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PubMed1985

Characterization of the ribosomal RNA gene clusters in Halobacterium cutirubrum.

We present a comprehensive and detailed analysis of the structure and organization of a cloned ribosomal RNA gene cluster from the archaebacterial species Halobacterium cutirubrum. With the exception of a region in the middle of the 23 S rRNA gene, the DNA sequence of the entire gene cluster has been determined. The gene organization is similar to that found in typical eubacteria with the 16, 23, and 5 S genes occupying the proximal, middle, and distal positions, respectively. There appears to be no equivalent to the eucaryotic 5.8 S gene in H. cutirubrum. The cluster also contains two putative tRNA genes, an alanine tRNA gene in the 16-23 S intergenic space, and a cysteine tRNA gene distal to the 5 S rRNA gene. The 16 and 23 S rRNA genes are surrounded by long nearly perfect inverted repeat sequences which are presumably utilized along with other structural features of the RNA for the processing of 16 and 23 S rRNA from a large precursor transcript. The 5' sequence flanking the 16 S rRNA gene contains two imperfect copies, followed by three perfect copies of a bipartite direct-repeat unit. The sequence AAGTAA, believed to be an important component of the Halobacterium promotor, is present in the highly conserved portion of the direct repeat unit. In the 3' region flanking the 5 S rRNA gene there are sequences, a short inverted repeat followed by T5, and a G/C-rich region followed by an A/T-rich region, which may function in transcription termination. Genomic southern hybridization experiments clearly indicate that the ribosomal RNA genes are unique single-copy DNA in H. cutirubrum.

Bacteriophage lambda↗
PubMed2001

The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression.

It has been suggested that sequences located within the 5' noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete approximately 151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5' noncoding region but that is inactive when inverted or when placed in the 3' noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5' splice site positioned at +51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3' to this splice site. Analysis of total cellular gag and pol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave the pol 5' splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5' noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3' noncoding sequence. Neither the R element nor the introduced 5' intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in cis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3' noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847--4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.

Gene Expression Regulation, Viral↗
PubMed2000

Machinery to support genome segment inversion exists in a herpesvirus which does not naturally contain invertible elements.

In many herpesviruses, genome segments flanked by inverted repeats invert during DNA replication. It is not known whether this inversion is a consequence of an inherently recombinagenic replicative mechanism common to all herpesviruses or whether the replication enzymes of viruses with invertible segments have specifically evolved additional enzymatic activities to drive inversion. By artificially inserting a fusion of terminal sequences into the genome of a virus which normally lacks invertible elements (murine cytomegalovirus), we created a genome composed of long and short segments flanked by 1,359- and 543-bp inverted repeats. Analysis of genomic DNA from this virus revealed that inversion of both segments generates equimolar amounts of four isomers during the viral propagation necessary to produce DNA for analysis from a single viral particle. We conclude that a herpesvirus which naturally lacks invertible elements is able to support efficient segment inversion. Thus, the potential to invert is probably inherent in the replication machinery of all herpesviruses, irrespective of genome structure, and therefore genomes with invertible elements could have evolved simply by acquisition of inverted repeats and without concomitant evolution of enzymatic activities to mediate inversion. Furthermore, the recombinagenicity of herpesvirus DNA replication must have some importance independent of genome segment inversion.

Animals↗
PubMed1989

The expression of the superoxide dismutase gene in Halobacterium cutirubrum and Halobacterium volcanii.

The gene encoding the Mn-containing superoxide dismutase (SOD) from Halobacterium cutirubrum has been cloned and sequenced. The deduced amino acid sequence is homologous to the sequences of Fe and Mn SODs from eubacteria. The high degree of amino acid identity between the archaebacterial and eubacterial proteins suggests that a SOD gene may have been laterally transferred between eubacteria and archaebacteria sometime after the accumulation of atmospheric oxygen. Consensus elements of halobacterial promoters are found upstream of the coding region, however, the spacing between them and the transcription start site is greater than in other genes. Termination of transcription occurs in five consecutive T residues that are preceded by a GC-rich sequence that has short inverted repeats. In addition to the authentic SOD gene, H. cutirubrum also contains a putative pseudogene. The SOD levels and growth rates of H. cutirubrum and Halobacterium volcanii were tested in response to treatment by paraquat, an intracellular generator of superoxide. In H. volcanii the growth rate slowed, and SOD was strongly induced throughout prolonged treatment with paraquat. In H. cutirubrum the same effects were noticed initially, but after 48 h exposure to the drug, the growth rate increased and the SOD level decreased. Production of paraquat resistant mutants of H. cutirubrum may play a part in this process, however, some type of physiological adaptation is also probably required.

Amino Acid Sequence↗
PubMed1987

Gene regulation by steroid hormones.

The location, orientation, and structure of the hormone regulatory elements (HRE) in nine hormonally modulated genes is described. Based on analysis of the contact points between the glucocorticoid receptor (GR) and the DNA double helix within the HREs, a model for the interaction is proposed in which a dimer of the receptor in head-to-head orientation binds to the inverted symmetry element of the HRE. The relationship between the regulatory elements for glucocorticoids and progesterone in the long terminal repeat region (LTR) of mouse mammary tumor virus (MMTV), and in the promoter region of the chicken lysozyme gene, indicates that the recognition mechanism for both receptors is similar but not identical. Curiously, the hormone ligand is not an absolute requirement for the GR to bind its HRE, though it influences the kinetics of the interaction. Other possible functions of the hormone in vivo are discussed, as well as the molecular mechanism responsible for transcriptional regulation after receptor binding to the HRE.

Animals↗
PubMed1989

Viral transcripts in cells infected with defective interfering particles of equine herpesvirus type 1.

Equine herpesvirus type 1 (EHV-1) preparations enriched in defective interfering particles (DIPs) have previously been demonstrated to mediate the coestablishment of persistent infection and oncogenic transformation in primary hamster embryo fibroblasts. In this study, it was demonstrated that infection of a rabbit kidney (RK) cell line with EHV-1 DIP-enriched preparations also results in the establishment of persistent infection. Viral transcription was characterized in RK cells infected with DIP-enriched stocks and compared to viral transcription in RK cells infected with standard (STD) EHV-1. During the first 8 hr of infection with the DIP-enriched EHV-1 preparation, viral DNA sequences which are conserved in the DIP genome were predominantly expressed. Thus, these transcripts originate from DNA sequences that contain the components of the defective genome which originates from DNA sequences mapping at 0.00-0.04 of the Long region terminus and within two portions of the Short region inverted repeats (IR), 0.78-0.79 and 0.83-0.865 of the internal IRs and 0.99-1.00 and 0.915-0.95 of the terminal IRs. The overwhelming majority of viral transcripts that were synthesized in the DIP-enriched infections appeared to correspond to transcripts expressed in STD infection as assessed by Northern hybridization analysis but the synthesis of transcripts originating from sequences not conserved in the defective genome was significantly delayed. However, some high molecular weight RNA species that were synthesized in STD infections were not detected in DIP-enriched infections. Studies utilizing metabolic inhibitors indicated that viral transcription in DIP-enriched infections, like that of STD cytocidal infection, is regulated in an immediate early, early and late manner.

Blotting, Northern↗
PubMed1997

Sequence and transcriptional analysis of groES and groEL genes from the thermophilic bacterium Clostridium thermocellum.

The groESL operon from Clostridium thermocellum (Ct) has been isolated and sequenced, revealing two ORFs of 285 and 1626 nt, separated by 48 nt. The first ORF encoded a 94-aa 10.6-kDa GroES homologue; the second encoded a 541-aa polypeptide of 57.6 kDa, that exhibited 61% and 77% sequence identity with GroEL from Escherichia coli (Ec) and Clostridium acetobutylicum (Ca), respectively. A putative tsp, preceded by -10 and -35 consensus promoters, was identified upstream of groES. This was followed by an inverted repeat observed previously in bacterial heat shock genes. A 15-nt palindrome characteristic of a Rho-independent transcription terminator, was located downstream of groEL. The first nt of the groES translational start codon was preceded (7 nt) by a putative RBS (AGGAGG); a second RBS sequence was located 8 nt upstream of the groEL start. Production of GroE homologues by Ct was constitutive, but was enhanced significantly during a temperature upshift from 60 degrees C to 70 degrees C. The Ct GroEL, expressed in Ec as a fusion protein with GST, was purified, free of contaminating Ec GroEL.

Blotting, Northern↗
PubMed2002

Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium.

Amino Acid Sequence↗
PubMed1988

Bidirectional transfer from a 24 bp border repeat of Agrobacterium tumefaciens.

T-region transfer from wild-type Agrobacterium strains is thought to be an orientated process, starting at the right border repeat and terminating at the left border repeat of the T-region. Here we demonstrate that a right border repeat in the inverted orientation relative to the onc-genes can also mediate transfer of the T-region to the plant cell, although with lower efficiency as a border repeat in the native orientation. Transfer mediated by an inverted right border repeat is stimulated by the presence of the T-region transfer enhancer. Similar single stranded molecules, comprising the bottom strand of the T-DNA, were isolated from acetosyringone induced bacteria, irrespective of the orientation of the right border. These findings show that border repeats work bidirectionally to some extent.

Cloning, Molecular↗
PubMed2000

A novel family of retrotransposons in Xenopus with a developmentally regulated expression.

SUMMARY: We have obtained a novel family of LTR-retrotransposons in Xenopus laevis, named Xretpos, from cDNA and genomic clones. Its long terminal repeats (LTRs) can be subdivided into U3, R, and U3 to U5 region, and are bounded by 6 bp inverted repeats. Xretpos contains primer binding site and polypurine tract, and multiple copies of Xretpos-related element are present in the genome. A long open reading frame (ORF) encodes the CCHC motif conserved in retroviral gag proteins and leucine zipper motif capable of forming the coiled-coil. However, no amino acid homology to usually conserved retroviral pol gene was revealed. We report that in vitro synthesized Xretpos complementary RNAs are translated to produce a predicted size of protein. We also show that zygotically activated Xretpos transcripts are restricted to ventro-posterior specific regions and induced by UV-irradiation and BMP-4 overexpression in cycloheximide-dependent way. genesis 26:198-207, 2000.

Amino Acid Sequence↗
PubMed1979

Sequence of 1019 nucleotides encompassing one of the inverted repeats from the yeast 2 micrometer plasmid.

A sequence of 1019 nucleotides encompassing one of the 600 base inverted repeats and non-repeated flanking regions has been determined in the type A yeast 2 micrometers plasmid cloned in pMB9. Methods are described for applying the Maxam-Gilbert sequencing procedure to DNA fragments labelled at the 3'-end using a T4-polymerase exchange/repair reaction and for sequencing 5'-end labelled fragments using dideoxy-nucleotides as chain terminators in the presence of E. coli DNA polymerase (nach Klenow). A notable feature of the sequence is its unusual content of symmetry elements. In one region of 140 nucleotides, 137 are involved in a complex arrangement of direct and inverted repeats linked by palindromic sequences.

Base Sequence↗
PubMed1991

The molecular biology of swinepox virus. I. A characterization of the viral DNA.

Swinepox virus (SPV), the prototype member of the Suipoxvirus genus, is uncharacterized at the molecular level. We have analyzed the DNA of SPV and demonstrate that the genome is 175 kb in size and like the more commonly studied Orthopoxvirus, Avipoxvirus, and Leporipoxvirus genera, is terminally cross-linked and contains inverted terminal repetitions (ITRs). In addition, the ITRs are unstable, probably due to the presence of a variable number of direct repeats of approximately 70 bp in length. Restriction enzyme cleavage maps for the enzymes HindIII, AvaI, HaeII, KpnI, BglI, SalI, and XhoI are also presented.

Animals↗
PubMed1997

[Structural features of the modified BARE-retroelement in the barley (Hordeum vulgare L.) genome].

The primary structure of the 4.2-kb BamHI-fragment occurring abundantly in the genome of barley Hordeum vulgare was determined. By means of computer analysis, considerable homology was found between this fragment and the copia-like BARE-l retrotransposon studied earlier. A unique distinction of the BamHI fragment is its symmetrical structure caused by the presence of two mutually inverted parts, each of which is homologous to a long region of BARE-l including a 5'-LTR (long terminal repeat) and the adjacent leader sequence. No sequences homologous to the coding domains of BARE-l were revealed in the fragment. However, potentially functional signals were found: TATA boxes and primer-binding sites (PBS) exhibiting statistically significant homology to the corresponding regulatory signals of known retroelements. Thus, we have revealed and characterized a repeated element of the H. vulgare genome that is a markedly modified derivative of the BARE family of retrotransposons of this genome.

Base Sequence↗
PubMed1984

Bovine leukemia virus: unique structural features of its long terminal repeats and its evolutionary relationship to human T-cell leukemia virus.

The nucleotide sequence of the long terminal repeat (LTR) of bovine leukemia virus, a unique oncogenic retrovirus of cattle, was determined. The LTR consisted of 530 base pairs (bp) with an inverted repeat of 6 bp at its 5' and 3' ends, flanked by a direct repeat of 6 bp of host cell origin. A tRNAPro binding site for minus-strand DNA synthesis followed the 5' LTR. The U3 region contained putative transcriptional promoters, "CAT" box and "TATA" box, but they had peculiar sequences (C-C-A-A-C-T and G-A-T-A-A-A-T). The U3 region also contained a potential enhancer element, whose sequence partially resembled those of other viral and cellular, especially of immunoglobulin, enhancers. The most striking structural feature of the LTR was an exceptionally long R region (228 bp), which separated a poly(A) addition signal (A-A-T-A-A-A) from a poly(A) site as far apart as 260 bp. The long R region was suggested to form a large stable hairpin structure on a nascent RNA chain, making the two transcription termination signals close together and thus ensuring normal termination of the chain. This structural feature of the bovine leukemia virus LTR was analogous to that of human T-cell leukemia virus LTR and, in fact, slight sequence homology (at most 50%) was observed between the R regions of these two retroviruses, indicating their evolutionary relationship. The unique structural feature of bovine leukemia virus and human T-cell leukemia virus LTRs may thus bear some relation to the biological features commonly shared by these retroviruses.

Base Sequence↗
PubMed1980

Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration.

Closed circular Moloney murine leukemia virus (M-MuLV) DNA was prepared from recently infected cells and cloned in a lambda vector. Four classes of cloned M-MuLV inserts were found: Class I, full length 8.8-kilobase (kb) inserts with two tandem long terminal repeats (LTRs) of 600 base pairs; class 2, 8.2-kb inserts with a single copy of a LTR; class 3, M-MuLV DNA inserts with various portions deleted; and class 4, an 8.8-kb insert with an internal sequence inversion. Determination of nucleotide sequence at the junction between the two LTRs from a class 1 insert suggested that circularization occurred by blunt-end ligation of an 8.8-kb linear DNA. The class 4 molecule had an inversion that was flanked by inverted LTRs, each of which had lost two terminal base pairs at the inversion end points. Also, four base pairs that were present only once in standard M-MuLV DNA were duplicated at either end of the inversion. This molecule was interpreted as resulting from an integrative inversion in which M-MuLV DNA has integrated into itself. Its analysis thus provided explicit information concerning the mechanism by which retrovirus DNA integrates into host cell DNA. Models of retrovirus integration based on bacterial DNA transposition mechanisms are proposed.

Bacteriophage lambda↗
PubMed1993

Isolation and characterization of a linear plasmid from the entomopathogenic fungus Ascosphaera apis.

An extrachromosomal DNA plasmid was isolated from both mating types of the entomopathogenic fungus Ascosphaera apis and named pAaL. The subculture in which pAaL was first identified originated from mummified honey bee larvae from an apiary in Wyoming. Very similar, homologous plasmids were found in 9 out of 10 of isolates collected from diverse geographic locations. The plasmid is found inside the mitochondria, has the same buoyant density as mitochondrial DNA in bisbenzamide--CsCl gradients, and does not contain sequences homologous to either mitochondrial DNA or genomic DNA. The plasmid is linear, double-stranded, of 12 kilobase pair, and has a higher copy number than the mitochondrial DNA. Endonuclease and exonuclease digestions suggest that an inverted repeat is probably present at each terminus and that pAaL has two blocked 5' ends, probably due to the presence of terminal binding proteins. Restriction site data showed pAaL to be AT-rich. There were no apparent differences in the growth rate, culture appearance, and reproductive cycle of plasmid-bearing or plasmid-free A. apis isolates. pAaL was stably inherited in the plasmid-free strains, but it was lost in the progeny of crosses and reciprocal crosses between the plasmid-free strain and plasmid-bearing strains. The biological function of this plasmid has not yet been determined.

Ascomycota↗
PubMed1999

A single amino acid, outside the AlcR zinc binuclear cluster, is involved in DNA binding and in transcriptional regulation of the alc genes in Aspergillus nidulans.

In Aspergillus nidulans, the transcriptional activator AlcR mediates specific induction of a number of alc genes. The AlcR DNA-binding domain is a zinc binuclear cluster that differs from the other members of the Zn2Cys6 family in several respects. Of these, the most remarkable is its ability to bind in vitro as a monomer to single sites, whereas only repeated sites (direct or inverted) are necessary and functional in vivo. Deletion of the first five amino acids (following the N-terminal methionine) upstream of the AlcR zinc cluster or mutation of a single residue, Arg-6, impairs the AlcR in vitro binding mainly to symmetrical sites. In vivo, the same mutations result in the inability of A. nidulans to grow on ethanol. The alc- phenotype results from a drastic decrease in activation of its own transcription and, in addition, that of the two structural genes, alcA and aldA, required for ethanol oxidation. This defect seems to be correlated to the inability of the Arg-6 AlcR mutant protein to bind to AlcR palindrome targets, which are essential in the three alc promoters. AlcR shows a unique pattern of binding and of transactivation among the Zn2Cys6 family.

Arginine↗
PubMed2000

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA.

Approximately 6000 specific DNA deletion events occur during development of the somatic macro-nucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis -acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetra-hymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis -acting sequences outside the deleted DNA.

Animals↗