Transcription factor C/EBP-ß induces tumor-suppressor phosphatase PHLPP2 through repression of the miR-17-92 cluster in differentiating AML cells.

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPß, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPß-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.

Microbial biogeochemistry of Boiling Springs Lake: a physically dynamic, oligotrophic, low-pH geothermal ecosystem.

Boiling Springs Lake (BSL) in Lassen Volcanic National Park, California, is North America's largest hot spring, but little is known about the physical, chemical, and biological features of the system. Using a remotely operated vessel, we characterized the bathymetry and near-surface temperatures at sub-meter resolution. The majority of the 1.2 ha, pH 2.2 lake is 10 m deep and 50-52 °C, but temperatures reach 93 °C locally. We extracted DNA from water and sediments collected from warm (52 °C) and hot (73-83 °C) sites separated by 180 m. Gene clone libraries and functional gene microarray (GeoChip 3.0) were used to investigate the BSL community, and uptake of radiolabeled carbon sources was used to assess the relative importance of heterotrophic vs. autotrophic production. Microbial assemblages are similar in both sites despite the strong temperature differential, supporting observations of a dynamic, convectively mixed system. Bacteria in the Actinobacteria and Aquificales phyla are abundant in the water column, and Archaea distantly related to known taxa are abundant in sediments. The functional potential appears similar across a 5-year time span, indicating a stable community with little inter-annual variation, despite the documented seasonal temperature cycle. BSL water-derived DNA contains genes for complete C, N, and S cycles, and low hybridization to probes for N and S oxidation suggests that reductive processes dominate. Many of the detected genes for these processes were from uncultivated bacteria, suggesting novel organisms are responsible for key ecosystem services. Selection imposed by low nutrients, low pH, and high temperature appear to result in low diversity and evenness of genes for key functions involved in C, N, and S cycling. Conversely, organic degradation genes appear to be functionally redundant, and the rapid assimilation of radiolabeled organic carbon into BSL cells suggests the importance of allochthonous C fueling heterotrophic production in the BSL C cycle.

Pilot study to assess the effects of early flea exposure on the development of flea hypersensitivity in cats.

This pilot study was to determine if early oral flea exposure reduces the incidence of flea allergy dermatitis (FAD) in cats. Eighteen kittens, assigned to three groups, received no flea exposure, oral flea exposure or flea infestation for 12 weeks. Then all the kittens were exposed continually to fleas for 31 weeks. Sensitization was monitored using intradermal testing (IDT), in vitro measurement of anti-flea saliva immunoglobulin E (IgE) and development of FAD. There was no statistically significant difference between groups in IDT reactions, in vitro data or clinical scores. The development of FAD was not associated with the presence of anti-flea saliva IgE. However, the development of a delayed reaction to flea bite was associated with symptoms after flea exposure. Although not statistically significant, the FAD scores in the oral group were lower than in the controls. Further studies are required to determine the role of oral flea exposure in the development of FAD in cats.

Viruses from extreme thermal environments.

Viruses of extreme thermophiles are of great interest because they serve as model systems for understanding the biochemistry and molecular biology required for life at high temperatures. In this work, we report the discovery, isolation, and preliminary characterization of viruses and virus-like particles from extreme thermal acidic environments (70-92 degrees C, pH 1.0-4.5) found in Yellowstone National Park. Six unique particle morphologies were found in Sulfolobus enrichment cultures. Three of the particle morphologies are similar to viruses previously isolated from Sulfolobus species from Iceland and/or Japan. Sequence analysis of their viral genomes suggests that they are related to the Icelandic and Japanese isolates. In addition, three virus particle morphologies that had not been previously observed from thermal environments were found. These viruses appear to be completely novel in nature.

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs.

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.

Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs.

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor.

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.

Viruses of the extremely thermophilic archaeon Sulfolobus.

Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses, and the primeval existence of common ancestors. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future.

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

pING family of conjugative plasmids from the extremely thermophilic archaeon Sulfolobus islandicus: insights into recombination and conjugation in Crenarchaeota.

A novel family of conjugative plasmids from Sulfolobus comprising the active variants pING1, -4, and -6 and the functionally defective variants pING2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly. In view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for Sulfolobus, we compared the functions and sequences of these variants and the previously described archaeal conjugative plasmid pNOB8 in order to identify open reading frames (ORFs) and DNA sequences that are involved in conjugative transfer and maintenance of these plasmids in Sulfolobus. The variants pING4 and -6 are reproducibly derived from pING1 in vivo by successive transpositions of an element from the Sulfolobus genome. The small defective but mobile variants pING2 and -3, which both lack a cluster of highly conserved ORFs probably involved in plasmid transfer, were shown to be formed in vivo by recombinative deletion of the larger part of the genomes of pING4 and pING6, respectively. The efficient occurrence of these recombination processes is further evidence for the striking plasticity of the Sulfolobus genome.

A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen.

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.

The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus.

A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co-transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading.

Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors.

Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15. 5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.

Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon.

The complete nucleotide sequence of the archaeal conjugative plasmid, pNOB8, from the Sulfolobus isolate NOB8-H2, was determined. The plasmid is 41,229 bp in size and contains about 50 ORFs. Several direct sequence repeats are present, the largest of which is a perfect 85-bp repeat and a site of intraplasmid recombination in foreign Sulfolobus hosts. This recombination event produces a major deletion variant, pNOB8-33, which is not stably maintained. Less than 20% of the ORFs could be assigned putative functions after extensive database searches. Tandem ORFs 315 and 470, within the deleted 8-kb region, show significant sequence similarity to the protein superfamilies of ParA (whole protein) and ParB (N-terminal half), respectively, that are important for plasmid and chromosome partitioning in bacteria. A putative cis-acting element is also present that exhibits six 24-mer repeats containing palindromic sequences which are separated by 39 or 42 bp. By analogy with bacterial systems, this element may confer plasmid incompatibility and define a group of incompatible plasmids in Archaea. Although several ORFs can form putative trans-membrane or membrane-binding segments, only two ORFs show significant sequence similarity to bacterial conjugative proteins. ORF630b aligns with the TrbE protein superfamily, which contributes to mating pair formation in Bacteria, while ORF1025 aligns with the TraG protein superfamily. We infer that the conjugative mechanism for Sulfolobus differs considerably from known bacterial mechanisms. Finally, two transposases were detected; ORF413 is flanked by an imperfect 32-bp inverted repeat with a 5-bp direct repeat at the ends, and ORF406 is very similar in sequence to an insertion element identified in the Sulfolobus solfataricus P2 genome.

Conjugation in archaea: frequent occurrence of conjugative plasmids in Sulfolobus.

We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeon Sulfolobus islandicus, which were obtained by plating of samples from Icelandic solfataras after liquid enrichment. They are related to each other and to the previously described CP pNOB8 from a Japanese Sulfolobus strain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences. All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells. Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions. Some of these CPs exclude superconjugation of the transcipients with closely related CPs. The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients. The stability of the CPs is higher in their original hosts or in related S. islandicus strains, than in Sulfolobus solfataricus strain PH1 as recipient. The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6. The dissection of genes and functions has been initiated by characterizing this incomplete variant.

Genetic elements in the extremely thermophilic archaeon Sulfolobus.

This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons.

Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface.

The bacterial enhancer-binding protein NtrC activates transcription when phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substitutions. The N-terminal domain of NtrC, which acts positively on the remainder of the protein, is homologous to a large family of signal transduction domains called receiver domains. Phosphorylation of an aspartate residue in a receiver domain modulates the function of a downstream target, but the accompanying structural changes are not clear. In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying constitutively activating substitutions. Combinations of such substitutions resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein. Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site. Interestingly, the activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face. Thus, the active site and the 3445 face appear to interact. Implications with respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainder of NtrC are discussed.

Unique long terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome.

Ovine pulmonary carcinoma (OPC) is a contagious lung cancer of sheep that is presumed to be caused by an exogenous retrovirus of sheep, jaagsiekte sheep retrovirus (JSRV). The sheep genome carries 15 to 20 copies of endogenous sheep retrovirus (ESRV) loci that hybridize to JSRV DNA probes. In order to clarity the etiologic roles of ESRV and an exogenous JSRV-like retrovirus (exJSRV) in OPC, we assessed sequence differences between ESRV and JSRV. Molecular characterization of six ESRV loci revealed restriction sites specific for JSRV. Nucleotide sequences of ESRVs from sheep of different breeds were similar to those of JSRV in structural genes but divergent in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for use in the PCR. Of 13 tumor DNAs tested by PCR with these exogenous-virus U3 primers, 8 produced DNA fragments that hybridized with the JSRV gag probe, but neither lung DNAs from healthy sheep nor DNAs from nontumor tissues of diseased sheep produced similar DNA fragments. exJSRV PCR products from tumor DNAs of sheep with OPC from three continents had restriction profiles similar to each other but different from those of ESRVs upon digestion with EcoRI, HindIII, NdeI, KpnI, and ScaI. These exjSRVs could be classified into two genotypes according to U3 sequences and restriction profiles. U3 sequences of exJSRV proviruses in tumors strongly resembled those of JSRV but differed from those of ESRVs, suggesting that exJSRVs, rather than ESRVs, are primarily associated with oncogenesis in OPC.

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.

The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep.

Sheep pulmonary adenomatosis ([SPA] ovine pulmonary carcinoma) is a transmissible lung cancer of sheep that has been associated etiologically with a type D- and B-related retrovirus (jaagsiekte retrovirus (JSRV]). To date it has been impossible to cultivate JSRV in vitro and therefore to demonstrate the etiology of SPA by a classical approach. In addition, the presence of 15 to 20 copies of endogenous JSRV-related sequences (enJSRV) has hampered studies at the molecular level. The aim of this study was to investigate whether the expression of exogenous JSRV was specifically associated with neoplasia in SPA-affected animals. Initially, we found that enJSRVs were transcribed in a wide variety of normal sheep tissues. Then, by sequencing part of the gag gene of enJSRV we established a ScaI restriction site in gag as a molecular marker for the exogenous form of JSRV. Restriction enzyme digestion of PCR products obtained from the amplification of cDNA from a total of 65 tissues collected from SPA-affected and unaffected control sheep revealed that the exogenous form of JSRV was exclusively and consistently present in tumor tissues and lung secretions of the affected animals. In addition, exogenous JSRV provirus was detected only in DNA from SPA tumors and not from nontumor tissues of the same animals. This study has demonstrated clearly that the exogenous form of JSRV is specifically associated with SPA tumors.