Tandem Reiterations Research Articles

Genetic variability in the region encompassing reiteration VII of herpes simplex virus type 1, including deletions and multiplications related to recombination between direct repeats.

A number of tandemly reiterated sequences are present on the herpes simplex virus type 1 (HSV-1) DNA molecule of 152 kbp. While regions containing tandem reiterations were usually unstable, reiteration VII, which is present within the protein coding regions of gene US10 and US11, was stable; hence, reiteration VII could be used as a genetic marker. In the present study, the nucleotide sequences (159–213 bp) of a region encompassing reiteration VII of 62 HSV-1 isolates were compared with that of strain 17 as the standard strain, and the genetic variability of base substitutions, deletions, and multiplications was revealed. Base substitution was observed in nine residues on the region flanking reiteration VII and sixty-two HSV-1 isolates were classified into twelve groups based on these base substitutions. Deletions, which were present in all sixty-two isolates, were classified into six groups. Multiplications, which were present in 19 isolates having the same deletion (named del-2), were classified into four groups. The sixty-two isolates were classified into twenty patterns based on variations in the region encompassing reiteration VII, and the region encompassing reiteration VII was considered to be useful for studies on the molecular epidemiology and evolution of HSV-1. The lengths of these deletions and multiplications were multiples of 3; thus, a frame-shift mutation was not induced, and a mechanism to maintain the functions of US10 and US11 was suggested. A series of multiplications, which consisted of the duplication, triplication, and tetraplication of the same sequence, were found. Since all isolates with a multiplication had del-2, multiplications were assumed to be generated after the generation of del-2, and an isolate with del-2 was considered to have the ability to generate a multiplication. Recombination between a pair of direct repeats in and around reiteration VII was accountable for the generation of deletions and multiplications, indicating the recombinogenic property of the region encompassing reiteration VII. A correlation was revealed between a set of 20 DNA polymorphisms widely present on the HSV-1 genome and the base substitutions and deletions of the region encompassing reiteration VII, using discriminant analyses.

Replication Initiator Protein NS1 of the Parvovirus Minute Virus of Mice Binds to Modular Divergent Sites Distributed throughout Duplex Viral DNA

To initiate DNA synthesis, the NS1 protein of minute virus of mice (MVM) first binds to a simple cognate recognition sequence in the viral origins, comprising two to three tandem copies of the tetranucleotide TGGT. However, this motif is also widely dispersed throughout the viral genome. Using an immunoselection procedure, we show that NS1 specifically binds to many internal sites, so that all viral fragments of more than approximately 170 nucleotides effectively compete for NS1, often binding with higher affinity to these internal sites than to sites in the origins. We explore the diversity of the internal sites using competitive binding and DNase I protection assays and show that they vary between two extreme forms. Simple sites with three somewhat degenerate, tandem TGGT reiterations bind effectively but are minimally responsive to ATP, while complex sites, containing multiple variably spaced TGGT elements arranged as opposing clusters, bind NS1 with an affinity that can be enhanced approximately 10-fold by ATP. Using immuno-selection procedures with randomized sequences embedded within specific regions of the genome, we explore possible binding configurations in these two types of site. We conclude that binding is modular, combinatorial, and highly flexible. NS1 recognizes two to six variably spaced, more-or-less degenerate forms of the 5'-TGGT-3' motif, so that it binds efficiently to a wide variety of sequences. Thus, despite complex coding constraints, binding sites are configured at frequent intervals throughout duplex forms of viral DNA, suggesting that NS1 may serve as a form of chromatin to protect and tailor the environment of replicating genomes.

The mitochondrial genome of the smaller tea tortrix Adoxophyes honmai (Lepidoptera: Tortricidae)

Almost complete mitochondrial genome from a representative of an insect order Lepidoptera, the smaller tea tortrix Adoxophyes honmai was determined. The 15,680 bp long A. honmai genome encodes 13 putative proteins, two ribosomal RNAs and 22 tRNAs. The nucleotide sequences of A. honmai mitochondrial DNA have been compared with those of five species from the Lepidoptera and insects in the other orders that are available in the databases. The orientation and gene order of A. honmai is almost same to that of other insects with a few minor exceptions in the order of tRNAs and distribution of non-coding regions. Nucleotide composition, amino acid composition and codon usage are in the range of values estimated from other insect mitogenomes. In AT rich region of A. honmai, tandem reiterations are observed with repeats of TAA. In a preliminary phylogenetic analysis based on the concatenated 7 protein coding genes, A. honmai, an apoditrysian tortricid moth joined basally within the monophyly of Lepidoptera, supporting its relationship with other more derived species including obtectomeran Ostrinia species.

Analysis of numbers of repeated units in R2 region among varicella–zoster virus strains

Background: A variable region, R2, on the varicella–zoster virus (VZV) genome contains a repeated 42-bp unit. Objective: The purpose of this study is the derivation of significance from tandem reiteration structure in the R2 region. Methods: Fifty-two specimens were collected from 52 patients with herpes zoster in Osaka and Tokyo, Japan. After treatment of the specimens to release viral DNA, the samples were amplified directly by polymerase chain reaction. In addition, 14 samples were collected from 7 of these zoster patients after valaciclovir or aciclovir therapy. Results: Analyses of the 52 specimens revealed that the number of repeats ranged from 4 to 13. Interestingly, the numbers of repeats among various VZV strains showed a normal distribution pattern, so that 6–9 repeats were found to be predominant in both Osaka (85%) and Tokyo (72%). The pre- and post-treatment strains taken from the same individuals showed the same numbers of repeats (7–9 in 6 cases and 11 in one). Conclusion: Our results suggest that the 6–9 repetitions of the 42-bp unit, with presumed stability, may offer these virus strains an advantage in virulence to human skin.

Characterization of acyclovir susceptibility and genetic stability of varicella-zoster viruses isolated during acyclovir therapy

We have characterized the susceptibility and genetic stability of varicella-zoster viruses (VZV) isolated from skin lesions of three patients with herpes zoster and six patients with varicella treated with conventional short-term acyclovir (ACV) administration. The susceptibilities to ACV of the serial isolates from the patients were examined, and there was no significant difference in the susceptibility to ACV among the isolates before and during the ACV treatment, indicating that conventional short-term ACV treatment did not generate ACV-resistant VZV infections. Polymerase chain reaction (PCR) analyses of these as well as seven thymidine kinase-deficient VZV strains derived from in vitro ACV treatment were carried out to examine their genomic stability. Five regions containing tandem direct reiterations (R1–R5) were amplified by PCR and compared, and the region containing the Pst I-site was also examined. PCR analyses demonstrated that the R1, R5 and the Pst I-sites were stable and useful in epidemiological studies even after ACV treatment. The R2, R3 and R4 sites were far less stable in these experimental conditions. In this paper we discuss the results of the PCR analyses with regard to the dynamics of VZV population in patients with VZV infection treated with conventional short-term ACV administration.

An analytical method for R5 repeated structure in varicella-zoster virus DNA by polymerase chain reaction

The tandem direct reiteration, R5, in varicella-zoster virus (VZV) genome consists of 88-bp and 24-bp elements. The R5 structure was examined by the polymerase chain reaction to differentiate between various strains. A sense primer was designed at the 88-bp element and an antisense primer at the site separated from R5, in which we expected to detect multiple bands corresponding to repeating numbers of 88-bp elements. Thirty-four wild strains and a vaccine strain were analyzed using this method. The 34 wild strains showed two to four bands corresponding to numbers of 88-bp elements, in which the strain showing three bands was found to be predominant. The vaccine strain also revealed three bands.

Analysis of strain variation of R1 repeated structure in varicella-zoster virus DNA by polymerase chain reaction

The tandem direct reiteration R1 in the varicella-zoster virus genome consists of two elements: units composed of 18 bp and those having 15 bp, both of whose numbers and types of combination patterns vary among strains. The variations of the R1 structure were examined in order to differentiate between the wild strains and a varicella vaccine (Oka) strain by the polymerase chain reaction using two primer sets. The results showed that the 31 wild strains were classified into nine patterns: the R1 structure consisted of a combination of 5 to 7 18-bp units and 8 to 12 15-bp units. The strain with a combination of 6 18-bp units and 10 15-bp units was found to be predominant in Japan, and the same pattern was found in the vaccine strain, so that differentiation between the vaccine strain and the wild strains in Japan merely by analysis of R1 in VZV genome is difficult. J. Med. Virol. 58:76–78, 1999. © 1999 Wiley-Liss, Inc.

Analysis of strain variation of R1 repeated structure in varicella-zoster virus DNA by polymerase chain reaction

The tandem direct reiteration R1 in the varicella-zoster virus genome consists of two elements: units composed of 18 bp and those having 15 bp, both of whose numbers and types of combination patterns vary among strains. The variations of the R1 structure were examined in order to differentiate between the wild strains and a varicella vaccine (Oka) strain by the polymerase chain reaction using two primer sets. The results showed that the 31 wild strains were classified into nine patterns: the R1 structure consisted of a combination of 5 to 7 18-bp units and 8 to 12 15-bp units. The strain with a combination of 6 18-bp units and 10 15-bp units was found to be predominant in Japan, and the same pattern was found in the vaccine strain, so that differentiation between the vaccine strain and the wild strains in Japan merely by analysis of R1 in VZV genome is difficult.

Molecular Mechanisms of Chromosomal Rearrangement in Fungi

Both sexual and asexual fungi undergo chromosomal rearrangements, which are the main cause of karyotype variability among the populations. Different recombination processes can produce chromosomal reorganizations, both during mitosis and meiosis, but other mechanisms operate to limit the extent of the rearrangements; some of these mechanisms, such as the RIP (repeat-induced point mutations) of Neurospora crassa, have been well established for sexual fungi. In laboratory strains, treatments such as mutation and transformation enhance the appearance of chromosomal rearrangements. Different DNA sequences present in fungal genomes are able to promote these reorganizations; some of these sequences are involved in well-regulated processes (e.g., site-specific recombination) but most of them act simply as substrates for recombination events leading to DNA rearrangements. In Penicillium chrysogenum we have found that short specific DNA sequences are involved in tandem reiterations leading to amplification of the cluster of the penicillin biosynthesis genes. In some cases, specific chromosomal rearrangements have been associated with particular phenotypes (as occurs in adaptive-like mutants of Candida albicans and Candida stellatoidea), and they may play a role in genetic variability for environmental adaptation.

The DNA Sequence of the RK Strain of Human Herpesvirus 7

The complete DNA sequence of human herpesvirus-7 (HHV-7) strain RK was determined following direct cloning of virion DNA fragments into a sequencing vector. The sequence was compared with the previously published complete sequences of HHV-7 strain JI and human herpesvirus-6 (HHV-6) strain U1102. Despite a very close relationship between the two HHV-7 strains, differences are apparent in regions containing tandem reiterations, particularly in the “telomeric” reiterations located near the termini of the large direct repeat at the genome ends, and in a total of 179 additional positions distributed throughout the genome (i.e., about one nucleotide difference per kbp). This extent of divergence implies that the two strains arose from an ancestral virus several thousands of years ago. Differences that affect coding potential do not cluster in particular protein-coding regions, indicating that specific HHV-7 genes have not been measurably subject to unusual evolutionary pressures since divergence. Reassessments of genetic content indicated that the HHV-7 genome contains 84 different genes, whereas the HHV-6 genome contains 85. All HHV-7 genes but 1 have direct HHV-6 counterparts, and all but 2 HHV-6 genes have HHV-7 homologues. Sequence comparisons between HHV-7 and HHV-6 provided evidence that the protein-coding regions of 11 genes are expressed by splicing.

Herpes zoster in three healthy children immunized with varicella vaccine (Oka/Biken); the causative virus differed from vaccine strain on PCR analysis of the IV variable region (R5) and of a PstI-site region.

This study was undertaken to determine whether the causative virus was a vaccine-derived or wild-type virus when zoster occurred in healthy children immunized with varicella vaccine (Oka/Biken). The DNAs of clinical isolated strains and vaccine strain (Oka/Biken) were analyzed by the polymerase chain reaction with two sets of primers for the variable region IV (R5 tandem direct reiterations, TDR) and for the region with a PstI site in a middle portion of the long unique segment of the varicella zoster virus genome. Of six zoster patients after vaccination with Biken, three clinical isolates were examined and had two copies in R5 TDR and were PstI-site positive. Therefore, these strains were different from the vaccine-type strain (Oka/Biken), which had two copies in R5 TDR and was PstI-site-negative. The mean age of onset of zoster was 4 years. The mean age of vaccination was 25 months. The mean interval between vaccination and onset of zoster was 22 months. Hence the results indicate that the causative virus of zoster in healthy children immunized with varicella vaccine (Oka/Biken) was wild type and differed from the vaccine strain. Some vaccinees probably do not have protective immunity for a long time after immunization because the mean interval between vaccination and onset of zoster was 22 months.

Molecular genetics as a tool to remove bottlenecks in the biosynthesis of ?-lactam antibiotics

Several strains of Penicillium chrysogenum with different productions of penicillin were characterized at the molecular level in order to establish the basis of the increased penicillin production rates. The cluster of penicillin biosynthetic genes was located in an amplified genomic region of 57.9 kb in a high-producing strain (E1) and 106.5 kb in two strains (AS-P-78 and P2) producing moderately high levels of penicillin. This region was shown to be present in multiple tandemly repeated copies with a different copy number depending on the strain. The sequence TTTACA appeared at the junction points between repeats and at the borders of the amplified region in strains AS-P-78 and P2, while its reverse complementary TGTAAA was found in strain E1. The tandem reiteration and deletion appear to arise by site-specific recombination induced by mutagenic treatments. Finally, the relationship between glucose repression and pH regulation was studied in strain AS-P-78.

Cloning of a DNA repeat element from horse: DNA sequence and chromosomal localization.

A DNA repeat element, revealed initially by digestion of horse DNA with TaqI, was cloned and characterized by Southern and in situ hybridization studies and nucleotide sequencing. The clone, e4/1, consisted of 32 tandem reiteration of a unit repeat of 21-22 bp, and produced multilocus DNA fingerprinting profiles that were useful for parentage analysis in horses. The tandem repeat element was shown by in situ hybridization to be localized in the centromeres of the acrocentric but not metacentric classes of horse chromosomes.

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences.

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

Drosophila melanogaster does not share the telomeric repeat sequence of another invertebrate, Ascaris lumbricoides

The DNA at the chromosomal termini of all eukaryotes from which it has been isolated contains a characteristic sequence motif consisting of tandem arrays of a regular or irregular repeat unit. These terminal repeats are thought to be essential for the maintenance of the chromosome ends. The sequences of the terminal repeats of all vertebrates studied thus far are identical and are similar enough to those of higher plants and some protozoans to cross-hybridize. However, previous studies have not detected cross-hybridization between the DNA of Drosophila melanogaster and the terminal DNA sequences of any of several organisms tested. Recently, the first terminal DNA clone from a multicellular invertebrate, that of Ascaris lumbricoides, was reported also to consist of a tandem reiteration of a short sequence similar to those previously identified for other eukaryotes. Here I show that a probe for this sequence from A. lumbricoides fails to hybridize detectably to the DNA of D. melanogaster. Thus, in contrast to their conservation among vertebrates, the terminal chromosomal sequences appear not to be shared by all metazoan invertebrates.

Intrahelical pseudoknots and interhelical associations mediated by mispaired human minisatellite DNA sequences in vitro

The human minisatellite arrays, 33.6 and 33.15, consist of tandem reiterations of a 37-nucleotide (nt) and a 16-nt repeat unit sequence, respectively, both of which contain a majority of purine bases on one strand. Knot-like tertiary structures, which mapped to the cloned arrays, were observed by electron microscopy (EM) in homoduplex molecules produced by denaturation and reannealing in vitro. They result from a primary hybridization between misaligned repeat units of the array, forming a slipped-strand structure with staggered single-stranded DNA loops, followed by a secondary hybridization between repeat units in the two loops. Depending on the relative alignment of the loops when they hybridize, a particular form of intrahelical pseudoknot is produced. Theta-shaped, figure-of-eight, and bow-shaped structures were the most common conformational isomers observed in homoduplexes flattened into two dimensions during EM preparation. At the site of a bow-shaped structure, a conformation-dependent bend of approximately 60° between the flanking DNA segments is induced; the other conformations generally do not deflect the line of the main DNA axis. Paired loops, similar to the bowshaped structure, were apically situated in some supercoiled plasmids containing the 33.6 array. Both plasmids formed intermolecular associations, consisting of two (or more) homoduplex molecules held together at or immediately adjacent to a nexus which mapped to the minisatellite sequences. These associations might arise either by interhelical hybridization between arrays or by knot-like structures interfering with branch migration of χ-form Holliday junctions.

The adenosine-uridine binding factor recognizes the AU-rich elements of cytokine, lymphokine, and oncogene mRNAs.

Selective mRNA degradation is an important control point in the transient expression of a variety of mRNAs coding for growth regulators. A variety of labile mRNAs coding for lymphokines, cytokines, and oncogenes contain within their 3'-untranslated region an AU-rich region shown to destabilize these messages. We recently identified a cytosolic protein, adenosine-uridine binding factor (AUBF), which complexes with four tandem AUUUA reiterations of a synthetic RNA transcript. We now show that AUBF forms RNase T1-resistant band-shifted complexes with a variety of in vitro transcribed mRNAs including granulocyte-macrophage colony-stimulating factor, interferon-gamma, interleukin-3, c-fos, and v-myc. Formation of complexes was specifically inhibited by AUUUA containing RNA, but not by irrelevant RNA. After brief ultraviolet light-induced cross-linking, AUBF.RNA complexes with the exception of c-fos comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mutations within the AUUUA motifs demonstrate that both nucleotide sequence and secondary structure are important in AUBF.AUUUA RNA complex formation. Based upon these data, we suggest AUBF may interact with a variety of labile mRNAs with multiple AUUUA reiterations or single reiterations within an AU-rich 3'-untranslated region.

Replication of an adenovirus type 34 mutant dna containing tandem reiterations of the inverted terminal repeat

A mutant of human adenovirus type 34 (Ad34) has been isolated which contains DNA molecules with tandem reiterations of from two to eight copies of a 131-bp sequence within the right-sided inverted terminal repetition. Terminal heterogeneity was not eliminated by repeated plaque purifications indicating that the population of DNA molecules with various numbers of reiterations could rapidly evolve from the DNA of a single virus particle. These enlarged DNA molecules were capable of replication both in vivo and in vitro. The nucleotide sequence of the mutant Ad34 inverted terminal repetitions contained most of the essential features of the Ad origin of DNA replication. These features include the ATAATATACC sequence which is present between the highly conserved bases 9–18 in all human adenoviruses, as well as the consensus sequences for the binding of nuclear factor I and nuclear factor III. However, the reiterated sequences lacked a dG appropriately placed on the template strand to serve as a potential site for internal initiation. It appears that the rapid amplification of two to eight copies of the reiterated terminal sequences does not arise from internal initiation during replication but probably from homologous recombination.

Heterogeneous genomic amplification inStreptomyces glaucescens: Structure, location and junction sequence analysis

Certain chromosomal markers in Streptomyces glaucescens behave unstably, being lost at high frequency as a result of extensive genomic deletion. Additionally, mutant strains possessing such deletions frequently display intense DNA amplification. With the help of a wild-type cosmid library we investigated the structure of the amplified DNA sequences (ADS) and the corresponding wild-type amplifiable units of DNA (AUD). The reiterations were heterogeneous in location, copy number and sequences involved and originated predominantly from a single 100 kb region of the chromosome called the AUD locus. All strains bearing reiterations possessed associated deletions which terminated either close to or at the ADS. The termini of four AUDs were sequenced in order to gain more knowledge about these heterogeneous amplifications. In three of the four cases investigated small, interrupted homologies were found bordering the AUDs. With the help of orthogonal-field-alternation gel electrophoresis (OFAGE) we were able to visualize a tandem reiteration of more than 1,500 kb in length.