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Insertion of tandem direct repeats consisting of avian leukosis virus LTR sequences into the inverted repeat region of Marek's disease virus type 1 DNA.

The BC-1 strain DNA of Marek's disease virus type 1 (MDV1) at high-passage in culture was found to contain tandem direct repeats of the complete long terminal repeat (LTR) sequence of avian leukosis virus RAV0 strain as the repeat unit within the short inverted repeats of the MDV1 DNA. Since the attenuated BC-1 strain grows well in cultured cells, the insertion site for retroviral DNA sequence within the short inverted repeat of MDV1 DNA is not essential for viral growth in culture.

Animals↗

Athila, a new retroelement from Arabidopsis thaliana.

An analysis of Arabidopsis thaliana heterochromatic regions allowed the identification of a new family of retroelements called Athila. These 10.5 kb elements, representing ca. 0.3% of the genome, present several features of retrotransposons and retroviruses. Athila elements are flanked by 1.5 kb long terminal repeats (LTR) that are themselves bounded by 5 bp perfect inverted repeats. These LTRs start and end with the retroviral consensus 5'TG...CA3' nucleotides. A putative tRNA-binding site and a polypurine tract are found adjacent to the 5' and 3' LTR respectively. The central domain is composed of two long open reading frames (ORFs) of 935 and 694 amino acids. Despite several indications of recent transposition activity, the translation of these ORFs failed to reveal significant homology with proteins associated to retrotransposition. We suggest that the Athila family could result from the transduction and dispersion of a cellular gene by a retrotransposon.

Amino Acid Sequence↗

Characterization and nucleotide sequence of a colicin-release gene in the hic region of plasmid ColE3-CA38.

Downstream from its colicin and immunity genes (col. imm), Escherichia coli plasmid ColE3-CA38 contains a 0.81-kb DNA segment, the hic region, which is required for high colicin production. Characterization of derived plasmids, carrying the col-imm operon but varying in the hic region, showed that the latter functions in lacuna production, colicin release, cell death, and lysis. The hic gene expression after induction was shown to be dependent on the col gene promoter. The nucleotide sequence of the 0.81-kb region was determined and the hic gene localized to its imm-distal portion following an open reading frame (ORF) with no known function. There are two overlapping ORFs in that portion of the sequence, one of which was identified as the hic gene by its partial homology to lysis gene H of CloDF13. The 3' half of the hic gene is non-essential and contains a terminator-like DNA sequence. Preceding the gene, there are also inverted repeats which may attenuate its transcription.

Amino Acid Sequence↗

Sequence analysis of the complete genome of rice black-streaked dwarf virus isolated from maize with rough dwarf disease.

The complete nucleotide sequences of 10 genomic segments (S1-S10) from an isolate of rice black-streaked dwarf virus causing rough dwarf disease on maize (RBSDV-Hbm) in China were determined, a total of 29,142 base pairs (bp). Each segment possessed the genus-specific termini with conserved nucleotide sequences of (+) 5'-AAGUUUUU......CAGCUNNNGUC-3' and a perfect or imperfect inverted repeat of seven to eleven nucleotides immediately adjacent to the terminal conserved sequence. While the coding strand of most RBSDV-Hbm segments contained one open reading frame (ORF), there were two non-overlapping ORFs in S7 and S9, and one small overlapping ORF downstream of the major ORF in S5. Homology comparisons suggest that S1 encodes a RNA-dependent RNA polymerase (RdRp), with 63.5% and 32.6% identity to the putative RdRp encoded by Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV), respectively. The proteins encoded by S2, S3, and S4 showed various degrees of similarity to those encoded by the corresponding segments of FDV or NLRV. In S5 and S6, low identities were found to those of FDV only, but not to NLRV. Sequence analyses showed that RBSDV-Hbm had the most similarities in the genome organizations and the coding assignments with a RBSDV isolated from rice in China, in which each pair of the corresponding segments shared sequence identities of 93.8-98.9% and 93.5-100% at nucleotide or amino acid levels, respectively. In addition, phylogenetic analyses suggested that RBSDV-Hbm had the closest evolutionary relationship to RBSDV in Fijivirus.

Conserved Sequence↗

Detection and cloning of new HTLV-related endogenous sequences in man.

Human T-cell leukemia virus (HTLV) type I-related endogenous sequences (HRES) have been cloned from a human genomic library. HRES-1/1 is present in DNA of all normal donors examined. By nucleotide sequence analysis, HRES-1/1 contains two potential open reading frames capable of encoding a p25 and a p15. A 684 bp flanking region 5' from the first ATG codon of p25 contains a TATA-box, a poly-adenylation signal, a putative tRNA primer binding site, and inverted repeats at locations which are typical of a retroviral long terminal repeat. Phylogenetic analysis suggests that HRES-1/1 entered the genome in primates, presumably as an exogenous retrovirus. From the deduced amino acid sequence of HRES-1/1 p25, residues 6-36 show a sequence homology of 32% and 39% to gag region segments of HTLV-I and HTLV-II, while residues 104-139 display a sequence homology of 33% and 28% to the gag regions of human immunodeficiency virus type 2 (HIV-2) and feline sarcoma virus (FSV), respectively. This suggests that the original exogenous virus infecting primate may be chimeric in structure. The HRES-1/1 genomic locus is transcriptionally active in lymphoid cells, melanoma cells, and embryonic tissues.

Amino Acid Sequence↗

Cloning and nucleic acid sequence of the Salmonella typhimurium pncB gene and structure of nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium, encoding nicotinate phosphoribosyltransferase (NAPRTase), was cloned on a 4.7-kb Sau3A fragment. The gene contains a 1,200-bp open reading frame coding for a 400-residue protein. Amino acid sequencing of the amino-terminal and two interior peptides of the purified protein confirmed the deduced sequence and revealed that the amino-terminal methionine residue was removed, giving a 399-residue mature protein of Mr 45,512. No signal sequence was observed in the predicted NAPRTase primary structure, suggesting that the enzyme is not periplasmic. The protein does not demonstrate clear sequence similarity to the other seven phosphoribosyltransferases of known primary structure and frustrates attempts to define a consensus 5-phosphoribosyl-1-pyrophosphate-binding region. The NAPRTase reaction is ATP stimulated, and the protein contains a carboxy-terminal sequence diagnostic of an ATP-binding site. An inverted repeat of the sequence TAAACAA observed in the proposed promoter region of pncB is also present in the promoter of nadA, which, like pncB, is also regulated by the NadR (NadI) repressor. The sequence may thus define an NadR repressor-binding site.

Amino Acid Sequence↗

[Studies on midecamycin 4"-O-propionyltransferase gene structure].

A BamHI-BamHI 8.0 kb DNA fragment which contains midecamycin propionyltransferase (mpt) gene was digested with different restriction enzymes and the restriction map was made. The mpt gene was localized in a EcoRI-EcoRI-PstI3.0 kb DNA fragment by Southern blot analysis using a 2.4 kb DNA fragment of the CarE gene as a probe. The 3.0 kb DNA fragment of mpt gene was cloned into E. coli/Streptomyces shuttle vector pWHM3 and a recombinant plasmid pWFPE was obtained. S. ambofaciens(pWFPE) and S. lividans(pWFPE) can convert endogenously synthesized or exogenously added spiramycin into 4"-O-propionylspiramycin, respectively. Sequence analysis of mpt gene demonstrated an open reading frame in the EcoRI-EcoRI-PstI3.0 kb DNA fragment, which starts with ATG and ends with TGA. Mpt gene encodes a product of 388 aa. G+C mol% of mpt is 68.0 and G+C mol% of 3rd codon position is 91.5. The putative product of mpt has a identity of 67.6% and a similarity of 86.4% with CarE product. A consensus RBS GAGGT in the 6bp upstream from ATG and a promoter region were found. An inverted repeat sequence in the downstream from TGA acts as transcriptional terminator.

Acyltransferases↗

Activity of the distal positive element of the peripherin gene is dependent on proteins binding to an Ets-like recognition site and a novel inverted repeat site.

The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at -173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (-2660 to -2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E. (1992) Mol. Cell. Biol. 12,2501-2513). Here we define a distal positive element (DPE, -2445 to -2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron-specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements.

Animals↗

Hybridization selection and cell-free translation of mRNA's encoded within the inverted terminal repetition of the vaccinia virus genome.

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.

Bacteriophage lambda↗

Analysis of the monkeypox virus genome.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

Animals↗

Visualization of an inverted terminal repetition in vaccinia virus DNA.

An inverted terminal repetition was observed in DNA molecules extracted from vaccinia virus. The repeated sequence was visualized by (i) nicking the hairpin loops present of the ends of vaccinia virus DNA, (ii) separating the strands of DNA by alkali denaturation, (iii) allowing the single strands to self-anneal, and (iv) examining the DNA with an electron microscope. Single-stranded circular molecules, each of which contained a duplex projection (3.54 +/- 0.12 micron) representing the terminal repetition, readily formed. Similar size projections were also seen in heteroduplex structures formed by crosshybridization of the separated strands of the two terminal HindIII restriction fragments. Based on contour length measurements and the electrophoretic mobility of the isolated inverted terminal repetition, a molecular weight of approximately 6.9 X 10(6), equivalent to about 10,500 nucleotide base pairs, was estimated. Evidence was obtained from DNA-RNA hybridization studies that the terminal repetition is transcribed.

Base Sequence↗

Proviral insertions within the int-2 gene can generate multiple anomalous transcripts but leave the protein-coding domain intact.

We examined the effects of mouse mammary tumor virus integration on the multiple RNA transcripts expressed from the int-2 proto-oncogene in virally induced breast tumors. Proviral insertion either upstream or downstream of the gene could simultaneously activate transcription from three dissimilar int-2 promoters. In some tumors, the activating provirus lies within the transcription unit and disrupts the structures of the various RNAs. Insertions in the 5' region of the gene had complex effects depending on the orientation and position of the provirus relative to the three promoters and intron-exon boundaries. RNase protection experiments identified transcripts initiated in the viral long terminal repeat, at normal and cryptic sites in the int-2 sequences, and from cryptic promoters in an inverted provirus. AT the 3' end, insertions occurred within the untranslated trailer and provided alternative termination signals that substituted for one or both of the normal the poly(A) addition sites. However, in no instance, of the 20 tumors analyzed in detail, did a provirus perturb the presumed open reading frame of the gene. These data strongly implicate the normal product of the int-2 gene, which is related to the fibroblast growth factor family, as a contributory factor in virally induced mammary tumors.

Animals↗

Nucleotide sequence analysis of the long terminal repeat of integrated bovine leukemia provirus DNA and of adjacent viral and host sequences.

The nucleotide sequence of the 3' long terminal repeat and adjacent viral and host sequences was determined for a bovine leukemia provirus cloned from a bovine tumor. The long terminal repeat was found to comprise 535 nucleotides and to harbor at both ends an imperfect inverted repeat of 7 bases. Promoter-like sequences (Hogness box and CAT box), an mRNA capping site, and a core enhancer-related sequence were tentatively located. No kinship was detected between this bovine leukemia proviral fragment and other retroviral long terminal repeats, including that of human T-cell leukemia virus.

Animals↗

Properties of Streptomyces phage SLE111.

Phage SLE111 infecting Streptomyces levoris 1331 was morphological different from most actinophages described and yielded very high titres (10(11) p.f.u/ml) after lytic growth. Typical morphological changes of infected hyphae were observed by phase contrast and electron microscopy of ultrathin sections. The double-stranded, linear DNA (44.2 +/- 1.3 kb) was characterized, according to electron microscopic analysis, by absence of cohesive ends, of terminal redundance, and circular permutation. A stem-loop structure containing inverted repeats of about 200 bp was identified by electron microscopy in two alternative positions in the SLE111 genome.

Bacteriophages↗

Dynamic balance of CRISPR-Cas immunity and resistance plasmid anti-immunity mediated by a bifunctional protein AcrIE10.

Despite targeting by CRISPR-Cas system, antimicrobial resistance plasmids are prevalent in clinical isolates of carbapenem-resistant Klebsiella pneumoniae which represent a major public health threat. A stable co-existence of plasmids and CRISPR-Cas systems is mediated by anti-CRISPR (Acr) proteins. Here, we report that previously identified AcrIE10 encoded by a resistance plasmid combines two functions: it inhibits CRISPR immunity by directly binding Cas7* subunit through its Acr domain, and acts as an Acr-associated (Aca) protein that self-represses the transcription of Acr locus. AcrIE10 is an example of an Aca protein that utilizes N-terminal ribbon-helix-helix (RHH) domain to specifically recognize the inverted repeat (IR) region in its own promoter. Crucially, a dimerization of AcrIE10 dimers is required for the effective binding to the IR and self-repression, while stoichiometry-dependent interaction with Cas7* facilitates transition to de-repressed state. These findings elucidate molecular mechanisms by which AcrIE10 operates as a dual functionAcr-Aca protein to achieve a delicate balance between host CRISPR-Cas immunity and plasmid anti-defense.

Klebsiella pneumoniae↗

Isolation of point mutations in bacteriophage Mu attachment regions cloned in a lambda::mini-Mu phage.

Twenty-one derivatives of a lambda::mini-Mu phage containing point mutations in the Mu attachment regions were isolated after mutD mutagenesis and selection for relief from Mu-specific replicative interference of lambda growth. DNA sequence analysis revealed that the single left-end mutant had suffered a T----C transition at position 1 of the Mu sequence, while the remaining 20 right-end mutants contained single base-pair insertions or deletions within the terminal 19 base pairs. A genetic assay showed that the right-end mutations revealed by sequencing were necessary for relief of the replicative inhibition of lambda growth. The properties of these mutants suggest that the terminal 2-base-pair and subterminal 8-base-pair inverted repeats are important for Mu-specific replicative transposition.

Bacteriophage lambda↗

Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum.

The rps12 gene in tobacco chloroplasts consists of three exons that code for a polypeptide with homology to Escherichia coli ribosomal protein S12. C-terminal exons 2 and 3 of rps12 are located in the inverted repeat regions of the tobacco chloroplast genome. Exon 1 of rps12 is 29 kilobase pairs downstream of the nearest copy of exons 2 and 3 and 69 kilobase pairs away from the distal copy of exons 2 and 3. RNA gel blot hybridization analysis and primer extension sequencing of cDNA to rps12 encoding RNAs indicate that exon 1 and exons 2 and 3 are encoded on separate transcripts. Exon 1 and exons 2 and 3 are covalently ligated in the correct reading frame in rps12 mRNA. These results indicate that a bimolecular (trans-) splicing event occurs during the formation of mature rps12 mRNA.

Chloroplasts↗

Dimerization interfaces of v-erbA homodimers and heterodimers with retinoid X receptor alpha.

The oncoprotein v-ErbA, a member of the zinc finger transcription factor superfamily, is a mutated version of thyroid hormone receptor alpha1 that is virtually incapable of binding T3. v-ErbA and other members of this family can bind as homodimers and heterodimers with retinoid X receptors to specific DNA sequences arranged as direct, inverted, or everted repeats. At least two regions in the C-terminal domain, the I box (10 and 11 helices in v-ErbA and thyroid hormone receptors) and the 20-amino acid region are involved in dimerization. However, it has not been entirely understood how these receptors dimerize on differently oriented core motifs and whether the domain(s) responsible for homodimerization and heterodimerization are identical. Therefore, deletions of the entire 20-amino acid region, the 10 helix, the 11 helix, and point mutations within these regions of v-ErbA were made by site-directed mutagenesis. The mutant proteins were tested for their ability to form v-ErbA homodimers and heterodimers with retinoid X receptor alpha on differently oriented core motifs by electrophoretic mobility shift assay. Transient transfections were performed to determine the dominant negative activity of the v-ErbA mutants. The data indicate that different dimerization interfaces are used for v-ErbA homodimerization and heterodimerization with retinoid X receptor alpha, and different dimerization interfaces are used on differently oriented core motifs. The data are of general interest because the information improves our understanding of the role of these dimerization interfaces in the mechanism of action not only of v-ErbA but also of other members of the superfamily.

Amino Acid Sequence↗