Comparative analysis of the genomic organization of Pax9 and its conserved physical association with Nkx2-9 in the human, mouse, and pufferfish genomes.

As a first step towards the identification of cis-regulatory elements of Pax9 by means of comparative genomics, we have analyzed genome regions encompassing the Pax9 gene in three vertebrate species, humans, mice (Mus musculus), and the Japanese pufferfish (Fugu rubripes). We show the genomic organization of Pax9 and its physical association with Nkx2-9 conserved in the three species. We discuss about possible implications of the conserved synteny between Pax9 and Nkx2-9 in a context of vertebrate evolution. This report also includes the first description of the primary structures of Fugu Pax9 and Nkx2-9. Furthermore, we report the identification of a novel upstream exon and putative transcription start sites in mouse Pax9. Our results suggest that transcription of Pax9 may be initiated at two alternative start sites and driven by TATA-less promoters.

Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses.

Persistent viruses have evolved multiple strategies to escape the host immune system. One important prerequisite for efficient viral reproduction in the face of an ongoing immune response is prevention of premature lysis of infected cells. A number of viruses achieve this goal by interfering with antigen presentation and recognition of infected cells by cytotoxic T cells (CTL). Another viral strategy aims to block apoptosis triggered by host defense mechanisms. Both types of strategies seem to be realized by human adenoviruses (Ads). The early transcription unit E3 of Ads encodes proteins that inhibit antigen presentation by MHC class I molecules as well as apoptosis induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand (FasL). Here, we will describe the organization of the E3 regions of different Ad subgroups and compare the structure and functions of the known immunomodulatory E3 proteins.

Infection of nonhuman primate cells by pig endogenous retrovirus.

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.

A polymerase chain reaction-based protocol for the detection of transmission of pig endogenous retroviruses in pig to human xenotransplantation.

BACKGROUND: Xenotransplantation of pig organs and tissues to humans bears the risk of infection of immunosuppressed recipients by porcine endogenous retrovirus (PERV) released from the transplanted tissue. However, when diagnosing potential PERV transmission, it is essential to exclude microchimerism, i.e., persisting pig cells in analyzed bioptic material of xenotransplanted patients, which give rise to false positive PERV signals. Polymerase chain reaction (PCR) is so far the only suitable method to diagnose a cross-species transfer of PERV, but the exclusion of microchimerism might be a serious problem because most of the presently employed primer pairs detect PERV sequences with higher sensitivity than primers used for the detection of contaminating pig sequences. METHODS: We designed and evaluated a novel and improved primer set for detection of pig sequences as well as complementing positive control primers on the basis of mitochondrial cytochrome B, an approved marker for phylogenetic studies. We further established primer pairs derived from the long terminal repeat/leader region of PERV isolated from a Duroc German Landrace cross-bred pig and tested their sensitivity in comparison with known PERV- and pig-specific PCR markers. RESULTS: In standard PCR assays, the new cytochrome B-derived primers are at least 10 times more sensitive than the presently used PERV retroviral polymerase gene and mammalian beta-actin primers. When tested in a tissue culture infection model, PERV transmission to human 293 cells can be unambiguously demonstrated, even in the presence of up to 10% pig cells. One of the primer combinations derived from the PERV DuxDL3791 long terminal repeat/leader region amplifies with even lower sensitivity than primers detecting porcine beta-globin, thus permitting the exclusion of microchimerism also via chromosomal loci. CONCLUSIONS: The availability of the new PCR markers allows the proposal of a rigorous setup for the routine detection of PERV transmission after xenotransplantation.

[Risk of infection causes by viruses in xenotransplantation]. / Infektionsrisiko durch Viren bei Xenotransplantation.

The risk of transferring exogenous pig viruses to man during organ xenotransplantation can be controlled by keeping the pig donors pathogen-free. A risk of mobilizing pig endogenous retroviruses and a "xenozoonosis"-infection in human organ recipients cannot be excluded according to recently reported virological cell culture experiments. The present state of research, however, does not allow an answer to the question of whether or not a disease might be caused by such an infection and whether such a virus might be contagious to third persons.

A pseudoautosomal boundary-like element adjacent to the SSAV1 locus at 18q21.

Pseudoautosomal boundary-like (PABL) elements have been found at transition sites between genomic regions with different GC-contents. A new PAB related sequence was found immediately adjacent to the S71 provirus on human chromosome 18q21.1-2 (officially designated the SSAV1 locus). The S71 PABL element was full-length as defined by comparison with elements identified at the pseudoautosomal boundaries of the sex chromosomes and in the MHC region. The 3'-ends of all PABL elements showed significant homology to functional CpG-islands, indicating that this similarity is a new common feature of PABL elements.

Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells.

BACKGROUND: The risk of interspecies transmission of retroviruses during xenotransplantation is suggested by reports of pig endogenous retrovirus (PERV) released from porcine cell lines productively infecting human cell lines in vitro and of infectious PERV being released from pig peripheral blood mononuclear cells after mitogenic stimulation. Endothelial cells are the main interface between a xenograft and the recipient's leucocytes and tissues. METHODS: We have analysed pig primary aortic endothelial cells (PAEC) together with other transplantation-relevant porcine cells and tissues for expression of PERV mRNA. Release of virus particles by PAEC was monitored by reverse transcriptase (RT) activity in the medium of cultured PAEC. Infectivity for human cells was tested by co-cultivation of irradiated PAEC with the human embryonal kidney cell line HEK293 and looking for virus release from the human cells. FINDINGS: PAECs, hepatocytes, lung, and skin from a variety of pig strains and breeds expressed PERV mRNA. PAEC released infectious particles. Co-cultivation of PAEC and HEK293 led to productive infection of the human cells and expression of PERV types A and B. INTERPRETATION: Release of infectious virus from PAEC occurred without mitogenic stimulation, suggesting a serious risk of retrovirus transfer after xenotransplantation.

Proviral structure, chromosomal location, and expression of HERV-K-T47D, a novel human endogenous retrovirus derived from T47D particles.

We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408-6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3' long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag, prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.

Kappa-casein polymorphisms in Indian dairy cattle and buffalo: a new genetic variant in buffalo.

We screened 57 Sahiwal cattle (Bos indicus) and 53 Murrah, 19 Nili-Ravi and 11 Egyptian buffaloes (Bubalus bubalis) to detect the polymorphisms at kappa-casein (CSN3) gene using the polymerase chain reaction (PCR). CSN3 A and B alleles were identified by PCR-RFLP using the restriction enzymes that detect the underlying nucleotide changes at codon 136 (Taql) and at codon 148 (HindIII or HinfI) in cattle. The frequency of CSN3 B allele in the Sahiwal cattle was estimated as 0.16 with no homozygous BB animal. Using the same set of primers as used in the Sahiwal cattle, a part of exon IV of buffalo CSN3 gene was amplified, but restriction enzyme analysis using HindIII/HinfI and TaqI did not reveal any polymorphism. However, DNA sequencing of the amplified fragment (GenBank Acc. No. U96662) revealed one polymorphism at codon 135 (ThrACC -->I1eATC) in buffalo; the frequencies of 135 Thr/Ile alleles were estimated as 0.88 and 0.12, respectively.

Identification of endogenous retroviral sequences based on modular organization: proviral structure at the SSAV1 locus.

The current genome sequencing projects reveal megabases of unknown genomic sequences. About 1% of these sequences can be expected to be of retroviral origin. These are often severely deleted or mutated. Therefore, identification of the retroviral origin of these sequences can be very difficult due to the absence of convincing overall sequence similarity. There are also many copies of solo-LTRs (long terminal repeats) distributed throughout genomic sequences. LTR and envelope sequences in general are among the most divergent parts of the retroviral genome and thus especially hard to detect in mutated endogenous sequences. We took advantage of the fact that these retroviral sections contain short highly conserved sequence regions providing retroviral hallmarks even after loss of overall similarity. We defined several sequence elements and peptide motifs within LTR and Env sequences and used these elements to construct models for LTRs and Env proteins of mammalian C-type retroviruses. We then used this strategy to identify successfully the hitherto missing LTRs and an env-like region in the S71 human retroviral sequence. Our approach provides a new strategy for identifying remotely related retroviral sequences in genomic DNA (especially human DNA), of potential significance for the interpretation of genomic sequences obtained from the current large-scale sequencing projects.

Cloning, sequencing and functional studies of the gene encoding human GTP cyclohydrolase I.

We have identified a genomic clone containing the 5' regulatory region of the gene GTP-CH encoding human GTP cyclohydrolase I. The transcription start point (tsp) was mapped by 5'-rapid amplification of cDNA ends (5'-RACE). The 2.6-kb region upstream from the tsp showed promoter activity when ligated upstream from a reporter gene. The truncation of approximately 2 kb of the promoter did not change expression activity, while a further removal of 243 bp halved the activity. The promoter contains CCAAT and TATA boxes. The GC-rich region close to the tsp, which contains several putative Sp1-responsive elements, is required for maximum promoter activity. Interferon-gamma treatment of B-cells transfected with reporter constructs had no influence on the expression activity.

Identification of S71-related human endogenous retroviral sequences with full-length pol genes.

The human genome contains sequences related to the simian sarcoma-associated virus SSAV. One of these endogenous retroviral elements, S71, is truncated in the pol gene and carries an insertion of a solitary HERV-K LTR. Using a PCR approach we have now identified further S71-related retroviral elements that lack the HERV-K LTR insertion and contain a full-length retroviral reverse transcriptase. Two of these sequences, pCRTK1 and pCRTK6, were cloned and further characterized. Clones pCRTK1 and pCRTK6 showed between 85 and 90% nucleotide homology to each other and to S71 within the "tether" region of the pol gene, indicating that pCRTK1 and pCRTK6 clearly belong to the S71 subgroup of C-type-related human endogenous retroviral elements. Some point mutations inactivating the reverse transcriptase are located at the same positions in pCRTK1 and pCRTK6. Therefore, we assume that these S71-related elements were dispersed in the human genome by reintegration as defective proviruses, probably using enzymes for retrotransposition provided in trans by other retrotransposons or by cellular genes. Examination of the presence of S71-related elements in apes and Old World monkeys revealed that the deletion of reverse transcriptase sequences in S71 has occurred in the lineage of primates prior to the insertion of the HERV-K LTR.

Multiple signal transduction pathways regulate discoidin I gene expression in Dictyostelium discoideum.

The expression of the discoidin I genes in Dictyostelium discoideum is regulated by the concerted action of the extracellular factors cyclic adenosine monophosphate (cAMP), folate, prestarvation factor (PSF) and conditioned media factor (CMF). However, the pathways by which these signals are transduced and the interactions between the pathways have been unexplored so far. We have analysed wild-type and mutant cells with defined lesions in signal transduction to elucidate these regulatory processes, and shown that different pathways are used for the down-regulation and induction of these genes. The cAMP receptor cARI is required for the cAMP-mediated down-regulation of discoidin I gene expression but not for the induction of discoidin I expression during development. Surprisingly, induction of the discoidin I genes requires G alpha 2, the G-protein subunit which is generally believed to couple to cARI, to control the expression of cAMP-inducible genes. Thus, our data suggest that G alpha 2 interacts with different receptors to regulate gene expression in early development. Furthermore, the analysis shows that discoidin induction in bacterially grown cells occurs in two sequential steps. The first is a low basal induction which occurs in late log-phase growth prior to starvation. PSF can induce the basal level, and the induction is independent of G alpha 2. The developmental induction following starvation is much stronger, dependent on G alpha 2 and probably signaled by CMF, which is secreted at that time.(ABSTRACT TRUNCATED AT 250 WORDS)

Folate responsiveness during growth and development of Dictyostelium: separate but related pathways control chemotaxis and gene regulation.

Folate-controlled gene expression and chemotaxis have been examined in Dictyostelium wild-type and mutant strains. We show that regulation of the discoidin genes is sensitive to folate in growing cells as well as in suspension development. The signal is transferred via the N10-methylfolate-sensitive folate receptor sites, which also appear to confer the chemotactic response. The strain HG5145 has previously been isolated as a mutant that does not display chemotactic movement towards folate. Nevertheless, these cells are fully functional in folate-mediated downregulation of discoidin I expression. The strain ga 93 has been isolated as an overproducer mutant of the cyclic nucleotide phosphodiesterase inhibitor. Simultaneously, these cells fail to downregulate discoidin I in response to folate but are fully functional in folate chemotaxis. Therefore we conclude that the pathways for chemotaxis and for gene regulation diverge downstream of a common receptor type.

Transcriptional regulation by folate: inducible gene expression in Dictyostelium transformants during growth and early development.

The Dictyostelium discoidin genes are induced in bacteria-grown cells shortly before the onset of development but are also highly expressed during growth in axenic medium. We here show that axenically growing cells strongly respond to the extracellular signal folate by suppressing discoidin synthesis while cell growth and development is not substantially affected. Repression occurs via two previously identified promoter elements, the dIE and the dAXE. Removal of the signal molecules or setting cells up for development results in rapid reactivation of the promoter. Based on this observation, we constructed the transformation vector pVEII and describe a convenient method which allows for controlled expression of a gene of interest in growing cells and also for external modulation in early development. Deletion constructs of the discoidin promoter can be used in addition to vary transcriptional activity over about one order of magnitude.

A cis-acting element responsible for early gene induction by extracellular cAMP in Dictyostelium discoideum.

We have analysed the promoter of the Dictyostelium discoideum alpha-L-fucosidase (ALF) gene, and have identified a 58 bp fragment responsible for transcriptional activation mediated by extracellular cAMP. Replacement of regulatory sequences in the cAMP-independent actin 15 promoter by this fragment confers cAMP regulation to the hybrid promoter. A cAMP analog with high affinity to the cell surface cAMP receptor can induce transcription from the endogenous as well as from the hybrid promoter. Gel-shift experiments show that the 58 bp fragment is a target for nuclear DNA-binding proteins, and that a specific complex is formed in response to cAMP stimulation. The major cAMP-dependent DNA.protein complex is formed with a 22 bp subfragment which we have termed DCRE (Dictyostelium cAMP responsive element).

Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.