Structure and genetic complexity of the genomes of herpesvirus defective-interfering particles associated with oncogenic transformation and persistent infection.

The complexity and structural organization of defective-interfering (DI) particle DNA of equine herpesvirus type 1 (EHV-1) have been elucidated by using restriction enzyme and Southern blot hybridization analyses. DI particles were generated by serial high-multiplicity passage of EHV-1 in L-M cells, and total viral DNA was extracted from virus purified from supernatants of these serial passages. EHV-1 DI particle DNA was quantitatively separated from standard (STD) DNA by several cycles of CsCl isopycnic banding in a vertical rotor. Restriction endonuclease digestion profiles of pure DI DNA were completely different from the mapped patterns observed for EHV-1 STD DNA. Digestion of pure defective DNA with restriction enzymes (Bg/II, EcoRI, and XbaI), for which there are few or no cleavage sites within the S (short) region of the EHV-1 STD genome, yielded high-molecular-weight supermolar DNA bands, suggesting that a large subgenomic repeat unit was present in defective DNA. DNA blot hybridization analysis with the Bg/II supermolar fragment of defective DNA, intact DI particle genomic DNA, and EHV-1 STD DNA restriction enzyme fragments as 32P-labeled probes indicated that the EHV-1 DI particle genome originates predominately from the STD DNA S region (0.77 to 1.00 map units) and to a lesser extent from the left terminus of the unique long (UL) region (0.00 to 0.05 map units). None of the EHV-1 DNA sequences associated to date with EHV-1 oncogenesis (0.32 to 0.38 map units; O'Callaghan et al. in B. Roizman [ed.], Herpesviruses, in press; Robinson et al., Cell 32:204-219, 1983, and Proc. Natl. Acad. Sci., U.S.A., 78:6684-6688, 1981) were detected in the DI particle DNA. The importance of these data with regard to DNA replication of DI particles and the role of DI particles in one model system of EHV-1 oncogenic transformation are discussed.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Genetic relatedness of the genomes of equine herpesvirus types 1, 2, and 3.

Genomic DNAs of equine herpesvirus type 1 (EHV-1), EHV-2 (equine cytomegalovirus), and EHV-3 were examined by reassociation kinetic and thermal denaturation analyses to determine the extent and degree of homology among the three viral DNAs. Results of reassociation analyses indicated a limited homology among the three EHV genomes. Homologous DNA sequences equivalent to 1.8 to 3.7 megadaltons between EHV-1 and equine cytomegalovirus, 7.6 to 8.2 megadaltons between EHV-1 and EHV-3, and 1.3 to 1.9 megadaltons between equine cytomegalovirus and EHV-3 were detected. Examination by thermal denaturation of the DNA homoduplexes and heteroduplexes formed during reassociation revealed a high degree of base pairing within the duplexes, suggesting that closely related sequences may be conserved among the genomes of EHV.     

Genome structure and virion polypeptides of the primate herpesviruses Herpesvirus aotus types 1 and 3: comparison with human cytomegalovirus.

Two serologically distinguishable primate herpesviruses, Herpesvirus aotus type 1 and type 3, were examined with regard to their genomes and structural polypeptides. The duplex DNA genomes of these two viruses were found to be essentially identical in molecular weight (Mr approximately equal to 145 X 10(6)) and guanine plus cytosine composition (55%). Both contained unique and inverted repeat nucleotide sequences of the same size and arrangement, which, as judged by DNA-DNA hybridization and restriction enzyme analyses, were at least 95% homologous. In addition, no differences were observed in electrophoretic profiles of virion polypeptides. Because of their great similarity with respect to these criteria, the two viruses ought to be considered independent isolates (or strains) of a single virus, which should be designated H. aotus type 1. The elevated molecular weight and presence of two sets of inverted repeat sequences closely resemble the structure of the human cytomegalovirus genome. However, no sequence homology (less than 5%) nor similarity in virion polypeptides was detected between H. aotus type 1 and human cytomegalovirus.     

Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost

We examined the arrangement of sequences common to seven angiosperm chloroplast genomes. The chloroplast DNAs of spinach, petunia and cucumber are essentially colinear. They share with the corn chloroplast genome a large inversion of approximately 50 kb relative to the genomes of three legumes--mung bean, pea and broad bean. There is one additional rearrangement, a second, smaller inversion within the 50 kb inversion, which is specific to the corn genome. These two changes are the only detectable rearrangements that have occurred during the evolution of the species examined (corn, spinach, petunia, cucumber and mung bean) whose chloroplast genomes contain a large inverted repeat sequence of 22-25 kb. In contrast, we find extensive sequence rearrangements in comparing the pea and broad bean genomes, both of which have deleted one entire segment of the inverted repeat, and also in comparing each of these to the mung bean genome. Thus there is a relatively stable arrangement of sequences in those genomes with the inverted repeat and a much more dynamic arrangement in those that have lost it. We discuss several explanations for this correlation, including the possibility that the inverted repeat may play a direct role in maintaining a conserved arrangement of chloroplast DNA sequences.     

Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed DeltaAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of DeltaAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of DeltaAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of DeltaAd. IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small DeltaAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for DeltaAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. DeltaAd.IR vectors were produced at a yield of approximately 10(4) particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. DeltaAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.     

A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Bam HI with ³²P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1-transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32–0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state.     

A 21 kilobase-pair deletion/addition difference in the inverted repeat sequence of chloroplast DNA from Chlamydomonas eugametos and C. moewusii

Summary Our recent physical mapping of chloroplast DNA (cpDNA) from Chlamydomonas moewusii, a unicellular green alga which is interfertile with Chlamydomonas eugametos, has revealed a two-fold size difference between the inverted repeat sequences of these algae. With a size of 42 kbp, the inverted repeat of C. moewusii is the largest yet identified in any chloroplast genome. Here we have compared the arrangement of conserved sequences within the two algal inverted repeats by hybridizing cloned restriction fragments representing over 90% of these repeats to Southern blots of cpDNA digests from the two algae. We found that the size difference between the two algal inverted repeats is due to the presence of an extra DNA segment of 21 kilobase pairs (kbp) in C. moewusii. Except for this sequence, the C. moewusii inverted repeat is highly homologous to the entire C. eugametos repeat and the arrangement of conserved sequences in the two repeats is identical. Southern hybridizations with specific gene probes revealed that the conserved sequences include the rDNA region and the genes coding for the large subunit of ribulose 1,5 bisphosphate carboxylase-oxygenase (rbcL) and for the 32 kilodalton thylakoid membrane protein (psbA). With respect to the conserved sequences, the extra 21 kbp DNA segment of C. moewusii lies in the region of psbA, most probably slightly downstream from this gene.     

Instability of long inverted repeats within mouse transgenes.

Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.     

Structural features of the integration site of foreign DNA in the transgenic mouse genome

The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome.     

石刁柏雄性偏向核质体DNA的克隆与分析

该研究以雌雄异株植物石刁柏为材料,利用基因组消减杂交技术对石刁柏雌雄核基因组中的性别差异核质体DNA（nuclear plastid DNA,NUPTs）进行了分离和分析。结果表明：（1）通过构建消减杂交文库共获得了52个雄性偏向序列,序列长度分布在63~297 bp之间,其中有19个差异序列属于叶绿体来源序列（命名为Ao1~Ao19）,且这些序列与石刁柏叶绿体基因组的相似性均大于84%,Ao19与石刁柏叶绿体基因组相似性为100%。（2）利用基因组半定量PCR对19个NUPTs序列的性别差异分析表明,有4条序列为稳定的雄性偏向NUPTs序列,分别为Ao1、Ao3、Ao10和Ao18。（3）序列比对表明,转移到核基因组的NUPTs主要来源于叶绿体基因组的反向重复区（包含IRa和IRb区）,说明石刁柏叶绿体基因组重复区序列更容易向核基因组进行转移形成雄性偏向的NUPTs序列。     

Eucaryotic transposable genetic elements with inverted terminal repeats

DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Bam HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction.     

Extensive Amplification and Transposition of a Novel Repetitive Element, Xstir, Together with Its Terminal Inverted Repeat in the Evolution of Xenopus

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis. Received: 5 April 2000 / Accepted: 3 August 2000     

Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.     

Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.     

Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences.

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.     

A second locus for the 5S multigene family in Secale L.: sequence divergence in two lineages of the family

The 5S RNA genes in Secale sp. are arranged as tandem arrays of a 460- and 480-bp repeating sequence. These size classes were initially discovered by restriction endonuclease analysis using BamHI and subsequently by DNA sequencing of cloned units. The length variation between short and long units originated from major deletion-insertion events in the noncoding spacer region of the 5S DNA repeat units. In situ hybridization with [3H]cRNA and biotin-labelled probes synthesized from both the short and long 5S DNA units of S. cereale localized the sites on chromosome 1R and a new site on a chromosome identified as 5R. We propose that the chromosome 1R locus, which has been mapped previously, be named 5SDna-R1 and the second locus, reported in the present paper, be referred to as 5SDna-R2. A preferential hybridization of a probe from the long unit to the 5SDna-R2 locus and of a probe from the short unit to the 5SDna-R1 locus is reported. The clustering of long units in the 5SDna-R2 locus was confirmed by restriction endonuclease digestion of DNA from rye chromosome 5R additions to wheat. Nucleotide sequence alignment of 5S DNA repeat units from a number of Secale species, using both phenetic and cladistic computer programmes, demonstrated that two clear lineages corresponding to the long and short units existed in this genus. The different Secale species could not be unambiguously differentiated using the 5S DNA sequences.     

Widespread occurrence of small inversions in the chloroplast genomes of land plants

Large inversions are well characterized in the chloroplast genomes of land plants. In contrast, reports of small inversions are rare and involve limited plant groups. In this study, we report the widespread occurrence of small inversions ranging from 5 to 50 bp in fully and partially sequenced chloroplast genomes of both monocots and dicots. We found that small inversions were much more common than large inversions. The small inversions were scattered over the chloroplast genome including the IR, SSC, and LSC regions. Several small inversions were uncovered in chloroplast genomes even though they shared the same overall gene order. The majority of these small inversions were located within 100 bp downstream of the 3' ends of genes. All had inverted repeat sequences, ranging from 11 to 24 bp, at their ends. Such small inversions form stem-loop hairpin structures that usually have the function of stabilizing the corresponding mRNA molecules. Intra-molecular recombination between the inverted sequences in the stem-forming regions are responsible for generating flip-flop orientations of the loops. The presence of two different orientations of the stem-loop in the trnL-F noncoding region of a single species of Jasminum elegans suggests that a short inversion can be generated within a short period of time. Small inversions of non-coding sequences may influence sequence alignment and character interpretation in phylogeny reconstructions, as shown in nine species of Jasminum. Many small inversions may have been generated by parallel or back mutation events during chloroplast genome evolution. Our data indicate that caution is needed when using chloroplast non-coding sequences for phylogenetic analysis.     

Electron microscopy of Achlya deoxyribonucleic acid sequence organization.

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.