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PubMed1987

Upstream regulatory regions controlling the expression of the yeast maltase gene.

The expression of the maltase (MALS) and the maltose permease (MALT) genes in Saccharomyces species is coregulated at the transcriptional level; they are coordinately induced by maltose in the presence of a positively acting regulatory (MALR) gene and carbon catabolite repressed by glucose. We generated a series of deletions in the upstream region of the MAL6S gene to examine the regulatory elements in detail. The results showed that inducible expression by maltose was lost when the region between 320 and 380 base pairs upstream of the translation initiation codon was deleted. This region contained an imperfect inverted repeat sequence (-361 to -327) or four copies of short direct repeats that might serve as components of the upstream activation site (UASM) for the maltase gene, or both. When a stretch of T-rich sequence (-253 to -237) was deleted, the susceptibility of the maltase gene to carbon catabolite repression was affected.

Chromosome Deletion↗
PubMed2000

New transposable elements identified as insertions in rice transposon Tnr1.

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.

Base Sequence↗
PubMed1993

Classification and relationships of rice strains with AA genome by identification of transposable elements at nine loci.

We analyzed the presence of p-SINE1 members at five loci in the rice strains belonging to seven species with AA genome in the Oryza genus by the methods including polymerase chain reaction (PCR). Four p-SINE1 members (p-SINE1-r3, r4, r5 and r7) were present at the corresponding loci in all the strains examined. One member (p-SINE1-r6) was, however, not present at the corresponding locus in most of the African strains of O. glaberrima and O. barthii, but was in the other strains. The PCR-amplified fragments containing p-SINE1-r4 in many strains were found to be larger due to insertion of either one of two transposable elements, named Tnr2 and Ret1, within or near p-SINE1-r4, respectively: Tnr2 is 157 bp in length with terminal inverted repeat sequences of about 56 bp; Ret1 is only 13 bp in length with a T stretch at its end. Tnr2 was not present in the corresponding locus in all the strains belonging to O. sativa Japonica and in some strains of O. rufipogon and O. longistaminata, while Ret1 was present only in the two strains of O. longistaminata. These results and previous ones obtained from the analysis of the other two p-SINE1 members (p-SINE1-r1 and r2) in the Wx gene indicate that the elements, such as p-SINE1-r6, Tnr2, Ret1 and p-SINE1-r2, have been inserted into the respective loci during divergence of the rice species with AA genome. The patterns for the presence and absence of the transposable elements at the respective loci enabled us to classify the rice strains with AA genome into ten groups and to infer their relationships.

Base Sequence↗
PubMed1999

Template secondary structure promotes polymerase jumping during PCR amplification.

Pairs of primers flanking known miniTn10 transposon insertion sites were used to confirm the presence of the transposon in DNA isolated from Legionella pneumophila mutants. It was expected that the polymerase chain reaction products derived from the mutant template would be larger than those from the wild-type (WT) template due to the presence of the 1.8-kb transposon. Instead, it was observed that the mutant template yielded a product of almost the same size as that yielded by WT template. We present evidence to indicate that the aberrant product from the mutant template is a direct result of secondary structure of the template resulting from an inverted repeat sequence present in the miniTn10 transposon.

Base Sequence↗
PubMed2026

Comparative genomics and phylogenetic analysis of three Malvaceae species on the basis of chloroplast genomes.

INTRODUCTION: The Malvaceae family shows rich species diversity and has substantial economic and medicinal value. However, the frequent interspecific hybridization among members of this family has resulted in confused phylogenetic relationships among the groups, limiting the usefulness of traditional classification methods. METHODS: This study aimed to investigate the phylogenetic relationships among selected taxa of Malvaceae by evaluating 23 chloroplast (CP) genomes, including three newly assembled CP genomes. Among these three genomes, the CP genome of Hibiscus schizopetalus L. was reported for the first time, while the CP genomes of Alcea rosea L. and Hibiscus grewiifolius L., which have been deposited in NCBI, were re-analyzed here alongside newly generated data for comparative purposes. In addition, 20 downloaded CP genomes encompassing 13 genera were analyzed using SNPs in whole CP genomes data. RESULTS: The results showed that the genomes ranged from 160,403 to 161,978 base pairs in length and consisted of small single copies (SSCs) and large single copies (LSCs) separated by two inverted repeat sequences (IRs), forming a typical quadripartite circular structure. The entire genome sequence showed relative conservation across species in terms of structure, GC content, codon usage, and gene composition. The mutation sites were mainly located in the LSC and SSC regions, and the variability in the non-coding regions was higher than that in the coding regions. The nucleotide polymorphism (Pi) analysis identified the non-coding regions such as ndhF-rpl32 and psbZ-trnG as high variable hotspots. A maximum likelihood phylogenetic tree was constructed based on SNPs in whole CP genomes data. The phylogenetic analysis divided these 23 species into five highly supported clades. It also revealed a close sister-group relationship between Abelmoschus and Hibiscus species, suggesting that Hibiscus may have a separate lineage from okra species. DISCUSSION: In conclusion, the increasing availability of CP genome resources will enhance our understanding of the classification and evolutionary patterns of the Malvaceae family. The development of molecular markers will provide important molecular evidence for precise identification and classification revision of plants in this family.

Malvaceae↗
PubMed1990

Studies on Clostridium acetobutylicum glnA promoters and antisense RNA.

The Clostridium acetobutylicum glnA gene has two transcript start sites under the control of promoters p1 and p2. Initiation of transcription was regulated by nitrogen and a downstream region was implicated in the regulation of transcript initiation by nitrogen in Escherichia coli. Putative antisense RNA was produced from a single downstream transcript start site under the control of p3. An up-promoter mutation in p3 resulted in lower levels of glutamine synthetase (GS) activity. Putative antisense RNA had a role in down-regulating GS expression but was not involved in regulation by nitrogen. Deletion of downstream inverted repeat sequences resulted in very low levels of GS activity.

Base Sequence↗
PubMed1990

Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site.

Formation of relaxosomes is the first step in the initiation of transfer DNA replication during bacterial conjugation. This nucleoprotein complex contains all components capable of introducing a site- and strand-specific nick at a cognate transfer origin (oriT) on supercoiled plasmid DNA, thus providing the substrate for generation of the strand to be transferred. Characterization of the terminal nucleotides at the oriT nick site revealed that relaxation occurs by hydrolysis of a single phosphodiester bond between a 2'-deoxyguanosyl and a 2'-deoxycytidyl residue. The relaxation nick site and a 19-base pair invert repeat sequence that is recognized by asymmetric binding of the RP4 TraJ protein are interspaced by 8 base pairs. The nicking reaction results in covalent attachment of the RP4 TraI protein to the 5'-terminal 2'-deoxycytidyl residue of the cleaved strand. The arrangement of the TraJ binding site and the relaxation nick site on the same side of the DNA double helix suggests that protein-protein interactions between TraJ and TraI are a prerequisite for oriT specific nicking. In accordance with the current model of transfer DNA replication, the 3' end remains accessible for primer extension by DNA polymerase I, enabling replacement strand synthesis in the donor cell by a rolling circle-type mechanism.

Bacterial Proteins↗
PubMed1990

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA.

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.

Animals↗
PubMed1985

Bleomycin cleaves DNA depending on DNA primary, secondary, and tertiary structures.

The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol was investigated by using 3'- or 5'-end-labeled DNA containing the region of the bacteriophage G4 origin of complementary strand synthesis as substrates. Bleomycin cleaved single-stranded DNA substrates preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, in addition to the primary sequence specificity previously reported. DNA sequences preferentially cleaved in the double-stranded substrate were resistant when they lay outside the stem regions. These results suggest the formation of three predicted stem-and-loop structures and other possible secondary structures near the replication origin. Changes of the degree of bleomycin-induced DNA cleavage in a NaCl concentration between 0 and 50 mM suggest that a subtle change of ionic conditions within the double helix, or of DNA conformation, or of both, may occur at 0-50 mM NaCl. Bleomycin appears to be a useful reagent for analyzing secondary and tertiary structures of DNA.

Base Sequence↗
PubMed1989

DNA-binding properties of the Hin recombinase.

The recombinase of the Salmonella inversion system, Hin, mediates site-specific recombination between two 26 base pairs (bp) inverted repeat sequences (hixL and hixR) which flank a 993-bp DNA segment. We have investigated Hin recognition of, and association with, the hix recombination sites. Nuclease and chemical protection studies with linear and supercoiled DNA substrates demonstrate that Hin initially binds hixL and hixR independently of binding of the other protein components of the inversion system, Fis and HU. DNA-binding assays with mutant recombination sites and methylation interference experiments indicate that the critical bases for Hin recognition of its DNA-binding site are within an 8-bp sequence covering adjacent major and minor grooves of the DNA helix in each of the 12-bp half-sites of the hix recombination sites. The nature of the Hin-hix complexes in these binding studies and the results of gel filtration assays with purified Hin suggests that Hin binds the recombination sites as a dimer. The implications of the nature of the interactions of Hin with its recombination sites on the mechanism of the recombination reaction and on the novel features of DNA recognition by Hin are discussed.

DNA Nucleotidyltransferases↗
PubMed1985

Method for the preparation of plasmid DNA suitable for physicochemical measurements.

A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.

Animals↗
PubMed1995

Latent phenotype analysis of three deletion variants of herpes simplex virus type 1 (HSV-1) in mouse model.

Latency analysis of three herpes simplex virus type 1 (HSV-I) strain 17 syn+ deletion variants (1704, 1705 and 1706) showed that they established, maintained and reactivated from latency. The kinetics of reactivation of 1705 and 1706 were similar to the parent HSV-1, 17 syn+, in which reactivation occurred 5-6 days post-explanation, but 1704 reactivated with delayed kinetics i.e. on the 12th day post-explantation. Since 1704 has deleted both copies of the latency associated transcripts (LATs) promoter region and one copy of the LAT coding region in internal inverted repeat sequence of long region (IRL), it was concluded that the LATs play a part in latency reactivation of 1704 from dorsal root ganglia (DRG) of spinal cord in mouse model. Restoration of the deleted sequences in the variant 1704 by marker rescue with the wild type BamHI b fragment resulted in a wild type genotype. This virus was designated as 1704R. Latency studies of 1704R revealed that the rate and frequency of reactivation was intermediate between 17 syn+ and 1704, suggesting a secondary undetected mutation affecting latency phenotype. Isolation of 1704LP-, during the same marker rescue experiment in which both copies of promoter region of the LAT are deleted and reactivation of this virus from latency with delayed kinetics confirms that the LATs play a role in reactivation from latency.

Amino Acid Sequence↗
PubMed1999

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.

Adenoviridae↗
PubMed1985

Distinct characteristics of loop sequences of two Drosophila foldback transposable elements.

A few foldback (FB) transposable elements have, between their long terminal inverted repeats, central loop sequences which have been shown to be different from FB inverted repeat sequences. We have investigated loop sequences from two such FB elements by analyzing their genomic distribution and sequence conservation and, in particular, by determining if they are normally associated with FB elements. One of these FB loop sequences seems to be present in a few conserved copies found adjacent to FB inverted repeat sequences, suggesting that it represents an integral component of some FB elements. The other loop sequence is less well-conserved and not usually associated with FB inverted repeats. This sequence is a member of another family of transposable elements, the HB family, and was found inserted in an FB element only by chance. We compare the complete DNA sequences of two HB elements and examine the ends of four HB elements.

Animals↗
PubMed1979

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3.

The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(1). The sequence, 38 base pairs long, is as follows: 5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG..(Tn3) 3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC.. The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not the same. Furthermore, there is no homologous structure around the integration point of Tn3.

Base Sequence↗
PubMed1990

Identification of the site of recombination in the generation of the genome of DI particles of equine herpesvirus type 1.

Defective interfering particles (DIPs) are generated by serial, undiluted propagation of equine herpesvirus type 1 (EHV-1). DIP-rich preparations of EHV-1 mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. The defective genomes consist of reiterations of sequences from the left terminus (0.00 to 0.04 map units) of the long (L) region covalently linked to sequences from the inverted repeats (0.78 to 0.79, 0.83 to 0.87, 0.91 to 0.95, and 0.99 to 1.00 map units) of the short (S) region of the standard genome. We have identified and determined the nucleotide sequences of these segments of the standard genome as well as the component of the defective DNA that contains the site at which these two viral sequences recombined. Comparison of these sequences revealed that there is an 8-nucleotide sequence that is common to both the left terminus sequences and the inverted repeat sequences. These 8-nucleotide identical sequences are located at 3.25 kbp from the left terminus and at 9 kbp downstream of the L-S junction. The recombination between the left terminus and the inverted repeat sequences occurred at the site of homology and resulted in the generation of a novel open reading frame. The last 97 amino acids of an open reading frame of 469 amino acids encoded by sequences within the inverted repeats were replaced by a sequence of 68 amino acids encoded by a 204-bp sequence mapping at 0.023 map units. It will be of interest to determine whether this altered open reading frame, generated by recombination of sequences separated by more than 110,000 bp in the standard genome, plays a role in the varied outcomes of infection mediated by EHV-1 DIPs.

Amino Acid Sequence↗
PubMed1990

Sequence and organization of the genomic termini of equine herpesvirus type 1.

The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DR1-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the 'a' sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV 'a' sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DR1-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DR1-Uc (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/packaging elements of other herpesviruses are discussed.

Base Sequence↗
PubMed1989

Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome.

As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.

Animals↗