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PubMed2006

Identification of transplanted human cells in animal tissues.

The potential of cell and gene therapy has generated extensive interest over the past several years. More recently, identification of stem cells of various types, especially embryonic stem cells, reinforced this interest. Systematic studies are now being launched to define the biology of various stem cells, including after transplantation of cells in mmunodeficient animals. This requires robust and unequivocal means to identify transplanted cells. Ideally, it should be possible to screen animal tissues for human cells with relatively simpler methods, followed by more precise localization of transplanted cells. We describe the application of conserved primate Charcot-Marie-Tooth disease type 1A repeat element for polymerase chain reaction-based screening of animal tissues for human cells. Similarly, direct polymerase chain reaction labeling of pancentromeric human alphoid sequences with digoxigenin-UTP generates in situ hybridization probes for identifying transplanted human cells. This pancentromeric probe identifies human cells irrespective of the original tissue source and can be combined with additional in situ methods to analyze cell differentiation. Incorporation of these strategies will facilitate translational studies aimed at understanding mechanisms concerning the trafficking, engraftment, proliferation, differentiation and function of human stem cells in animals.

Animals↗
PubMed2004

Transforming growth factor beta enhances the glucocorticoid response of the mouse mammary tumor virus promoter through Smad and GA-binding proteins.

Tissue-specific transcription is advantageously investigated by using viral promoters, which are selected for compact regulatory elements. Mouse mammary tumor virus (MMTV) has adapted to specialized cell types and targets initially B lymphocytes. We previously showed that, in B-cell lines, glucocorticoid-induced MMTV transcription requires an ETS family factor, GA-binding protein (GABP), bound in tandem to the MMTV DNA next to the glucocorticoid receptor (GR). We now report that transforming growth factor beta (TGF-beta) superinduces this response up to 10-fold through binding of its effectors, Smads, between the GABP-binding motifs. The basal level was unaffected. The TGF-beta-glucocorticoid cooperation also depended on GR and GABP binding, was transferable to another promoter, and occurred both with transiently transfected and with integrated templates. Smad3 associated in vitro with GR, with GABPalpha (via the MH2 domain), and with GABPbeta, Smad4 only with GABPalpha. Interactions of Smad3 with GABP (when coexpressed or endogenous to B cells) were shown by coprecipitation and by mammalian two-hybrid assay. This composite DNA element integrates three signaling pathways deriving from TGF-beta, glucocorticoid hormones, and a unique ETS factor, and may allow MMTV to exploit factors from the milk. It may as well indicate novel possibilities for cellular regulatory networks.

Animals↗
PubMedDate not supplied

IS elements as constituents of bacterial genomes.

We provide here an overview of our present understanding of the distribution of different insertion sequences (ISs) within bacterial genomes (both chromosomes and plasmids). This is at present fragmentary and a significant effort is needed in the analysis of the increasing number of genomes whose sequence has been determined. We also consider some of the properties of ISs which are important in their role of assembling, reassorting, and transmitting groups of genes.

Bacteria↗
PubMed2026

Chromosome-level genome assembly of the hemiparasitic Taxillus sutchuenensis (Loranthaceae).

Taxillus sutchuenensis, an ecologically and medicinally important hemiparasitic plant that parasitizes diverse woody hosts, was sequenced to generate a high-quality chromosome-level genome assembly. PacBio HiFi long reads, RNA-seq transcriptome data, and Hi-C data were used to assemble a 406.32 Mb genome anchored onto nine pseudo-chromosomes, with a scaffold N50 of 45.59 Mb. The assembly showed high completeness and accuracy, supported by BUSCO (93.6%) and Merqury QV (70.6) assessments. The LTR Assembly Index (LAI) of 13.98 indicated excellent continuity. A total of 21,795 protein-coding genes were predicted, with 94.46% functionally annotated. Repetitive sequences accounted for 50.05% of the genome, primarily LTR retrotransposons. This genome provides a valuable resource for investigating the evolution, functional genomics, and parasitic mechanisms of hemiparasitic plants.

Genome, Plant↗
PubMed2005

CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K(HML-2).

A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) transcription remains poorly understood. We investigated in detail the role of CpG methylation on the transcriptional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-K(HML-2) proviral 5' LTRs are methylated at different levels in the cell line Tera-1. Methylation levels correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5' LTR reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5' LTR activity, as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in evolutionary old HERV-K(HML-2) 5' LTRs suggests that CpG methylation had already silenced HERV-K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG methylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.

Cell Line, Tumor↗
PubMed1997

BCL6 can repress transcription from the human immunodeficiency virus type I promoter/enhancer region.

The gene BCL6 encodes a zinc finger protein with similarities to transcription factors. We previously reported that a number of viral genomes, including human immunodeficiency virus type I (HIV-1), contain sequences which are similar to the BCL6 DNA-binding consensus in their promoter regions. Electrophoretic mobility shift assays showed that the full-length BCL6 protein extracted from transfected COS cells and a bacterially expressed truncated protein containing the BCL6 zinc fingers can bind specifically to DNA from the U3 promoter/enhancer region of HIV-1. Transient transfections were performed to analyze the effects of the BCL6 protein on luciferase expression driven by the HIV-1 long terminal repeat (LTR) sequences. Full-length BCL6 significantly repressed luciferase activity compared with multiple controls. We conclude that the BCL6 protein can bind to the HIV-1 promoter-enhancer region and contains a domain upstream of its zinc fingers that can repress transcription from the HIV-1 LTR.

Animals↗
PubMed1995

Keratanase digestion of keratan sulphates: characterization of large oligosaccharides from the N-acetyllactosamine repeat sequence and from the non-reducing terminal chain caps.

Keratan sulfate (KS) chains prepared from both bovine tracheal rings and bovine femoral head cartilage were digested with the enzyme keratanase from Pseudomonas species; large repeat-sequence and non-reducing terminal oligosaccharides were fractionated and purified using high-performance ion-exchange chromatography. The main beta-linked pentasulfated hexasaccharide repeat segment, [R6], GlcNAc(6S)1-1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal-ol and the asialo beta-linked capping pentasulfated heptasaccharide, [C7], Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) 1-3Gal-ol have been completely characterized by high-field NMR spectroscopy using one- and two-dimensional methods. Partial 1H assignments are summarized for three homologous series of higher oligosaccharides: GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-5(1-3)Gal-0l [R8,R10,R12] Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]3-5(1-3)Gal-ol [C9,C11,C13] NeuAc alpha 2-3Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-4(1-3)Gal-ol [C8,C10,C12] obtained from keratan sulfate by keratanase cleavage. The first shows that the unsulfated galactose residues within the repeat sequence region of KS may be separated by fully sulfated segments which have a wide distribution of lengths. The others, viz. those with sialylated caps, and the related galactose capped asialo-segments (derived from a KS digestion in which the keratanase also exhibited sialidase activity) represent an homologous series of epitopes in which the first internal unsulfated galactose is located at a position which may be up to five or more fully sulfated N-acetyllactosamine disaccharide repeat units along from the non-reducing terminus of the KS polymer.

Animals↗
PubMed2004

Stable and strictly controlled expression of LTR-flanked autoregulated expression cassettes upon adenoviral transfer.

An autoregulatory bidirectional expression cassette encoding all components necessary for regulated gene expression in a one-step gene transfer was evaluated for use in adenoviral vectors. Adenoviral vectors transducing this cassette provide about 1000-fold regulation. Regulation could be further improved by integrating the cassette as a retroviral vector into the adenoviral backbone. Moreover, with these adeno/retroviral hybrid vectors, the frequency of chromosomal integration is enhanced and about 1% of infected cells show stable chromosomal integration of the autoregulated cassette. In these stably transduced cells high regulation capacity is maintained. To elucidate the molecular mechanism underlying this unexpected observation we investigated the regulation capacity of these cassettes in a viral and non-viral vector background after stable integration into the host's DNA. While naked cassettes show regulated expression that is strongly influenced by the chromosomal surrounding sequences the regulatory capacity of LTR flanked cassettes is highly comparable amongst different cell clones. This strict regulation with little influence from the flanking sequences is obtained when LTR-flanked cassettes are transduced as DNA, by retroviral or by adenoviral infection.

Adenoviridae↗
PubMed2000

The complete unique long sequence and the overall genomic organization of the GA strain of Marek's disease virus.

We have determined the DNA sequence of the unique long (UL) region and the repeat long (RL) region in the genome of serotype 1 GA strain of Marek's disease virus (MDV), a member of the alpha-herpesvirus family. With this information, the complete nucleotide sequence of GA-MDV is now known. The entire GA-MDV genome is predicted to be about 174 kbp in size, with an organization of TRL-UL-IRL-IRS-US-TRS, typical of a alpha-herpesvirus. The UL sequence contains 113,508 bp and has a base composition of 41.7% G + C. A total of 67 ORFs were identified completely within the UL region, among which 55 are homologous to genes encoded by herpes simplex virus-1. Twelve of them are unique with presently unknown functions. The sequence of RL reported here together with those published earlier reveal the major structural features of the RL. Virtually all of the ORFs encoded by RL are specific to serotype I of MDV. These ORFs are likely to contribute to some of the unique biological properties of MDV. Among the proteins encoded by MDV-specific ORFs are Meq, a jun/fos family of transcriptional factor implicated in transformation and latency, virus-encoded interleukin-8, a CXC chemokine, and pp38 and pp24, two phosphoproteins with undefined functions. There is also a putative lipase gene (LORF2) that has homologies in HPRS-24 (serotype II) strain of MDV and in various avian adenoviruses. An additional unique feature of MDV is the presence of long terminal repeat remnant sequences of avian retrovirus reticuloendotheliosis virus. These remnant sequences are derived from the U3-enhancer region through ancestral insertions by reticuloendotheliosis virus proviruses.

DNA Replication↗
PubMed2006

The proteins encoded by the pogo-like Lemi1 element bind the TIRs and subterminal repeated motifs of the Arabidopsis Emigrant MITE: consequences for the transposition mechanism of MITEs.

MITEs (miniature inverted-repeated transposable elements) are a particular class of defective DNA transposons usually present within genomes as high copy number populations of highly homogeneous elements. Although an active MITE, the mPing element, has recently been characterized in rice, the transposition mechanism of MITEs remains unknown. It has been proposed that transposases of related transposons could mobilize MITEs in trans. Moreover, it has also been proposed that the presence of conserved terminal inverted-repeated (TIR) sequences could be the only requirement of MITEs for mobilization, allowing divergent or unrelated elements to be mobilized by a particular transposase. We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements. This suggests that Lemi1 could mobilize Emigrant elements and makes the Lemi1/Emigrant couple an ideal system to study the transposition mechanism of MITEs. Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs. The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases.

Arabidopsis↗
PubMed1989

Interactions between the termini of adeno-associated virus DNA.

The adeno-associated virus (AAV) genome is a linear, single polynucleotide chain with inverted terminal repeats of 145 bases. In order to test whether the terminal repeats at opposite ends of the genome have to be able to completely base-pair during DNA replication, we have created chimeric genomes in which an 11 base symmetrical sequence has been deleted from the terminal repeat at one end of the genome and replaced by a different 12 base symmetrical sequence. We have used these chimeric constructs either as a duplex insert in pBR322 or as purified duplex virion DNA to transfect adenovirus-infected HeLa cells. When chimeric duplex virion DNA was used, all of the progeny virions obtained after two cell passages contained DNA with wild-type sequences in both terminal repeats. When plasmid clones were used, the structure of virion DNA depended on the original orientation. If the mutant terminal repeat was originally at the right end of the genome (terminus of genetic map), all progeny terminal repeat sequences were again wild-type. However, if the original construct contained the mutant sequence in the left terminal repeat, the majority of progeny molecules were parental in type (i.e. mutant left and wild-type right terminal repeat). We conclude (1) although the terminal repeats at opposite ends of the genome may interact during DNA replication, it is not necessary that they be perfectly complementary. (2) In direct competition, the wild-type sequence displays an advantage over the mutant allele. (3) In a plasmid clone, the terminal repeat on the left end of the genome is at an advantage in a competitive situation. We note that the left terminal repeat is adjacent to a transcriptional promoter.

Base Sequence↗
PubMed2000

Enhanced binding of the TATA-binding protein to TATA boxes containing flanking cisplatin 1,2-cross-links.

The TATA-binding protein (TBP) is essential for transcription initiation in eukaryotes. TBP recognizes and binds to the minor groove of a consensus sequence, TATAAA, known as the TATA box or TATA element. DNA binding is affected largely by hydrophobic contacts and through the intercalation of two sets of adjacent phenylalanine residues. The resultant duplex is sharply kinked, bending toward the major groove. Inspired by prior structural information showing intercalation of a phenylalanine side chain of a high mobility group (HMG) domain into the site of a cisplatin 1, 2-intrastrand d(GpG) cross-link, a series of DNA probes was prepared with one or two such adducts flanking the TATA box positions at or near the sites of TBP intercalation. The platinum adducts bend the DNA toward the major groove and result in as much as a 175-fold increase in binding affinity of the TBP over the unmodified target sequence. Kinetic studies indicate that the enhanced binding to the modified TATA box is predominantly a consequence of a >30-fold slower dissociation rate of the protein-platinated DNA complex. This work demonstrates that it is feasible to design rationally and to synthesize an enhanced affinity-binding site for a sequence-specific DNA-binding protein by appropriate chemical modification of flanking sequences. It also has implications for the mechanism of action of cisplatin.

Binding, Competitive↗
PubMed1998

Comparative analyses of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication.

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3' long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the env antisense RNA, followed by the pol complementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and env antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the transdominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

3T3 Cells↗
PubMed1999

Introduction of a cis-acting mutation in the capsid-coding gene of moloney murine leukemia virus extends its leukemogenic properties.

Inoculation of newborn mice with the retrovirus Moloney murine leukemia virus (MuLV) results in the exclusive development of T lymphomas with gross thymic enlargement. The T-cell leukemogenic property of Moloney MuLV has been mapped to the U3 enhancer region of the viral promoter. However, we now describe a mutant Moloney MuLV which can induce the rapid development of a uniquely broad panel of leukemic cell types. This mutant Moloney MuLV with synonymous differences (MSD1) was obtained by introduction of nucleotide substitutions at positions 1598, 1599, and 1601 in the capsid gene which maintained the wild-type (WT) coding potential. Leukemias were observed in all MSD1-inoculated animals after a latency period that was shorter than or similar to that of WT Moloney MuLV. Importantly, though, only 56% of MSD1-induced leukemias demonstrated the characteristic thymoma phenotype observed in all WT Moloney MuLV leukemias. The remainder of MSD1-inoculated animals presented either with bona fide clonal erythroid or myelomonocytic leukemias or, alternatively, with other severe erythroid and unidentified disorders. Amplification and sequencing of U3 and capsid-coding regions showed that the inoculated parental MSD1 sequences were conserved in the leukemic spleens. This is the first report of a replication-competent MuLV lacking oncogenes which can rapidly lead to the development of such a broad range of leukemic cell types. Moreover, the ability of MSD1 to transform erythroid and myelomonocytic lineages is not due to changes in the U3 viral enhancer region but rather is the result of a cis-acting effect of the capsid-coding gag sequence.

3T3 Cells↗
PubMed2005

Telomere-specific non-LTR retrotransposons and telomere maintenance in the silkworm, Bombyx mori.

Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6-8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.

Animals↗
PubMed2005

Characterization of the human CD5 endogenous retrovirus-E in B lymphocytes.

All T lymphocytes and some B lymphocytes express CD5. This coreceptor is encoded by one gene that consists of 11 exons. We have previously described a B cell-specific alternative exon 1, leading to the synthesis of a protein, devoid of leader peptide, and, therefore, retained in the cytoplasm. The novel exon 1 originates from a human endogenous retrovirus (HERV) at a time interval between the divergence of New World monkeys from Old World monkeys, and prior to the divergence of humans from Old World monkeys. Based on sequence similarity to gamma-retroviruses, it was categorized as class I: based on the specificity of its primer binding site, it was allotted to the subclass E, and based on its location within the cd5 gene, named HERV-E.CD5. Alternative transcripts were detected in lymphoid organs including fetal liver (not adult liver), more particularly in CD5-negative cell surface B-1b than in CD5-positive cell surface B-1a, and not at all in B-2 cells. By alignment of 5' long terminal repeats, HERV-E.CD5 was distinguished from similar proviruses. This could be central to the regulation of membrane expression of CD5 in human B lymphocytes, and, thereby, to the strength of the B-cell antigen receptor signaling.

B-Lymphocytes↗
PubMed2026

Hijacking pre-tRNA enables LTR-retrotransposon-initiated constitutive heterochromatin formation.

Pericentric heterochromatin serves as a fundamental component of eukaryotic chromosomes, endowing specialized genomic architecture with broad functional consequences. Although it is universally marked by H3K9me3 modification, the underlying pericentric DNA sequences diverge substantially across species. Here, by leveraging a transposition reporter system combined with a genome-wide RNA interference (RNAi) screen, we identified a specialized mechanism for recruiting SUV39H methyltransferase to initiate pericentric heterochromatin formation. This pathway depends on a highly ordered complex comprising the Puf68, pre-transfer RNAs (tRNAs), and the primer binding site (PBS). Puf68 binds with high affinity to poly-U tracts in pre-tRNA 3' trailer, forming a Puf68/pre-tRNA complex that subsequently base-pairs with the PBS of nascent long terminal repeat (LTR)-retrotransposons. Through direct interaction, Puf68 recruits Su(var)3-9 to these regions, catalyzing H3K9 trimethylation. Notably, Puf68 is sufficient to initiate de novo heterochromatin assembly both at pericentric and ectopically integrated LTR-retrotransposon regions. Our findings not only uncover a previously unrecognized mechanism of heterochromatin initiation but also resolve a long-standing question of how hosts harness nascent LTR-retrotransposon transcripts.

Heterochromatin↗
PubMed1988

A repetitive DNA sequence in the salmonid fishes similar to a retroviral long terminal repeat.

We describe the characteristics of a repetitive DNA sequence from the rainbow trout and related salmonid fishes that is similar to a retroviral long terminal repeat (LTR). The repeat is 160 bp long and contains a region of homology to the LTR of the avian sarcoma virus. Two clones with this repeat from the chum salmon also have a polypurine tract and tRNA binding site, respectively, and these clones may represent the two LTRs of a retrovirus or retroviral-like repetitive element. Copies of the repeat are also adjacent to rainbow trout and chum salmon protamine genes. These repeats may be "solo" LTRs. There appears to be some polymorphism in restriction sites between individual rainbow trout and considerable differences between salmonid fish species when the repeat is used as a probe.

Amino Acid Sequence↗