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PubMed1988

Q-mediated late gene transcription of bacteriophage lambda: RNA start point and RNase III processing sites in vivo.

The location of the RNA start point of in vivo Q-activated late gene RNA of bacteriophage lambda has been determined to be identical to the start point of in vitro 6 S RNA. The 6 S RNA is made early in infection and is efficiently antiterminated by Q. Two RNase III cut sites are located within Q-dependent RNA sequences, 209 and 270 bp from the beginning of the late transcript, and lie on the stem of an inverted repeat which has features in common with previously described RNase III processing sites. This is the third example of RNase III cut sites immediately downstream of transcription termination points in lambda, the others being antiterminated N gene mRNA and int gene mRNA.

Bacteriophage lambda↗
PubMed1995

Mating variation by DNA inversions of shufflon in plasmid R64.

Gene organization of the 54-kb transfer region of IncI1 plasmid R64 was deduced from the DNA sequence. Forty-eight ORFs were found in this region. A unique DNA rearrangement designated shufflon is located at the downstream region of an operon responsible for synthesis of thin pilus. The shufflon of R64 consists of four DNA segments, designated as A, B, C, and D, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination mediated by the product of the rci gene between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of seven C-terminal segments of the pilV genes. The products of pilV genes were shown to be components of thin pilus which was required for liquid mating. Seven R64 derivatives where the pilV genes were fixed in the seven C-terminal segments were constructed and their transfer frequencies in liquid mating were measured using various bacterial strains as recipients. Transfer frequencies of R64 in liquid mating strongly depended on the combination of C-terminal segments of the pilV genes in donor cells and bacterial strains of recipient cells, suggesting that the shufflon determines the recipient specificity in liquid mating of plasmid R64.

Biophysical Phenomena↗
PubMed1983

Sequence and hairpin structure of an inverted repeat series at termini of the Physarum extrachromosomal rDNA molecule.

The termini of the 61 kb palindromic rDNA molecules of Physarum polycephalum possess a series of multiple inverted repeats in which are located specific single-strand gaps and tightly attached protein. After treating rDNA with S1 nuclease, we have cloned several 5 kb Eco RI terminal restriction fragments. Sequencing of more than 800 nucleotides from the end of one such clone reveals the presence of six to ten tandemly repeated units averaging 140 +/- 4 bp in length and flanked by Hae III sites. Each 140 nucleotide repeat unit can form thermodynamically stable hairpin structures based on complex internal palindromic components. When the specific gap sequence CCCTA is present, it is located near the apex of a hairpin component. These secondary structures are formed in growing plasmodia, as seen in electron micrographs of native rDNA molecules, which also reveal apparent recombination forms involving rDNA ends and noncontiguous DNA segments. Recombination initiated at terminal single-strand hairpin loops can result in genetic exchange of ribosomal gene sequences and can lead to completion of 5' nucleotide sequences at ends of newly replicated rDNA molecules.

Base Sequence↗
PubMed2003

Molecular analysis of Fiji disease Fijivirus genome segments 1 and 3.

Fiji disease fijivirus (FDV) genomic segments 1 (S1) and 3 (S3) were completely sequenced. FDV S1 comprised 4,532 nt and was predicted to encode a 170.6 kDa protein. FDV S3 comprised 3,623 nt and was predicted to encode a 135.5 kDa protein. The terminal sequences of S1 and S3 were 5' AAGUUUUU......CAGCUAGCGUC 3' and 5' AAGUUUUU......CAGCAGAUGUC 3', respectively, and located immediately adjacent to these sequences were 12 bp imperfect inverted repeats. The predicted translation product of FDV S1 showed highest similarity to Rice black-streaked dwarf virus (RBSDV) S1 and is thought to encode the viral RNA-dependent RNA polymerase (RdRp). The predicted translation product of FDV S3 was found to be most similar to RBSDV S4 which is thought to encode the 'B-spike' protein. The FDV sequence contained an ATP/GTP binding motif and a leucine zipper motif, but these motifs were not found in the RBSDV sequence. Phylogenetic analysis based on the amino acid sequences of the RdRp of FDV S1 and other reoviruses revealed that the fijiviruses form a cluster close to the oryzaviruses. The RdRp sequences were grouped into genera that were consistent with the current reovirus classification scheme that is based on physico-chemical and biological properties.

Amino Acid Sequence↗
PubMed1996

Construction of human factor IX expression vectors in retroviral vector frames optimized for muscle cells.

Development of a highly refined human factor IX (hFIX) expression vector system is critical for establishing a durable hemophilia B gene therapy. Here we report construction of a series of retroviral vectors and identification of an optimal basic structure and components for expressing hFIX in skeletal muscle cells. These vectors, which are derived from Moloney murine leukemia virus (MoMLV) with its enhancer sequence in the 3' long terminal repeat (LTR) deleted, contained internal hFIX expression units inserted in forward configuration without or with a viral vector intron sequence (pdL or pdLIn vector frame, respectively) or in inverted configuration without a viral vector intron sequence (pdLi frame). Internal expression units contained a hFIX cDNA or hFIX minigene (hIXm1 or hIXm2) derived from the hFIX cDNA by insertion of a shortened first intron sequence of the hFIX gene. Regardless of the promoter and vector frame used, both hIXm1 and hIXm2 gave 10- to 14-fold higher hFIX expression compared to those with hFIX cDNA. Internal hFIX transcriptional control units of these vectors were composed of various promoters linked with or without the muscle creatine kinase enhancer (Me) sequence. Promoters tested included those of alpha-actin (alpha A775), beta-actin (beta A280), cytochrome oxidase (CO1250 and CO650), myogenin (Mg1031 and Mg353), and Rous sarcoma virus (RSV). beta A200, which was derived from beta A280 by eliminating potential polyadenylation sites, was also tested. As extensively examined with the myogenin promoter, presence of one or multiple copies of Me in the vectors elevated the expression activity in myotubes by 4.5- to 19-fold over those without Me, but not significantly in myoblasts. Similar enhancements in expression activity with Me were also observed with other promoters, except those of RSV and CO. The latter two showed only modest enhancements in the presence of Me. As assayed with myotubes in culture, the general order of hFIX expression activity of various promoters with four copies of Me in the three different vector frames was beta A280 approximately beta A200 > Mg353 > Mg1031 approximately RSV approximately CO650 approximately alpha A775 > CO1250. One exception was that CO650 showed significantly less activity in pdLi-type vectors than in the pdLIn vectors. Based on the systematic analyses of various structural components, a group of pdLi vectors consisting of beta A200, two to four copies of Me, and hIXm2 was identified to have the optimal basic vector structure to be used in retrovirus for hFIX expression in differentiated skeletal muscle cells. The present studies provide the critical first step for establishing a highly refined hemophilia B gene therapy based on skeletal muscle-targeted hFIX gene transfer.

Animals↗
PubMed2000

Heat shock proteins and inflammatory acne vulgaris: molecular cloning, overexpression and purification of a propionibacterium acnes GroEL and DnaK homologue.

Propionibacterium acnes is associated with inflammatory acne. The genes encoding two putative mediators of inflammation, the heat shock proteins GroEL and DnaK, were cloned from this organism and sequenced. groEL and dnaK encode proteins of 56.8 and 66.4 kDa, respectively, which show a high degree of homology (>75% similarity) to the GroEL and DnaK proteins of mycobacteria and streptomycetes. The promoter regions of both genes contain inverted repeat sequences believed to be involved in the transcriptional regulation of heat shock genes. Recombinant P. acnes GroEL and DnaK were overexpressed in Escherichia coli with C-terminal histidine tags. The recombinant proteins were purified from E. coli by metal affinity chromatography. These proteins will now be used in immunological investigations to determine their role in inflammatory acne.

Acne Vulgaris↗
PubMed1986

Nucleotide sequence analysis of squirrel monkey retrovirus reveals a novel primer-binding site for tRNALys1,2.

Nucleotide sequences of a DNA fragment containing the long terminal repeat (LTR) of squirrel monkey retrovirus (SMRV) were determined. Sequence analysis showed that the SMRV LTR is 456 base pairs (bp) long and is bounded by 2-bp inverted repeats. Within the U3 region, there are two 43-bp repeats and two 42-bp repeats which are homologous to each other. These repeats are likely to provide enhancer activities commonly observed in other enhancer sequences. Following the repeats are transcriptional regulatory sequences including a CAT box, a Goldberg-Hogness box, and a polyadenylation signal, all positioned within the U3 region of SMRV LTR. A 22-nucleotide sequence immediately downstream from the LTR was found to be complementary to tRNALys1,2, suggesting that tRNALys1,2 serves as the primer for the reverse transcription of SMRV viral RNA.

Animals↗
PubMed1977

Deletions affecting the transposition of an antibiotic resistance gene.

The structural gene for plasmid-mediated ampicillin resistance resides upon a 3.2 X 10(6) dalton transposable sequence (TnA) flanked by short inverted repeated sequences that accompany its insertion. TnA was transposed to pMB8, a 1.8 X 10(6) dalton derivative of the colicingenic plasmid ColE1. Random deletions were introduced in the resultant 5 X 10(6) dalton recombinant plasmid by a combination of nuclease treatments in vitro. From this set of deletions a subset was isolated that contained deletions affecting the transposition of TnA. The deletions were mapped by digestion with restriction nucleases and electron microscopic analysis of DNA hetero-duplexes and were found to include one of the inverted repeated sequences or lie in the central portion of TnA. Complementation experiments were attempted between these plasmids and another compatible plasmid carrying a deletion in TnA that abolished its ampicillin resistance. The results of the deletion data indicate that approximately 2 X 10(6) daltons of TnA is required for transposition; the complementation experiments suggest that the terminal inverted repetition and the central region of TnA play different essential roles in TnA transposition.

Crosses, Genetic↗
PubMed2006

Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences.

The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.

Base Sequence↗
PubMed2003

Divergence and transcriptional analysis of the division cell wall (dcw) gene cluster in Neisseria spp.

Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3' of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox-like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5' of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw-associated CREE, the first demonstration of active promoter generation at the 5' end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components.

Bacterial Proteins↗
PubMed1994

DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64.

The shufflon is a unique DNA rearrangement found in plasmid R64. R64 shufflon consists of four DNA segments, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any inverted repeats results in a complex DNA rearrangement where four DNA segments invert independently or in groups. The shufflon is a biological switch to select one of the seven C-terminal segments of the pilV gene. To examine the biological significance of the shufflon, R64 derivatives, where the pilV gene was fixed in seven C-terminal segments, were constructed and used as donor cells for liquid mating. The transfer frequencies depended markedly on the combinations of recipient bacterial strains and C-terminal segments of the pilV gene in donor cells, indicating that the shufflon determines the recipient specificity in liquid mating of R64. The products of the pilV genes were found to be a component of thin pili produced by R64.

Conjugation, Genetic↗
PubMed1998

The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability.

A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract. To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3' UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs.

Adenosine Triphosphatases↗
PubMed1982

Genetic and molecular characterization of Tn21, a multiple resistance transposon from R100.1.

Tge transposon Tn21 has been transposed from R100.1 to plasmid pACYC184 and, from the resulting recombinants, to plasmid R388. The sites of insertion and the orientation of the element in several pACYC184::Tn21 recombinants have been examined. Restriction enzyme analysis of these recombinants has resulted in a detailed map of Tn21; this is compared with the published maps of the relevant part of R100.1. Heteroduplex analysis has shown short inverted repeat sequences at the ends of the element. With various in vitro-generated deletion mutants of Tn21, the internal gene necessary for transposition (tnpA) was localized within the terminal 4.3 kilobases of the right-hand end of the element. Genetic analysis of transposition of Tn21 suggests that the process proceeds via cointegrates. Since the end products of transposition are simple recombinants of the element and the recipient replicon, Tn21 must contain a gene that codes for a resolvase type of activity (tnpR gene).

Conjugation, Genetic↗
PubMed1988

Wolffish antifreeze protein genes are primarily organized as tandem repeats that each contain two genes in inverted orientation.

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.

Amino Acid Sequence↗
PubMed1986

[Structure of long terminal repeats of transcriptionally active and inactive copies of the mobile dispersed gene MDG3 in Drosophila melanogaster].

The nucleotide sequences of long terminal repeats (LTRs) and adjacent regions are determined in the transcribed and non-transcribed variants of a mobile dispersed genetic element MDG3. MDG3 is similar to other mdg elements. A 4 bp duplication of the host DNA is generated upon its integration. MDG3 is flanked by a 5 bp inverted repeat. The length of the LTRs varies in different MDG3 variants, the difference being connected mainly with duplications of certain sequences in U3 and R regions. The copies with 267 bp LTR are the most abundant ones and perfectly conservative in their primary structure. They are transcribed in 67J25D cell culture and are not transcribed in Kc cell line, where another variant with LTR length 293 bp is transcriptionally active. S1-mapping of transcription initiation and termination sites has demonstrated that in both MDG3 variants they are situated in the same positions, and the LTR itself may be subdivided into U3, R and U5 regions, like retroviral LTRs. Possible factors involved in the regulation of mdg transcription are discussed.

Animals↗
PubMed1985

Orthopoxvirus DNA: a comparison of restriction profiles and maps.

Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus-cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAs of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus.

Animals↗
PubMed2005

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo.

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.

DNA, Viral↗
PubMed1995

Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenzae Rd genome.

The naturally transformable, Gram-negative bacterium Haemophilus influenzae Rd preferentially takes up DNA of its own species by recognizing a 9-base pair sequence, 5'-AAGTGCGGT, carried in multiple copies in its chromosome. With the availability of the complete genome sequence, 1465 copies of the 9-base pair uptake site have been identified. Alignment of these sites unexpectedly reveals an extended consensus region of 29 base pairs containing the core 9-base pair region and two downstream 6-base pair A/T-rich regions, each spaced about one helix turn apart. Seventeen percent of the sites are in inverted repeat pairs, many of which are located downstream to gene termini and are capable of forming stem-loop structures in messenger RNA that might function as signals for transcription termination.

Base Composition↗