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PubMed2003

Sequence analysis of porcine endogenous retrovirus long terminal repeats and identification of transcriptional regulatory regions.

Porcine cells express endogenous retroviruses, some of which are infectious for human cells. To better understand the replication of these porcine endogenous retroviruses (PERVs) in cells of different types and animal species, we have performed studies of the long terminal repeat (LTR) region of known gammaretroviral isolates of PERV. Nucleotide sequence determination of the LTRs of PERV-NIH, PERV-C, PERV-A, and PERV-B revealed that the PERV-A and PERV-B LTRs are identical, whereas the PERV-NIH and PERV-C LTRs have significant sequence differences in the U3 region between each other and with the LTRs of PERV-A and PERV-B. Sequence analysis revealed a similar organization of basal promoter elements compared with other gammaretroviruses, including the presence of enhancer-like repeat elements. The sequences of the PERV-NIH and PERV-C repeat element are similar to that of the PERV-A and PERV-B element with some differences in the organization of these repeats. The sequence of the PERV enhancer-like repeat elements differs significantly from those of other known gammaretroviral enhancers. The transcriptional activities of the PERV-A, PERV-B, and PERV-C LTRs relative to each other were similar in different cell types of different animal species as determined by transient expression assays. On the other hand, the PERV-NIH LTR was considerably weaker in these cell types. The transcriptional activity of all PERV LTRs was considerably lower in porcine ST-IOWA cells than in cell lines from other species. Deletion mutant analysis of the LTR of a PERV-NIH isolate identified regions that transactivate or repress transcription depending on the cell type.

Animals↗
PubMed1984

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.

Animals↗
PubMed1999

Replication of Mus dunni endogenous retrovirus depends on promoter activation followed by enhancer multimerization.

Mus dunni endogenous virus (MDEV) is an apparently intact retrovirus that normally lies transcriptionally silent in cultured M. dunni cells, but the provirus can be activated by treatment of the cells with hydrocortisone or 5-iodo-2'-deoxyuridine. Sequence analysis of a molecular clone of the replicating virus revealed a simple retrovirus with a chimeric VL30/GALV-like structure. Interestingly, in the region of the long terminal repeat (LTR) that typically contains the retroviral transcription enhancers, we found over six 80-bp repeats with only a single mismatch, indicating that acquisition of the repeats was a recent event. Here we provide evidence for the following model of MDEV activation and replication. The MDEV provirus in M. dunni cells has a chimeric structure similar to that of the molecular clone but has only 1.15 copies of the 80-bp repeat sequence found in the molecular clone. Activating chemicals directly stimulate transcription from the LTR, allowing a low level of virus replication. Copying errors made during reverse transcription allow multimerization of the 80-bp enhancer region, resulting in viruses with higher transcriptional rates and improved fitness, but increased enhancer copy number is likely balanced by the natural instability of retroviral repeats and constraints imposed by virion packaging limits. The resultant population of replicating MDEV is widely heterogeneous, having from 2.15 to 13.15 enhancer repeats in the LTR. These results reveal a novel mechanism for regulation of transcription and replication of an endogenous retrovirus, in terms of both activation of the virus by the steroid hydrocortisone and the large number and variation in enhancer repeats observed.

Animals↗
PubMed2004

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres.

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.

Binding Sites↗
PubMed2001

Organization and distribution pattern of MGLR-3, a novel retrotransposon in the rice blast fungus Magnaporthe grisea.

A specific telomere was deleted in spontaneous, gain-of-virulence mutants derived from a rice pathogen of Magnaporthe grisea. Three different types of transposons, including Pot2, Mg-SINE, and a novel, 6-kb-long LTR (long terminal repeat)-type retrotransposon designated MGLR-3, were identified on this chromosomal end. The 114-bp-long telomeric repeat is immediately followed by the 3' LTR of MGLR-3. A truncated copy of Pot2 immediately flanks the 5' LTR, suggesting that this telomere was generated by a transposition event of MGLR-3 into this Pot2 element, causing the breakage of a chromosome. The subsequent addition of a telomeric repeat to the 3' LTR of MGLR-3 most probably repaired the broken end of the chromosome. Mg-SINE is located 25 bp away from the truncated Pot2 element. MGLR-3 exhibited strong homology to various gypsy-class retrotransposons, including grh and MAGGY in M. grisea. MGLR-3 is ubiquitous regardless of the host of origin.

Amino Acid Sequence↗
PubMed1997

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.

Cell Line, Transformed↗
PubMed1985

Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as lambda-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directly repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTRs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGly. Functional analysis of the 3' LTR present in lambda-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhancer domains in the AGM provirus.

Acetyltransferases↗
PubMed1997

T- and B-cell responses of malaria immune individuals to synthetic peptides corresponding to non-repeat sequences in the N-terminal region of the Plasmodium falciparum antigen Pf155/RESA.

While the C-terminal repeat region of Pf155/RESA, a Plasmodium falciparum vaccine candidate has been extensively studied for B- and T-cell reactivities, little is so far known about the non-repeat region in this respect. The present study aimed at investigating the non-repeat sequence 171-227 of Pf155/RESA for T- and B-cell epitopes. Eight overlapping peptides were synthesised and assayed for their ability to stimulate peripheral blood mononuclear cells obtained from P. falciparum-immune donors to proliferate and to induce secretion of interferon-gamma (IFN-gamma) and/or interleukin 4 (IL-4) using the ELISPOT assay. The plasmas of the corresponding donors were tested for antibody reactivity with the same peptides in ELISA. The individual cellular responses to the different peptides varied and in general they were not correlated, emphasising the importance of including several parameters for T-cell activation. The most frequent T-cell responses (proliferation, IFN-gamma and/or IL-4) were seen with two partially overlapping peptides corresponding to the sequences 171-185 and 181-195 that induced responses in 71 and 62% of the donors, respectively. Although, the frequency of responders was high, the magnitude of the responses was generally low. Two overlapping peptides corresponding to the sequence 186-206 bound antibodies from a large number of plasma samples. IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide. However, there was no absolute correlation and many donors having anti-peptide antibodies could also be induced to produce IFN-gamma. In conclusion, the non-repeat region of Pf155/RESA contains several epitopes inducing functionally distinct T-cell responses. The sequence 171-206 was found to contain both B- and T-cell epitopes recognised by almost all individuals naturally primed to malaria. Thus, this sequence should be a useful tool in future immuno-epidemiological studies and/or for inclusion into a subunit vaccine against the asexual blood stages of the P. falciparum parasite.

Adolescent↗
PubMed1998

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.

Animals↗
PubMed2001

Evolutionary history of Cer elements and their impact on the C. elegans genome.

We report the results of sequence analysis and chromosomal distribution of all distinguishable long terminal repeat (LTR) retrotransposons (Cer elements) in the Caenorhabditis elegans genome. Included in this analysis are all readily recognizable full-length and fragmented elements, as well as solo LTRs. Our results indicate that there are 19 families of Cer elements, some of which display significant subfamily structure. Cer elements can be clustered based on their tRNA primer binding sites (PBSs). These clusters are in concordance with our reverse transcriptase- and LTR-based phylogenies. Although we find that most Cer elements are located in the gene depauperate chromosome ends, some elements are located in or near putative genes and may contribute to gene structure and function. The results of RT-PCR analyses are consistent with this prediction.

Animals↗
PubMed2001

DNA binding and modulation of gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is highly expressed in these malignancies and has been shown to play an important role in episomal maintenance, presumably by binding to a putative oriP. In addition, LANA modulates cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these features are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome maintenance, binds to oriP, and strongly activates transcription from two promoters, including its own. We have previously shown that LANA can transactivate its own promoter and therefore asked whether LANA, like EBNA-1, activates transcription by direct binding to DNA. By using recombinant LANA expressed from vaccinia virus vectors for electrophoretic mobility shift assays, we found that LANA does not bind to its own promoter. In contrast, LANA binds specifically to sequences containing an imperfect 20-bp palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domain of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibits transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when linked to a heterologous promoter. These data suggest that LANA, although fulfilling functions similar to those of EBNA-1, does so by very different mechanisms.

Animals↗
PubMed1991

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.

Actins↗
PubMed1991

Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences.

During transcription of the human immunodeficiency virus type 1 provirus, polyadenylation signals present in the 5' long terminal repeat (LTR) are disregarded while the identical polyadenylation signals present in the 3' LTR are utilized efficiently. As both transcribed LTR sequences contain all signals known to be required for efficient polyadenylation, the basis for this differential utilization has been unclear. Here, we describe experiments that suggest that transcribed sequences present within the human immunodeficiency virus type 1 LTR U3 region act in cis to enhance polyadenylation within the 3' LTR.

Base Sequence↗
PubMed1982

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

Amino Acid Sequence↗
PubMed1988

Structure and function of endogenous feline leukemia virus long terminal repeats and adjoining regions.

The nucleotide sequence of the 5' long terminal repeat (LTR) of three independent loci (CFE-6, CFE-16, and CF-14) of endogenous feline leukemia virus (FeLV) DNAs of the domestic cat genome was determined. The 3' LTR of the CFE-6 clone was also sequenced. The endogenous FeLV LTRs, which were very similar to each other in sequence and in organization of the functional domains, differed considerably from the exogenous FeLV LTR in the U3 region. The major differences in U3 included variations in sets of small (14 to 19 base pair) direct repeats, altered location of the simian virus 40 core enhancer-like sequence, and occurrence of three segments of largely nonhomologous sequences. There was extensive homology between endogenous and exogenous FeLV LTRs in sequences beginning from the TATA box through the R region down to the 3' end of the U5 region. The DNA sequence downstream of the 5' LTR encompassing the primer-binding site, leader, and almost to the end of the p15gag coding region, a point up to which the sequencing was carried out, also revealed a high degree of conservation. However, the detection of frameshift and nonsense mutations in this region of a nearly full-length endogenous provirus sequence (CFE-6) predicted its defectiveness and correlated with the lack of infectivity of this DNA. The functional studies of the endogenous LTRs, based on linkage to the bacterial cat gene and transient expression in feline cell lines, indicated that although the basic characteristics for promotion and enhancement of transcription were retained in each LTR, there was a significant variation in the activity of the cat constructs. Reconstruction and deletion analyses with the CFE-6 5' LTR revealed the presence of strong transcription regulatory sequences in the 702-base-pair region immediately upstream of the 5' boundary of the endogenous LTR. These and related data suggest that in addition to the transcription-modulating elements occurring within the LTR, the cis-acting nucleotide sequences in the upstream cellular DNA may determine the overall efficiency of transcription of the defective endogenous FeLV provirus loci of the felid genome.

Amino Acid Sequence↗
PubMed2003

Ribonuclease III-mediated processing of specific Neisseria meningitidis mRNAs.

Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26-27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis- mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly.

Base Sequence↗
PubMed2002

A cis-acting element that directs circular adeno-associated virus replication and packaging.

A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB'), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential cis-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.

Base Sequence↗
PubMed1999

Wake up of transposable elements following Drosophila simulans worldwide colonization.

Transposable elements (TEs) make up around 10%-15% of the Drosophila melanogaster genome, but its sibling species Drosophila simulans carries only one third as many such repeat sequences. We do not, however, have an overall view of copy numbers of the various classes of TEs (long terminal repeat [LTR] retrotransposons, non-LTR retrotransposons, and transposons) in genomes of natural populations of both species. We analyzed 34 elements in individuals from various natural populations of these species. We show that D. melanogaster has higher average chromosomal insertion site numbers per genome than D. simulans for all TEs except five. The LTR retrotransposons gypsy, ZAM, and 1731 and the transposon bari-1 present similar low copy numbers in both species. The transposon hobo has a large number of insertion sites, with significantly more sites in D. simulans. High variation between populations in number of insertion sites of some elements of D. simulans suggests that these elements can invade the genome of the entire species starting from a local population. We propose that TEs in the D. simulans genome are being awakened and amplified as they had been a long time ago in D. melanogaster.

Animals↗