Sequence arrangements of the Escherichia coli chromosome and of putative insertion sequences, as revealed by electron microscopic heteroduplex studies.

Total Escherichia coli DNA from strain DK445 (which is CSH50 F^?, R^?, deletion lac and pro, lysogenized with lambda cIts857 Sam7 lac5: :Mu cI⁺) was denatured, reannealed, and observed by electron microscopy. The single-strand DNA lengths ranged from about 50 to 150 kilobases (kb). In some molecules a short duplex region with a single-stranded fork at each end was observed. The duplex lengths were 0.75 kb, 1.30 kb, 5.22 kb, 5.62 kb, which correspond to those of IS1; of IS2, IS3, or IS4; of the ribosomal RNA genes; and of the γδ sequence, respectively. Duplexes of 1.0 kb and 0.5 kb were also found. Most of the duplexes of 0.5, 0.75, 1.0 and 1.3 kb were observed as intramolecular stem-loop structures and were therefore interpreted to be sequence duplications in inverted order on the same DNA strand. The most frequent separations of the putative inverted insertion sequences were around 22 and 27.5 ± 1.5 kb. About 14% of the E. coli chromosome is estimated to be involved in the sequence arrangements that give rise to stem-loop structures upon denaturation and reannealing. The copy numbers of the putative insertion sequences and other elements that form the “stems” of the stem-loop structures are also estimated.     

Molecular Relatedness of the Prevalent Cryptic Plasmids of Bacteroides Species Isolated in Hungary

The molecular relatedness of plasmids of 44 previously-reported plasmid-bearing Bacteroides strains were examined by Southern hybridization. The identity of 5.5 kb plasmids, which occur most frequently (34.8%) in Hungarian clinical isolates, and 4.2 kb plasmids, the second most prevalent (21.7%) plasmid type were analyzed. Homology with class II and class III plasmids from North American Bacteroides isolates was also investigated. These experiments revealed that the 5.5 kb plasmids are highly homologous and belong in class III, and the 4.2 kb plasmids belong in class IIA. One plasmid belonging to class IIB was observed, and two 4.2 kb plasmids displayed homology to the 5.5 kb group. In geographically closely situated regions, the frequency of occurrence of these plamids is similar, while in distant regions their prevalences differed.     

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.     

Genetic variance of Trichomonas vaginalis isolates by Southern hybridization

In the present study, genomic DNAs were purified from Korean isolates (KT8, KT6, KT-Kim and KT-Lee) and foreign strains (CDC85, IR78 and NYH 286) of Trichomonas vaginalis, and hybridized with a probe based on the repetitive sequence cloned from T. vaginalis to observe the genetic differences. By Southern hybridization, all isolates of T. vaginalis except the NYH286 strain had 11 bands. Therefore all isolates examined were distinguishable into 3 groups according to their banding patterns; i) KT8, KT6 and KT-Kim isolates had 11 identical bands such as 1 kb, 1.2 kb, 1.6 kb, 1.9 kb, 2.3 kb, 2.7 kb, 3.2 kb, 3.4 kb, 3.8 kb, 4.9 kb and 6.0 kb. ii) The metronidazole-resistant IR78 strain had the same bands as KT-Lee isolate at bands of 1 kb, 1.2 kb, 1.6 kb, 1.8 kb, 2.1 kb, 2.5 kb, 2.7 kb, 2.9 kb, 3.4 kb, 5.0 kb and 6.0 kb. Bands of CDC85, metronidazole-resistant strain, were similar to those of IR78 and KT-Lee, except that 3.2 kb replaced 2.9 kb. iii) NYH286 particularly had 12 bands and band patterns were similar to IR78 with a few exceptions as follows: i) 6.2 kb in place of 6.0 kb, ii) 2.0 kb and 2.2 kb instead of 2.1 kb. Through the results obtained, genetic variance of T. vaginalis isolates was demonstrated by Southern hybridization.     

Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping.

The ColE1-Km plasmids pCR1 and pCR11 have been characterized by electron microscope techniques. Their sizes have been determined to be 13.1 and 9.2 kb, respectively, and heteroduplex studies show that the plasmids differ in the presence of a 3.9 kb deletion in the ColE1 region of pCR11. Both contain a nontandem inverted duplication of a 1.06 kb sequence. The single HindIII site, 3.5 kb from the EcoR1 site, lies in the 0.97 kb of DNA between the inverted repeat sequences. Since DNA insertions at the HindIII site destroy kanamycin resistance, it can be concluded that the kanamycin phosphotransferase gene is contained in this region. Electron microscopy of self-annealed plasmids treated with restriction endonucleases shows that each inverted duplication sequence contains one HindII site and at least two SmaI sites.     

Cloning and Expression in Escherichia coli of the Polysaccharide Depolymerase Associated with Bacteriophage-Infected Erwinia amylovora

The bacteriophage-encoded polysaccharide depolymerase produced in Erwinia amylovora has been cloned and expressed in Escherichia coli. The bacteriophage ERA103 genome was observed to consist of five EcoRI fragments, labeled as follows: A, 7.5 kilobases (kb); B, 5.0 kb; C, 2.7 kb; D, 2.1 kb; and E, 1.8 kb. A restriction map for ERA103 was also prepared. Each of the fragments were cloned into the positive-selection vector pOP203(A₂⁺) and pBR322.     

Retroviral Recombination Rates Do Not Increase Linearly with Marker Distance and Are Limited by the Size of the Recombining Subpopulation

Recombination occurs at high frequencies in all examined retroviruses. The previously determined homologous recombination rate in one retroviral replication cycle is 4% for markers 1.0 kb apart in spleen necrosis virus (SNV). This has often been used to suggest that approximately 30 to 40% of the replication-competent viruses with 7- to 10-kb genomes undergo recombination. These estimates were based on the untested assumption that a linear relationship exists between recombination rates and marker distances. To delineate this relationship, we constructed three sets of murine leukemia virus (MLV)-based vectors containing the neomycin phosphotransferase gene (neo) and the hygromycin phosphotransferase B gene (hygro). Each set contained one vector with a functional neo and an inactivated hygro and one vector with a functional hygro and an inactivated neo. The two inactivating mutations in the three sets of vectors were separated by 1.0, 1.9, and 7.1 kb. Recombination rates after one round of replication were 4.7, 7.4, and 8.2% with markers 1.0, 1.9, and 7.1 kb apart, respectively. Thus, the rate of homologous recombination with 1.0 kb of marker distance is similar in MLV and SNV. The recombination rate increases when the marker distance increases from 1.0 to 1.9 kb; however, the recombination rates with marker distances of 1.9 and 7.1 kb are not significantly different. These data refute the previous assumption that recombination is proportional to marker distance and define the maximum recombining population in retroviruses.     

Characterization of the gene encoding murine heparin-binding epidermal growth factor-like growth factor

The mouse gene (mHB-EGF) encoding heparin-binding epidermal growth factor-like growth factor was isolated from a mouse 129SVJ genomic library. DNA sequence analysis confirmed that the clone contained six exons (I–VI) and five introns (A–E), and spanned approx. 14 kb of DNA. PCR analysis showed that introns A–E of mHB-EGF are 203 bp, 2.5 kb, 5.5 kb, 825 bp and 272 bp in length, respectively. These results establish that mHB-EGF is similar in organization to human HB-EGF (hHB-EGF). However, DNA sequence analysis of introns A–E of mHB-EGF failed to show significant overall homology with those of hHB-EGF     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

pBLI is a conjugative linear extrachromosomal element of 43 kb previously isolated after interspecific mating between Streptomyces bambergiensis and S. lividans. Cloning experiments using the non-conjugative, circular Streptomyces vector pIJ702 allowed the identification of a 5.74 kb region from pBL1 which facilitates plasmid transfer. Insertion and deletion mutagenesis, gene disruptions, and sequence data suggest that at least five previously unknown genes of pBL1 are required for efficient plasmid transfer and its regulation.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region,the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.