SAg Genes Research Articles

Strain Discrimination of Staphylococcus aureus Using Superantigen Profiles.

Staphylococcus aureus is one of the major bacterial species that may cause clinical infection and food-poisoning cases. Strains of this species may produce a series of superantigens (SAgs). Due to the importance of staphylococcal infections, reliable methods for the discrimination of strains of this species are important. Such data may allow us to trace the infection origins and be used for epidemiological study. For strain discrimination, genotyping methods, such as pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), and multi-locus sequence typing (MLST), etc., could be used. Recently, toxin gene profiles, which can be used for the elucidation of the genetic and pathogenic relatedness between strains, also have been used to improve the strain discrimination. For S. aureus, as more SAg genes were discovered, the SAg profiles become more useful for the strain discrimination of S. aureus. In this chapter, a method for the discrimination of S. aureus strains using superantigen profiles will be described in detail.

Association of Retinal Artery and Other Inner Retinal Structures With Distribution of Tapetal-like Reflex in Oguchi's Disease.

To report novel ophthalmoscopic features of patients with Oguchi's disease, and to describe how they may be related to the unusual tapetal-like fundus appearance. Twenty-one eyes of 11 patients who were diagnosed with Oguchi's disease were investigated. Genetic screening of seven cases showed homozygous mutations in the SAG gene (c.926delA). The retinal appearance was retrospectively assessed in the fundus photographs, and the optical coherence tomographic (OCT) and fundus autofluorescence (AF) images. In 11 eyes of 7 patients, clearly demarcated dark regions without tapetal-like reflex were observed in the midperipheral retinal regions. In the dark regions, OCT showed lower reflectances in the photoreceptor layer but the AF images had normal reflectances. In nine eyes of six patients, the dark regions were partially demarcated by retinal arteries but not by veins. In nine eyes of five patients, the extent of the dark regions either increased or decreased during the course of the disease process, and these changes were not due to the state of adaptation or a posterior vitreous detachment. In all eyes, the peripheral retinal arteries but not veins had either high or low reflective regions along one side. Although the alterations of the outer retinal layers are believed to be most responsible for the abnormal tapetal-like reflex in patients with Oguchi's disease, these ophthalmoscopic features cannot be explained solely by the abnormality of the outer retina. Our findings suggest that the appearance of tapetal-like reflex is strongly affected by alterations of structures in the inner retinal layers.

Down-regulation of catalase activity contributes to senescence induction in wheat leaves exposed to shading stress

In shaded wheat (Triticum aestivum L.) leaves, the suppression of blue radiation (BR) triggers senescence. This phenomenon is correlated to an increase in oxidative stress symptoms and a decrease of catalase (CAT) activity, among other traits. Previous data suggest that the radiation signal transduction pathway may involve changes in Ca2+ and H2O2 homeostasis. For better a understanding of the interaction among the spectral composition of radiation, Ca2+ availability, and the antioxidant metabolism in the regulation of shade-induced senescence, detached wheat leaves were placed in a growth chamber and exposed to either blue (B, high BR transmittance) and/or green (G, very low BR transmittance) Lee® filters in the absence or presence of 0.8 mM verapamil (a Ca2+ channels blocker), 4.0 mM EGTA (a Ca2+chelator), or 8.0 mM 3-amino-1,2,4-triazole (a CAT inhibitor). At defined time points, the leaf samples were analyzed for changes in chlorophyll content, specific activities of CAT, ascorbate peroxidase (APX), and guaiacol peroxidase (POX), CAT isozymes, and gene expression of CAT1, CAT2, and two senescence markers (TaSAG1 and TaSAG3). BR transmittance decreased the chlorophyll degradation rate and SAG genes expression either in leaves continuously exposed under the B filter, as well as in leaves previously exposed under the G filter. The effect of BR was associated with the maintenance of a high CAT (but not APX and POX) activity, and it was suppressed either in the presence of 3-AT or when Ca2+ availability was decreased. BR altered the CAT activity both at the transcriptional and at the posttranscriptional level. Nevertheless, different responses of CAT isozymes and CAT genes expression profiles to specific treatment combinations indicate that they differed in their regulatory pathways.

Superantigens in Staphylococcus aureus isolated from prosthetic joint infection

Staphylococcus aureus is a common cause of prosthetic joint infection (PJI). The prevalence of superantigens (SAgs) among PJI-associated S. aureus is unknown. Eighty-four S. aureus isolates associated with PJI isolated between 1999 and 2006 were studied. SAg genes, sea, seb, sec, sed, see, seg, seh, sei, and tst, were assayed by PCR. Seventy-eight (92.9%) isolates carried at least 1 SAg gene studied, with 61 (72.6%) harboring more than 1. seg was most commonly (70.2%), and seh was least frequently (4.8%) detected. tst-positive isolates were associated with early infection and increased erythrocyte sedimentation rate at diagnosis (P=0.006 and P=0.021, respectively). seg and sei were associated with methicillin resistance (P=0.008 and P=0.002, respectively). A majority of PJI-associated isolates studied produced biologically active SAgs in both planktonic and biofilm growth modes. SAg genes are prevalent in S. aureus causing PJI.

The association of apoptotic protein expressions sensitive to apoptosis gene, p73 and p53 with the prognosis of cervical carcinoma.

ObjectiveTo evaluate the expressions of several apoptotic pathway proteins in relation to clinical parameters and survival in patients with cervical carcinoma.MethodsA total of 20 patients with clinically advanced staged carcinoma of cervix (International Federation of Gynecology and Obstetrics [FIGO] stage IIB-IVA) aged from 40 to 75 years were included in this study. The expression profile of anti-apoptotic protein (sensitive to apoptosis gene [SAG]), mitochondrial apoptotic proteins (B-cell lymphoma-extra-large [Bcl-xL] and Bcl-2 homologous antagonist/killer [Bak]), and tumor suppressor proteins (p73 and p53) were examined by real-time polymerase chain reaction experiments along with their relation to clinical parameters and survival analyses during follow-up for 5 to 8 years.ResultsNo significant difference was found in the expressions of SAG, Bcl-xL, Bak, p73 and p53 proteins with respect to stage and grade of tumor. A significant positive correlation was noted between SAG and Bcl-xL genes (r=0.752, P<0.001) and between SAG and Bak genes (r=0.589, P=0.006). Among genes determined to be significantly associated with overall survival in the univariate analysis (P=0.026 for SAG, P=0.002 for Bcl-xL, and P=0.027 for p53), only p53 was identified as the significant predictor in the multivariate analysis (hazard ratio: 8.53, 95% confidence interval: 1.34–54.2, P=0.023).ConclusionIn conclusion, our findings demonstrated a reverse correlation of SAG, Bcl-xL, and p53 expressions with overall survival of patients. No association of apoptotic pathway proteins with clinicopathological characteristics of cervical carcinoma patients was noted. Low SAG, Bcl-xL, and p53 expression levels revealed to be useful as prognostic predictors in patients with cervical carcinoma.

Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences

The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3′ long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

Identification of β‐haemolysin‐encoding genes in Streptococcus anginosus

Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for β-haemolysis we performed genomic mutagenesis of the β-haemolytic S.anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. Integration site analysis of 15 non-haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S (SLS) encoding sag gene cluster of S.pyogenes. The gene cluster harbours 10 open reading frames displaying significant similarities to the S.pyogenes genes sagA-sagI, with the identities on protein level ranging from 38 to 87%. Complementation assays of S.anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for β-haemolysin production and suggest the presence of post-translational modifications in S.anginosus SLS similar to SLS of S.pyogenes. Characterization of the S.anginosus haemolysin in comparison to the S.pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S.pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S.anginosus in culture supernatants. Inhibition of β-haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore-forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of Streptococcus constellatus, a closely related streptococcal species that belongs to the S.anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S.anginosus and demonstrate its importance for β-haemolysin production in S.anginosus.

Evolutionary paths of streptococcal and staphylococcal superantigens

BackgroundStreptococcus pyogenes (GAS) harbors several superantigens (SAgs) in the prophage region of its genome, although speG and smez are not located in this region. The diversity of SAgs is thought to arise during horizontal transfer, but their evolutionary pathways have not yet been determined. We recently completed sequencing the entire genome of S. dysgalactiae subsp. equisimilis (SDSE), the closest relative of GAS. Although speG is the only SAg gene of SDSE, speG was present in only 50% of clinical SDSE strains and smez in none. In this study, we analyzed the evolutionary paths of streptococcal and staphylococcal SAgs.ResultsWe compared the sequences of the 12–60 kb speG regions of nine SDSE strains, five speG+ and four speG–. We found that the synteny of this region was highly conserved, whether or not the speG gene was present. Synteny analyses based on genome-wide comparisons of GAS and SDSE indicated that speG is the direct descendant of a common ancestor of streptococcal SAgs, whereas smez was deleted from SDSE after SDSE and GAS split from a common ancestor. Cumulative nucleotide skew analysis of SDSE genomes suggested that speG was located outside segments of steeper slopes than the stable region in the genome, whereas the region flanking smez was unstable, as expected from the results of GAS. We also detected a previously undescribed staphylococcal SAg gene, selW, and a staphylococcal SAg -like gene, ssl, in the core genomes of all Staphylococcus aureus strains sequenced. Amino acid substitution analyses, based on dN/dS window analysis of the products encoded by speG, selW and ssl suggested that all three genes have been subjected to strong positive selection. Evolutionary analysis based on the Bayesian Markov chain Monte Carlo method showed that each clade included at least one direct descendant.ConclusionsOur findings reveal a plausible model for the comprehensive evolutionary pathway of streptococcal and staphylococcal SAgs.

Staphylococcal superantigens in colonization and disease

Superantigens (SAgs) are a family of potent immunostimulatory exotoxins known to be produced by only a few bacterial pathogens, including Staphylococcus aureus. More than 20 distinct SAgs have been characterized from different S. aureus strains and at least 80% of clinical strains harbor at least one SAg gene, although most strains encode many. SAgs have been classically associated with food poisoning and toxic shock syndrome (TSS), for which these toxins are the causative agent. TSS is a potentially fatal disease whereby SAg-mediated activation of T cells results in overproduction of cytokines and results in systemic inflammation and shock. Numerous studies have also shown a possible role for SAgs in other diseases such as Kawasaki disease (KD), atopic dermatitis (AD), and chronic rhinosinusitis (CRS). There is also now a rich understanding of the mechanisms of action of SAgs, as well as their structures and function. However, we have yet to discover what purpose SAgs play in the life cycle of S. aureus, and why such a wide array of these toxins exists. This review will focus on recent developments within the SAg field in terms of the molecular biology of these toxins and their role in both colonization and disease.

Similar Superantigen Gene Profiles and Superantigen Activity in Norwegian Isolates of Invasive and Non‐Invasive Group A Streptococci

Group A streptococcus (GAS) harbours several virulence factors, including M protein (coded by the emm gene) and superantigens (SAgs). SAgs are extracellular toxins that directly activate the immune system by cross-binding to the HLA class II molecule and T cell receptor (TCR), thereby causing activation of up to 30% of the T cells and subsequent massive secretion of cytokines. Forty-eight GAS strains isolated from patients at Norwegian hospitals between 1988 and 2004 were included in this study. Of these, 24 were invasive streptococcal toxic shock syndrome (STSS) or necrotizing fasciitis (NF) isolates and 24 were non-invasive pharyngitis isolates, matched for having the same T-type and year of isolation as the invasive isolates. The isolates were characterized by emm sequence typing, multilocus sequence typing (MLST) and SAg gene profiles. A correlation between T-type, emm type, sequence type and SAg gene profile was revealed. No difference between invasive and non-invasive isolates regarding serotype or genotype was demonstrated. Selected invasive and non-invasive isolates with identical SAg gene profiles were analysed for SAg activity in bacterial growth culture media with and without human cell culture media added. A human T cell proliferation assay was used as measurement for SAg activity and simultaneously we also measured the cytokine content in normal human peripheral blood leucocyte cell culture media. The results revealed that invasive and non-invasive isolates did not differ significantly in SAg activity as it is present in semipurified bacterial culture medium.

Mizuo-Nakamura phenomenon in Oguchi disease due to a homozygous nonsense mutation in the SAG gene

Mizuo-Nakamura phenomenon in Oguchi disease due to a homozygous nonsense mutation in the SAG gene

Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens

Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.

Characterization of Infecting Strains and Superantigen-Neutralizing Antibodies in Staphylococcus aureus Bacteremia

Staphylococcus aureus superantigens (SAgs) are highly potent T cell mitogens. Antibodies against non-enterotoxin gene cluster (non-egc) SAgs are common in healthy adults, whereas neutralizing antibodies against egc SAgs are rare. We investigated the infecting S. aureus strains and the anti-SAg antibody response during S. aureus bacteremia (SAB). This prospective clinical study (www.clinicaltrials.gov, NCT00548002) included 43 injection drug users (IDUs) and 44 group-matched nonaddicts with SAB. spa genotypes and SAg gene patterns (multiplex PCR) of the S. aureus isolates were determined. The neutralizing capacities of sera obtained at the acute phase and the convalescent phase of SAB were tested against the SAg cocktail of the respective infecting strain and a panel of recombinant SAgs. The lineages CC59 and CC30 were more prevalent among bacteremia strains from IDUs than among strains from nonaddicts. SAg gene patterns in isolates from IDUs and nonaddicts were similar. At the acute phase of bacteremia, IDUs had more neutralizing antibodies against non-egc SAgs than did nonaddicts. Antibody titers frequently increased during infection. In contrast, there were no neutralizing antibodies against egc SAgs at disease onset and such antibodies were not induced by SAB. SAB triggers an antibody response only against non-egc SAgs. Preimmunization in IDU patients is probably due to previous exposure to the infecting strain.

Detection of Multiple Superantigen Genes in Stools of Patients with Kawasaki Disease

To investigate whether superantigens (SAgs) are involved in the development of Kawasaki disease (KD) by examining SAg genes in the stool of patients with KD. Stool specimens were obtained from 60 patients with KD and 62 age-matched children (36 children with acute illness and 26 healthy children). Total DNA was extracted from these stool samples. Using polymerase chain reaction, we examined genes of 5 SAgs: streptococcal pyrogenic exotoxin-A (SPE-A), SPE-C, SPE-G, SPE-J, and toxic shock syndrome toxin-1. At least 1 of the 5 SAg genes was detected in 42 (70%) specimens from patients with KD, 14 (38.9%) from the febrile group, and 7 (26.9%) from the healthy group. The detection rate between subjects with and without KD was of at least 1 of the 5 SAg genes (P < .001), and more than 2 SAg genes were significantly different (P = .002). SAg may be involved in the development of KD; data suggest that multiple SAgs may trigger KD.

PCR detection of Staphylococcal enterotoxins (SEs) N, O, P, Q, R, U, and survey of SE types in Staphylococcus aureus isolates from food-poisoning cases in Taiwan

Staphylococcal enterotoxins (SEs) are superantigenic toxins. They are five major classical types, i.e., SEA, SEB, SEC, SED, SEE, and new SEs or SE-like superantigens, such as SEG to SEU. Only the staphylococcal superantigens (SAgs) that induce emesis following oral administration in a monkey model are designated as SEs while other related toxins are called SE-like (SEl) superantigens. To survey the enterotoxin genotypes for S. aureus strains isolated from food-poisoning cases in Taiwan, we developed PCR primers specific for SEN, SEO, SEP, SEQ, SER, and SEU genes. The complete SE sequences and their expression potential for strains positive to sen, seo, sep, seq, ser, and seu specific primers were also determined. These strains were used as reference strains. With the PCR primers specific for all SEs or SAgs, including toxic shock syndrome toxin I (TSST-1), we assayed the genotypes of 147 S. aureus strains isolated from patients associated with staphylococcal food-poisoning outbreaks occurred during 2001–2003. For these147 strains, 135 (91.8%) were found positive for one or more SE or SAg genes. For classical enterotoxin and TSST-1 types, the major one was tsst-1 (59.1%) following by sea (29.2%), seb (19.7%), sec (6.8%), and sed (2.0%). For new SE and SAg types, the major one was sei (29.9%) and sep (27.9%) followed by, sek (16.3%), seo (14.3%), seu (14.2%), sem (11.6%), sen (10.9%), seq (10.9%), seh (8.2%), sel (6.8%), and ser (5.4%) etc. This report reveals the whole SE and SAg genotypes for S. aureus strains isolated from staphylococcal food-poisoning cases in Taiwan.

A new closed-tube multiplex real-time PCR to detect eleven superantigens of Streptococcus pyogenes identifies a strain without superantigen activity

Superantigens (SAgs) are very potent microbial toxins that are involved in severe diseases such as necrotizing fasciitis and toxic shock syndrome. There are currently 11 different SAgs that have been identified from Streptococcus pyogenes. In the present study, two sets of multiplex PCRs were developed for detection of these 11 SAg genes. The first group comprises spea1−3 +5, spec, speg, spej, spek, and spel. The second group consists of spea1−4, speh, spei, spem, ssa, and smez. The presence of Streptococcus pyogenes SAg genes can be immediately identified using a real-time method with SYBR-Green, thus providing an excellent tool in clinical diagnostics. After testing more than 300 clinical isolates, we identified one strain without any SAg gene. This finding contrasts with previous reports describing SAg genes located on every Streptococcus pyogenes genome. This SAg gene-negative strain also did not show any mitogenic activity. It is hypothesized that clinical isolates from patients may overrepresent bacterial strains with pathogenic factors, such as SAgs.

Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea

A multiplex PCR assay that allows for the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs) ( sea to see, and seg to sei), SE-like (SEl) toxins ( sej to ser, and seu), and toxic shock syndrome toxin-1 (TSST-1) ( tst) was developed in this study. These toxins are included in the pyrogenic toxin superantigen (PTSAg) family and are responsible for many diseases such as staphylococcal food poisoning (SFP) and TSS. The primers were designed based on dual priming oligonucleotide (DPO) technology to detect all of the 19 SAg genes in three sets of PCR. The developed multiplex PCR was applied to 143 Staphylococcus aureus strains isolated from pork and chicken meat in Korea. Almost 50% of the strains possessed at least one of the 19 SAg genes. The most frequently found genes were seg, sei, sem, and sen (53 isolates, 37%), which were often found simultaneously in the same isolate. In those isolates, the seo (39 isolates, 27%) or seu (6 isolates, 4%) genes were frequently found together and this combination ( seg, sei, sem, sen, and seo or seu) was considered to be a part of the enterotoxin gene cluster ( egc). The sea gene (10 isolates, 7%) was the gene most frequently detected out of all the classical SE genes ( sea to see). Although these classical SEs are considered to be major etiological factors in SFP, newly described SE or SEl genes ( seg to ser, and seu) were more frequently detected than the classical SE genes in this study. There was no isolate detected containing the seb, sec, sek, sel, or seq genes. S. aureus possessing mobile genetic elements known to encode these SAg genes, such as egc, were presumed to be widely distributed among pork and chicken meats in Korea. The multiplex PCR developed in this study could be applied to the investigation of SAg genes in S. aureus strains isolated from various sources.

Superantigen types inStaphylococcus aureus isolated from patients with cystic fibrosis

The screening of 17 SAg genes of S. aureus isolated from the sputum of cystic fibrosis (CF) patients revealed that among 47 genetically different strains, 39 (83 %) carried SAg genes. Superantigens forming enterotoxin gene cluster were detected in 20 strains. The 2nd most common superantigen type was selk detected in 13 strains. In 9 strains, selk occurred together with the sea gene. Out of 74 strains recovered from nasal carriers, 56 (75 %) were found to carry SAg genes, 38 carried egc genes, while selk was detected in 5 strains. The predominant SAg types in both investigated S. aureus populations were egc and selk/sea, but selk gene frequency was significantly higher in the CF-derived strains.

INDUCTION OF MOUSE MAMMARY TUMOR VIRUS SUPERANTIGEN IN THE SPLEEN AFTER BURN INJURY

A significant portion (~8 to 10%) of human and mouse genome consists of endogenous retroviruses (ERVs). In particular, there are two major types of ERVs in the mouse genome; murine leukemia virus (MuLV) and mouse mammary tumor virus (MMTV). Recent studies from several laboratories including ours suggest that ERVs are involved in various disease processes, such as injury response, multiple sclerosis, and diabetes. We hypothesized that endogenous MMTVs participate in the systemic immune response after burn injury through the modulation of their superantigen (Sag) gene expression. The recognition of MMTV Sag by T cell receptors with certain Vβ chains leads to cell proliferation and cytokine production. Female C57BL/6J mice were subjected to 18% TBSA flame burn followed by tissue collection at 3 hours and 1 day postburn. RT-PCR analysis using a primer set spanning the MMTV Sag splicing junction was performed to investigate the postburn changes in the MMTV Sag expression in the liver and spleen. Subsequently, the MMTV Sag transcripts were further characterized by cloning, sequencing, multiple alignment, and in silico genomic mapping. There was a substantial and transient induction of MMTV Sag expression in the spleen, but not in the liver, at 3 hours postburn. Two MMTV Sag transcripts were isolated from the band induced in the spleen. Sequencing analysis demonstrated that they were unique among the pool of diverse MMTV Sag sequences, both genomic as well as transcripts from 17 different tissues. In addition, a single copy of endogenous MMTV harboring these Sag sequences with more than 98% homology was mapped to chromosome 4. The biological roles of these MMTV Sag genes isolated from the spleen in postburn systemic response are currently being determined. The data obtained from these studies provide a new insight into the understanding of postburn pathologic changes through the biology of MMTV Sag genes.

Clonal isolation of different strains of mouse mammary tumor virus-like DNA sequences from both the breast tumors and non-Hodgkin's lymphomas of individual patients diagnosed with both malignancies.

In a previous study, we had detected the presence of mouse mammary tumor virus (MMTV)-like envelope (ENV) gene sequences in both the breast tumors and non-Hodgkin's lymphoma tissue of two of our breast tumor patients who had been diagnosed simultaneously with both malignancies. The aim of this study was to determine if MMTV-like DNA sequences are present in the breast tumors and non-Hodgkin's lymphomas of additional patients suffering from both malignancies and if so to characterize these sequences in detail. DNA was extracted from formalin-fixed, paraffin-embedded tissue sample blocks of breast tumors and non-Hodgkin's lymphomas from patients suffering from both malignancies. A 250-bp region of the MMTV ENV gene and a 630-bp region of the MMTV long terminal repeat (LTR) open reading frame (ORF) that encodes the MMTV superantigen (sag) gene were amplified by PCR from the isolated DNA. Amplified products were analyzed by Southern blotting, cloned, and sequenced. MMTV-like ENV and LTR sequences were detected in both the breast tumors and non-Hodgkin's lymphomas of 6 of 12 patients suffering from both malignancies. A novel mutant of the MMTV ENV gene was identified in these patients. Characterization of the MMTV-like LTR highly variable sag sequences revealed total or nearly total identity to three distinct MMTV proviruses from two different branches of the MMTV phylogenetic tree. The presence of MMTV-like ENV and LTR sequences in both the breast tumors and non-Hodgkin's lymphomas of 6 additional patients suggests a possible involvement of these sequences in these two malignancies. MMTV-like LTR sequence homology to different MMTV proviruses revealed the presence of more than one strain of MMTV-like sequences in each individual suggesting the possibility of multiple infections in these patients.