishable. Together, the results suggest that the dimer consists of two monomer genomes. Reannealing of equal amounts of fragmented light monomer and heavy dimer complementary strands isolated from alkaline buoyant cesium chloride gradients leads to a high molecular weight duplex with a buoyant density indistinguishable from the parent duplexes. It is concluded from this result that the degree of homology between monomer and dimer is at least 90%. Denaturation of a mixture of lightly nicked monomer and dimer duplexes, followed by reannealing in formamide solutions, leads to mixtures of circular monomer and dimer duplexes and heteroduplexes which contain one dimer strand and two monomer strands. These heteroduplexes occur in the form of a “figure 8” molecule, a new DNA structure which we have called the fused dimer. Neither heterologous regions, nor insertions, nor deletions were found in a careful examination of the electron micrographs. It is concluded from this part of the work that heterologous regions, insertions, or deletions exceeding 50 to 100 nucleotides in length do not occur in the heteroduplexes. At this level of insight, the dimer is a circular concatamer of two monomer genomes. In addition, the results show that leukemic leukocyte mitochondrial DNA monomers (as well as dimers) are substantially homogeneous in base sequence. The electron microscope studies also show that monomer genomes in the dimer are connected in a head-to-tail structure, rather than in a head-to-head structure.