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PubMed2004

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed.

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.

Animal Feed↗
PubMed2003

Delineation of sequences important for efficient packaging of feline immunodeficiency virus RNA.

We have used systematic deletion analysis of the 5' untranslated region (UTR) of the feline immunodeficiency virus (FIV) genome, both in the presence and absence of various amounts of gag, to define the cis-acting sequences responsible for efficient RNA packaging. Our analyses revealed that the primary FIV packaging signal consists of two essential core elements located within the first 90-120 bp of the 5'UTR and the first 90 bp of the gag gene. Interestingly, the region between the major splice donor (SD) and gag, including approximately 130-160 bp upstream of the SD, is dispensable for encapsidation. Finally, other determinants of packaging were found to be present in the viral LTR and/or within the 3' end of the viral genome. Taken together, our results suggest that the primary packaging determinants of FIV are multipartite and discontinuous, composed of two elements within the 5'UTR and gag gene.

5' Untranslated Regions↗
PubMed2004

Retrotransposon-adenovirus hybrid vectors: efficient delivery and stable integration of transgenes via a two-stage mechanism.

It has long been recognized that the mechanisms mediating retrotransposition might be adapted for genomic integration and long-term expression of foreign genes. In particular, long interspersed nuclear elements (LINEs), an abundant class of retrotransposons that are the most active mobile genetic elements in the human genome, have been largely ignored as candidates for development as an integrating vector system because there has been no suitable method for efficiently introducing them into target cells. We have recently developed a LINE-based retrotransposon-adenovirus hybrid vector, in which a helper-dependent adenovirus (HDAd) is utilized as the platform for delivery of a human L1 element and its linked heterologous transgene cassette into the host cell nuclei. While a major drawback to the use of HDAd vectors has been their lack of specific mechanisms to achieve permanent integration into the host genome, the inserted retrotransposon sequences overcome this limitation. The L1-HDAd hybrid thus represents a single vector capable of mediating long-term gene expression by a two-stage mechanism: in the first (adenovirus) stage, the helper-dependent adenovirus serves as a carrier for efficient delivery and transient expression of its encoded L1/transgene cassette, and in the second (retrotransposon) stage, the L1 retro-element and its associated transgene then permanently integrate into the genome of the adenovirus-transduced cells. We propose that this novel retrotransposon-adenovirus hybrid vector system will be useful both as a vehicle for efficient delivery and long-term stable transduction of therapeutic genes, as well as a tool to elucidate aspects of retrotransposon biology that have previously been difficult to study.

Adenoviridae↗
PubMedDate not supplied

[A new KIAA1245 gene family with or without HERV-K LTRs in their introns].

A transcript containing the long terminal repeat (LTR) and the sequence homologous to the KIAA1245 mRNA fragment were revealed among the transcribed LTRs of human endogenous viruses of the K family in normal and tumor tissues. Ten other sequences with a high level of homology to the KIAA1245 mRNA were found in the GenBank. The intron-exon structures were determined for all the sequences, and their exon sequences were compared. The comparison showed that they differ both in the extent of the exon homology and in the presence or absence of the HERV-K LTR in the third intron. The revealed sequences form a new gene family that comprises at least four subfamilies. Two of these subfamilies have the LTR, and the other two do not. We showed by PCR that the LTR was integrated into the introns after the divergence of the orangutan evolutionary branch from other hominoids but before the divergence of the gorilla branch, i.e., 8-13 million years ago. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Animals↗
PubMed2002

Genetic variations of human herpesvirus 7 by analysis of glycoproteins B and H, and R2-repeat regions.

Clinical isolates of human herpesvirus 7 (HHV-7) from the saliva of healthy individual were investigated for genetic variations in the regions of two immediate-early (IE) genes, the glycoprotein B (gB) and glycoprotein H (gH) genes, and in R2-repeat. The genomic DNA of 24 isolates from citizens of Thailand, Japan, and the United States was amplified to detect size variations in the IE-1 and IE-2 loci, but none was observed, suggesting that there was no deletion or insertion in these genes, in contrast with an IE gene of human herpesvirus 6 (HHV-6). The sequences of the gB gene from isolates acquired from 5 Japanese and 8 Thai subject were then compared with those of American strains JI and RK with respect to codons that are known to differentiate gB alleles. All the isolates were found to have gB allele C except for the JI strain, which has allele F. Variability was also observed in five specific gH codons, resulting in 6 different groups. The HHV-7 isolates might be classified into two major genetic variants by combining their gB and gH allelic groupings. In the present study, only JI belonged to variant 1, while the rest of the isolates appeared to belong to variant 2. In the R2-repeat region, size heterogeneities were observed among the 24 isolates, due to different repeat numbers (17, 15, 14, 13, or 12 repeats). Therefore, we used the R2-repeat to identify the origins of isolates in a study of HHV-7 transmission, and found HHV-7 to be transmitted within a family from both mothers and fathers to their children.

Adult↗
PubMed2003

Gene transfer into genomes of human cells by the sleeping beauty transposon system.

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Blotting, Western↗
PubMed2001

Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus.

Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 x 10(5) infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.

Adult↗
PubMed2005

Use of endogenous retroviral sequences (ERVs) and structural markers for retroviral phylogenetic inference and taxonomy.

BACKGROUND: Endogenous retroviral sequences (ERVs) are integral parts of most eukaryotic genomes and vastly outnumber exogenous retroviruses (XRVs). ERVs with a relatively complete structure were retrieved from the genetic archives of humans and chickens, diametrically opposite representatives of vertebrate retroviruses (over 3300 proviruses), and analyzed, using a bioinformatic program, RetroTector, developed by us. This rich source of proviral information, accumulated in a local database, and a collection of XRV sequences from the literature, allowed the reconstruction of a Pol based phylogenetic tree, more extensive than previously possible. The aim was to find traits useful for classification and evolutionary studies of retroviruses. Some of these traits have been used by others, but they are here tested in a wider context than before. RESULTS: In the ERV collection we found sequences similar to the XRV-based genera: alpha-, beta-, gamma-, epsilon- and spumaretroviruses. However, the occurrence of intermediates between them indicated an evolutionary continuum and suggested that taxonomic changes eventually will be necessary. No delta or lentivirus representatives were found among ERVs. Classification based on Pol similarity is congruent with a number of structural traits. Acquisition of dUTPase occurred three times in retroviral evolution. Loss of one or two NC zinc fingers appears to have occurred several times during evolution. Nucleotide biases have been described earlier for lenti-, delta- and betaretroviruses and were here confirmed in a larger context. CONCLUSION: Pol similarities and other structural traits contribute to a better understanding of retroviral phylogeny. "Global" genomic properties useful in phylogenies are i.) translational strategy, ii.) number of Gag NC zinc finger motifs, iii.) presence of Pro N-terminal dUTPase (dUTPasePro), iv.) presence of Pro C-terminal G-patch and v.) presence of a GPY/F motif in the Pol integrase (IN) C-terminal domain. "Local" retroviral genomic properties useful for delineation of lower level taxa are i.) host species range, ii.) nucleotide compositional bias and iii.) LTR lengths.

Endogenous Retroviruses↗
PubMed2000

The role of CpG motifs in immunostimulation and gene therapy.

The mammalian immune system has evolved mechanisms to recognize and respond to 'danger' signals arising from pathogens. Among those danger signals are the unmethylated CpG dinucleotide motifs found in bacteria. At least some of the recognition of these sequences is through cellular components of the innate immune system, such as macrophages. Cytokines released by these cells in response to CpG motifs in turn activate other immune cells, such as NK cells and T cells, and can drive the development of adaptive immune responses. These proinflammatory, Th1 responses can also be generated intentionally with small oligodeoxynucleotides containing stimulatory CpG motifs, and have beneficial properties as vaccine adjuvants and in cancer immunotherapy. These proinflammatory responses have also been seen in gene therapy applications, especially in systemic delivery systems in which plasmid DNA vectors have been introduced with a vehicle such as a cationic lipid. For many gene therapy applications, finding ways to counter the immunostimulatory properties of plasmid DNA vectors is an important approach designed to enhance the vector safety profile, thereby increasing its effective therapeutic index.

B-Lymphocytes↗
PubMed2003

RNA interference is required for normal centromere function in fission yeast.

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.

Centromere↗
PubMed2004

Molecular characterization of clinical Saccharomyces cerevisiae isolates and their association with non-clinical strains.

We assessed the molecular characterization of 96 clinical isolates of S. cerevisiae from a Spanish medical institution and we compared them with 6 non-clinical strains isolated from wine, beer and bread and 1 S. boulardii strain collected from a commercial preparation. The strains were subjected to HinfI mtDNA restriction analysis and PCR amplification of delta sequences. Although both techniques are appropriate for routine clinical analysis, that based on PCR turned out to be the most discriminating. This study, apart from providing tools for clinical application, deals with the relationships between clinical and non-clinical strains. The two baker's yeasts analysed shared mtDNA and PCR patterns with a group of 31 clinical isolates. An exogenous entry of a strain was also reflected in the case of 19 clinical isolates and the therapeutic strain S. boulardii. Both baker's yeasts and S. boulardii were identified respectively among 32.3% and 19.8% of the clinical isolates and there seemed to be a connection between their ability to colonize humans and their ability to cause vaginal infection. The rest of food isolates were not grouped with clinical strains.

DNA, Fungal↗
PubMed2005

A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles.

Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme. The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production.

Animals↗
PubMed2005

A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid.

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.

Carbohydrate Conformation↗
PubMed2002

A minimal genome simian foamy virus type 1 vector system with efficient gene transfer.

Foamy viruses have several inherent features for the opportunity to develop efficient and versatile vectors for gene therapy. We have constructed a series of vectors and helper plasmids based on simian foamy virus type 1 (SFV-1) to establish the minimum vector genome required for efficient gene transduction. To characterize the efficiency of gene transduction by these vectors, the green fluorescent protein (GFP) coding sequence is linked to the human cytomegalovirus immediate gene promoter. Several deletion analyses of SFV-1 vectors revealed that the minimum genome with efficient GFP transduction contained the 5' untranslated region extending to the first 637 nucleotides of the gag gene, a 596 nucleotides of pol sequence from position 3137-3733, the 3' pol region at position 5200-5693, the 3' end polypurine tract, and the 3' LTR. An additional 1131 nucleotides can be removed from the 3' end LTR without affecting the efficiency of vector transduction. SFV-1 vector can therefore accommodate a minimum 8930 base-size heterologous DNA fragment. Furthermore, the efficiency of SFV-1 vector transduction was analyzed using different packaging plasmids. GFP transduction with packaging plasmid that contained the 5' R-U5 region of the LTR was compared with helper plasmids that had deletions in this region except for 22 nucleotides (positions 21-41), the first 61, 77, or 140 nucleotides of the R of the LTR. Transduction efficiencies were significantly reduced with the deletion mutations implicating that for optimum SFV-1 vector productions a packaging construct that includes the 5' R-U5 is required.

5' Untranslated Regions↗
PubMed2005

Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) after a long latent period. Among accessory genes encoded by HTLV-I, the tax gene is thought to play a central role in oncogenesis. However, Tax expression is disrupted by several mechanims including genetic changes of the tax gene, deletion/hypermethylation of 5'-LTR. To clarify the role of epigenetic changes, we analyzed DNA methylation and histone modification in the whole HTLV-I provirus genome. RESULTS: The gag, pol and env genes of HTLV-I provirus were more methylated than pX region, whereas methylation of 5'-LTR was variable and 3'-LTR was not methylated at all. In ATL cell lines, complete DNA methylation of 5'-LTR was associated with transcriptional silencing of viral genes. HTLV-I provirus was more methylated in primary ATL cells than in carrier state, indicating the association with disease progression. In seroconvertors, DNA methylation was already observed in internal sequences of provirus just after seroconversion. Taken together, it is speculated that DNA methylation first occurs in the gag, pol and env regions and then extends in the 5' and 3' directions in vivo, and when 5'-LTR becomes methylated, viral transcription is silenced. Analysis of histone modification in the HTLV-I provirus showed that the methylated provirus was associated with hypoacetylation. However, the tax gene transcript could not be detected in fresh ATL cells regardless of hyperacetylated histone H3 in 5'-LTR. The transcription rapidly recovered after in vitro culture in such ATL cells. CONCLUSION: These results showed that epigenetic changes of provirus facilitated ATL cells to evade host immune system by suppressing viral gene transcription. In addition, this study shows the presence of another reversible mechanism that suppresses the tax gene transcription without DNA methylation and hypoacetylated histone.

Carrier State↗
PubMed1999

Heterologous gene expression in avian cells: potential as a producer of recombinant proteins.

We have explored the possibility of using avian cells for the expression of human proteins. We found that various avian cells including quail fibrosarcoma cells (QF), duck embryo cells (DE) and primary chicken embryo fibroblasts (CE) could efficiently be transfected with DNA by calcium phosphate coprecipitation, and that promoters which are transcriptionally active in mammalian cells also functioned well in these avian cells. Among the promoters we tested, the major immediate early promoter of human cytomegalovirus drove the highest level of chloramphenicol acetyl transferase (CAT) expression, outperforming the SV40 early promoter and the RSV LTR. Using the bacterial CAT gene as a reporter, we found that levels of CAT activity were higher in QF and DE cells than in mammalian cells such as CHO, HeLa, Vero and 293T cells. We further cloned a sequence encoding human erythropoietin (EPO) and compared its expression in QF and mammalian cells. Consistent with the CAT data, in transient transfection assays, QF cells produced higher levels of EPO than the mammalian cell lines tested. QF cells which can be passaged permanently were stably transfected with an EPO expression vector. The subcloned QF line was able to produce up to 1,700 U/ml EPO from 3 x 10(6) cells in 72 h. Purified QF-produced EPO showed a broad but discrete protein band, ranging from 33 to 41 kD and was as biologically active as CHO-produced EPO. Although a number of factors still remain to be optimized, our results demonstrate the potential of avian cells such as QF as producers of heterologous proteins.

Animals↗
PubMed2000

Molecular misreading in non-neuronal cells.

+1 Frame-shifted proteins such as amyloid precursor protein(+1) and ubiquitin-B(+1) have been identified in the neuropathological hallmarks of Alzheimer's disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed 'molecular misreading'. A key question is whether this process is confined to neurons or whether it could also occur in non-neuronal cells. A transgenic mouse line (MV-B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV-LTR promotor was used to screen non-neuronal tissues for molecular misreading by means of detection of the rat vasopressin(+1) protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non-neural tissues too, could be affected by cellular derangements caused by molecular misreading.

Animals↗
PubMed2005

Genetic structure and diversity of European flint maize populations determined with SSR analyses of individuals and bulks.

Landraces of maize represent a valuable genetic resource for breeding and genetic studies. Using simple sequence repeat (SSR) markers, we analysed five flint maize populations from Central Europe that had played an important role in the pre-hybrid era in Germany. Our objectives were to (1) investigate the molecular genetic diversity within and among the populations based on the SSR analysis of individuals, (2) compare these results of the SSR analysis based on individuals with those based on bulks, (3) examine genotype frequencies for deviations from Hardy-Weinberg equilibrium (HWE) at individual loci, and (4) test for linkage disequilibrium (LD) between pairs of loci within populations. Thirty individuals and their bulked DNA per population were fingerprinted with 55 SSR markers. Across all populations, 46.7% of the SSR markers deviated significantly from HWE, with an excess of homozygosity in 97% of the cases. This excess of homozygosity can largely be explained by experimental errors during the amplification of SSRs apart from genuine genetic causes. Allele frequencies of the SSR analyses of individuals and bulks were significantly correlated (r=0.85, P< 0.01), suggesting that SSR analysis of bulks is very cost-effective for large-scale molecular characterisation of germ plasm collections. No evidence for genome-wide LD among pairs of loci was observed, indicating that the populations are well suited for high resolution association mapping studies.

Europe↗