Xenotransplantation of porcine organs might provide an unlimited source of donor organs to treat endstage organ failure diseases in humans. However, pigs harbour retroviruses with unknown pathogenic potential as an integral part of their genome. While until recently the risk of interspecies transmission of these porcine endogenous retroviruses (PERV) during xenotransplantation has been thought to be negligible, several reports on infection of human cells in vitro and spread of PERV from transplanted porcine islets in murine model systems have somewhat challenged this view. Here, we compile available data on PERV biology and diagnostics, and discuss the significance of the results with regard to the safety of clinical xenotransplantation.
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.
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.
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.
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.
The early transcription unit 3 (E3) of adenoviruses (Ads) encodes immunomodulatory functions. We previously described a novel gene of 49K within the E3 region of Ad19a, an Ad of subgenus D that is similar to Ad8 and Ad37 causes epidemic keratoconjunctivitis (EKC). Interestingly, 49K was reported not to be present in Ad9 and Ad17, other subgenus D Ads not causing EKC. Therefore, we investigated whether 49K is selectively expressed in EKC-causing Ads. Using specific DNA probes, we detect 49K-homologous genes in all subgenus D Ads tested. Moreover, 49K-specific antibodies recognize a high molecular weight protein in cells infected with all subgenus D serotypes irrespective of their ability to cause EKC. Sequencing of several 49K genes reveals a high homology without a distinct feature recognizable for those of EKC-associated Ad strains. Thus, E3/49K is a subgenus D specific E3 protein whose expression does not correlate with the EKC-causing phenotype and thus may rather be implicated in illnesses commonly caused by this subgenus. Interestingly, the 49K sequences of Ad19a and Ad37 are identical. To estimate the extent of the sequence identity between these two viruses, we initially sequenced the right ITR and the hexon. This analysis revealed that the right ITR of Ad19a is identical to Ad37, while the hexon sequence is Ad19p-like. This suggested that the region of identity is much larger and that Ad19a arose by recombination of Ad37 with an Ad19p-like Ad. Further sequencing mapped the crossover within the DNA binding protein. Thus, Ad19a contains a large sequence block ( approximately 13 kb), from the 100K gene to the right ITR, identical to Ad37. The implications of these findings in light of the temporal appearance of the EKC-causing Ad strains are discussed.
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