Characteristics of Bacillus thuringiensis phages with circular permutation in the DNA molecule

Bacteriophages Tm2 and Tg27 of different origins but identical in biological properties have been compared. Physicochemical characteristics of bacteriophages have revealed the existence of end repeats and circular permutation of phage DNA. Phages Tm2 and Tg27 share the same dimensions of incapsulated DNA, differing in the sizes of phage genome and end repeats. Bacteriophage Tm2 genome is 45.2 kb. long with the end repeats containing 5.7%. The genome of Tg27 is 42.53 kb and 11.8% of end repeat. The bacteriophages relation has been confirmed by heteroduplex and restriction analysis. Tm2 and Tg27 share 84% of homology. Two regions of nonhomology are found representing a single-stranded loop and equishouldered vesicle with the sizes 2.19 kb and 5.03 kb, respectively.     

A novel transposon-like repeat interrupted by an LTR element occurs in a cluster of B1 repeats in the mouse c-mos locus.

The mouse genomic locus containing the oncogene c-mos was analyzed for repetitive DNA sequences. We found a single B1 repeat 10 kb upstream and three B1 repeats 0.6 kb, 2.7 kb, and 5.4 kb, respectively, downstream from c-mos. The B1 repeat closest to c-mos contains an internal 7-bp duplication and a 18-bp insertion. Localized between the last two B1 repeats is a copy of a novel mouse repeat. Sequence comparison of three copies of this novel repeat family shows that they a) contain a conserved BglII site, b) are approximately 420 bp long, c) possess internal 50-bp polypurine tracts, and d) have structural characteristics of transposable elements. They are present in about 1500 copies per haploid genome in the mouse, but are not detectable in DNA of other mammals. The BglII repeat downstream from c-mos is interrupted by a single 632-bp LTR element. We estimate that approximately 1200 copies of this element are present per haploid genome in BALB/c mice. It shares sequence homology in the R-U5 region with an LTR element found in 129/J mice.     

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

Organization of the human myoglobin gene.

Cross-hybridization of the grey seal myoglobin gene to human DNA detected a single human myoglobin gene plus an extensive family of sequences apparently related to the central exon of this gene. The functional human gene is 10.4 kb long and has a haemoglobin-like three exon/two intron structure with long non-coding regions similar to its seal homologue. At least 300 bp of 5'-flanking region are closely homologous between the two genes, with the exception of a divergent purine-rich region 68-114 bp upstream of the cap site. A diverged tandem repetitive sequence based on (GGAT)165 is located 1100-1750 bp upstream from the gene; internal homology units within this sequence suggest sequence homogenization by gene microconversions. A second 33-bp tandem repeat element in the first intron is flanked by a 9-bp direct repeat, shares homology with other tandem repetitive elements in the human genome and may represent a novel form of transposable element.     

Structural analysis of repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region.

Repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region have been mapped and subjected to nucleotide sequence analysis. Two of the four repetitive DNA segments found are located in the 5'-flanking region, and one each within the intervening sequences. Each repetitive DNA segment contains one to three highly homologous unit sequences with an approximate length of 120 base pairs. All the unit sequences are flanked on the 3' side by tandem repeats. There are about 10(5) copies of the repetitive DNA in the bovine genome. Comparison of the bovine repetitive sequences with those of other mammalian species reveals the presence of a homologous segment of approximately 40 base pairs. This segment and the region preceding it in the bovine repetitive DNA exhibit sequence homology with the region encompassing the origin of DNA replication in papovaviruses.     

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.     

Interruption of a human nuclear sequence homologous to mitochondrial DNA by a member of the KpnI 1.8 kb family.

We reported that several DNA sequences homologous to mitochondrial DNA (mtDNA) are present in the human nuclear genome (Tsuzuki et al. (1983) Gene 25, 223-229). Detailed Southern blot analyses revealed that one of such sequences is interrupted by a repetitive sequence about 1.8 kb long, and that the insert is one member of the dispersed repeated DNA sequences of the KpnI 1.8 kb family. Nucleotide sequence analysis showed that the KpnI 1.8 kb DNA is flanked with imperfect 15-base pair (bp) direct repeats of mtDNA. This KpnI 1.8 kb DNA has an A-rich sequence at its 3'-end, and has a considerable homology with one of the published cDNA sequences homologous to one of the human KpnI families and also to one of the African green monkey KpnI families, KpnI-LS1. These structural features suggest that the KpnI 1.8 kb DNA is a movable element and is inserted within the mtDNA-like sequence by an RNA-mediated process.     

The U1 snRNA gene repeat from the sea urchin (Strongylocentrotus purpuratus): the 70 kilobase tandem repeat ends directly 3'' to a U1 gene.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.     

Preferential recombination between GC clusters in yeast mitochondrial DNA.

Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome.     

New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene.

A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination.     

Intein harbouring large tandem repeats in replicative DNA helicase of Trichodesmium erythraeum

Protein splicing inteins can be small as approximately 130 aa or up to approximately 600 aa when harbouring an endonuclease domain. Here we report the identification and characterization of an unusually large intein, 1650 aa long and the largest of known inteins, encoded by the replicative DNA helicase gene dnaB of the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. This Ter DnaB-1 intein co-exists with a 177-aa mini-intein in the same host protein and harbours large tandem repeats in which an 84-aa sequence is repeated 16 times. Comparison between this tandem repeats and the recently reported tandem repeats of Ter DnaE-1 intein revealed differences and similarities. The two tandem repeats, residing in different inteins of different host proteins, differ by 50% in size and have little sequence similarity. Tandem repeats in the Ter DnaB-1 intein were required for the protein splicing activity when tested in Escherichia coli, in contrast to tandem repeats of the Ter DnaE-1 intein that inhibited protein splicing. On the other hand, tandem repeats of both inteins are located in the same corresponding region of the intein sequence and have the same number of repeating units. These suggest that the two tandem repeats could be related but have diverged greatly in size, sequence and effect on protein splicing. Alternatively, they could have independent origins but evolved certain similarities because of common constraints in structure and maintenance.     

Identification and nucleotide sequences of two similar tandem direct repeats in Epstein-Barr virus DNA

Epstein-Barr virus DNA is known to have partially homologous segments, designated DL and DR, near the left and right ends of the long unique region (Raab-Traub et al., Cell 22:257-267, 1980). DL and DR are each partially composed of tandem direct repeat sequences. DL contains 11 to 14 repeats of a 124-base-pair sequence designated IR2. DR contains approximately 30 direct repeats of a 103-base-pair sequence designated IR4. The DL and DR sequences have colinear partial homology for approximately 2.4 and 1.5 kilobase pairs to the right of IR2 and IR4, respectively. IR2 and IR4 are similar sequences and evolved in part from a common ancestor. Both sequences are 84% guanine and cytosine and have limited homology to Epstein-Barr virus IR1 and to the herpes simplex virus type 1 inverted terminal repeat "a" sequence. IR2 encodes part of an abundant 2.5-kilobase persistent early EBV RNA expressed in productively infected cells, but does not encode part of the 3-kilobase Epstein-Barr virus RNA which is transcribed from the adjacent IR1-U2 region of the Epstein-Barr virus genome in latently infected cells.     

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences

The rye-specific R173 family of repeated DNA sequences consists of ca. 15 000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins >20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheatGlu-D1 gene.     

Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.     

Homologous recombination in real time: DNA strand exchange by RecA

Homologous recombination, the exchange of strands between different DNA molecules, is essential for proper maintenance and accurate duplication of the genome. Using magnetic tweezers, we monitor RecA-driven homologous recombination of individual DNA molecules in real time. We resolve several key aspects of DNA structure during and after strand exchange. Changes in DNA length and twist yield helical parameters for the protein-bound three-stranded structure in conditions in which ATP was not hydrolyzed. When strand exchange was completed under ATP hydrolysis conditions that allow protein dissociation, a "D wrap" structure formed. During homologous recombination, strand invasion at one end and RecA dissociation at the other end occurred at the same rate, and our single-molecule analysis indicated that a region of only about 80 bp is actively involved in the synapsis at any time during the entire reaction involving a long ( approximately 1 kb) region of homology.     

Concerted evolution of the tandem array encoding primate U2 snRNA occurs in situ, without changing the cytological context of the RNU2 locus.

In primates, the tandemly repeated genes encoding U2 small nuclear RNA evolve concertedly, i.e. the sequence of the U2 repeat unit is essentially homogeneous within each species but differs somewhat between species. Using chromosome painting and the NGFR gene as an outside marker, we show that the U2 tandem array (RNU2) has remained at the same chromosomal locus (equivalent to human 17q21) through multiple speciation events over > 35 million years leading to the Old World monkey and hominoid lineages. The data suggest that the U2 tandem repeat, once established in the primate lineage, contained sequence elements favoring perpetuation and concerted evolution of the array in situ, despite a pericentric inversion in chimpanzee, a reciprocal translocation in gorilla and a paracentric inversion in orang utan. Comparison of the 11 kb U2 repeat unit found in baboon and other Old World monkeys with the 6 kb U2 repeat unit in humans and other hominids revealed that an ancestral U2 repeat unit was expanded by insertion of a 5 kb retrovirus bearing 1 kb long terminal repeats (LTRs). Subsequent excision of the provirus by homologous recombination between the LTRs generated a 6 kb U2 repeat unit containing a solo LTR. Remarkably, both junctions between the human U2 tandem array and flanking chromosomal DNA at 17q21 fall within the solo LTR sequence, suggesting a role for the LTR in the origin or maintenance of the primate U2 array.