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At least 541 records · Page 30Linked to original sources
PubMed2006

Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria.

BACKGROUND: Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNAala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNAala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNAala genes. RESULTS: The three tRNAala genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNAalaU and tRNAalaV containing the attB site, a single base difference was observed between the two species. We observed that the tRNAalaU gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNAalaV gene was used as the target. No integration occurred in the BCG tRNAalaU T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNAala T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNAalaU and tRNAalaV T-loops of the same BCG chromosome. CONCLUSION: Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNAala genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies.

Attachment Sites, Microbiological↗
PubMed1997

Marking progress toward service integration: learning to use evaluation to overcome barriers.

While federal statutes defining categorical programs and funding streams are most often cited as barriers to service integration efforts, there is evidence that service integration efforts are also being stymied by factors at the state and local levels as well. This case study examines five types of barriers to service integration that have been evident in Georgia's Family Connection and describes how an evaluation instrument and process was developed to assess progress toward service integration at the local level. The findings of a pilot study of the use of this instrument suggest that it can help to: (a) define starting points for service integration efforts; (b) identify specific service integration activities that are feasible in the context of a unique community; (c) specify the time needed to be allocated to service integration efforts; (d) identify the local costs and benefits of engaging in service integration; and (e) provide specifications for conducting a self-assessment of progress at the local level toward service integration goals.

Benchmarking↗
PubMedDate not supplied

Primary care clinicians evaluate integrated and referral models of behavioral health care for older adults: results from a multisite effectiveness trial (PRISM-e).

BACKGROUND: Recent studies have shown that integrated behavioral health services for older adults in primary care improves health outcomes. No study, however, has asked the opinions of clinicians whose patients actually experienced integrated rather than enhanced referral care for depression and other conditions. METHOD: The Primary Care Research in Substance Abuse and Mental Health for the Elderly (PRISM-E) study was a randomized trial comparing integrated behavioral health care with enhanced referral care in primary care settings across the United States. Primary care clinicians at each participating site were asked whether integrated or enhanced referral care was preferred across a variety of components of care. Managers also completed questionnaires related to the process of care at each site. RESULTS: Almost all primary care clinicians (n = 127) stated that integrated care led to better communication between primary care clinicians and mental health specialists (93%), less stigma for patients (93%), and better coordination of mental and physical care (92%). Fewer thought that integrated care led to better management of depression (64%), anxiety (76%), or alcohol problems (66%). At sites in which the clinicians were rated as participating in mental health care, integrated care was highly rated as improving communication between specialists in mental health and primary care. CONCLUSIONS: Among primary care clinicians who cared for patients that received integrated care or enhanced referral care, integrated care was preferred for many aspects of mental health care.

Adult↗
PubMed2004

Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV), avian sarcoma-leukosis virus (ASLV), and murine leukemia virus (MLV). Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.

Avian Sarcoma Viruses↗
PubMed2006

Retroviral DNA integration: viral and cellular determinants of target-site selection.

Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

Attachment Sites, Microbiological↗
PubMed2005

Information integration in molecular bioscience.

Integrating information in the molecular biosciences involves more than the cross-referencing of sequences or structures. Experimental protocols, results of computational analyses, annotations and links to relevant literature form integral parts of this information, and impart meaning to sequence or structure. In this review, we examine some existing approaches to integrating information in the molecular biosciences. We consider not only technical issues concerning the integration of heterogeneous data sources and the corresponding semantic implications, but also the integration of analytical results. Within the broad range of strategies for integration of data and information, we distinguish between platforms and developments. We discuss two current platforms and six current developments, and identify what we believe to be their strengths and limitations. We identify key unsolved problems in integrating information in the molecular biosciences, and discuss possible strategies for addressing them including semantic integration using ontologies, XML as a data model, and graphical user interfaces as integrative environments.

Animals↗
PubMed1998

Facilitating an integrated approach to primary health care in Saskatchewan.

It is unlikely that truly integrated primary health care will become a reality unless health professionals actively strive to foster integration. Dialogue about integration barriers and opportunities has begun between the nursing, pharmacy, social work, emergency medical services, and medical professions. This dialogue should be expanded to include all of the professions with a potential role in an integrated system. The dialogue will also need to include government leaders as key public policy decisions will significantly influence the success or failure of integrated primary health service delivery strategies. As well, dialogue needs to occur with communities so that citizens can understand, value and help shape and maintain this kind of service delivery. Public education needs to emphasize that an integrated approach to primary health care in Saskatchewan is the best kind of service that can be provided. The Integrated Primary Health Care Working Group believes that the principles of primary health care require health professionals in Saskatchewan to develop an integrated approach to health services delivery in Saskatchewan. An exploration of possible barriers to this integrated approach is an important first step in eliminating barriers and facilitating effective health care service delivery to meet population health needs.

Delivery of Health Care, Integrated↗
PubMedDate not supplied

Integration site selection by retroviruses.

Integration into the host-cell genome is a critical step in the retrovirus life cycle. In particular, the choice of the integration site is crucial for retroviral replication, since integration at a site incompatible for high-level transcription may impair production of the progeny virus. Integration is not sequence specific, thus all chromosomal sites could potentially host integration events. However, this is not what is observed in vivo, where integrated viruses are preferentially detected in chromatin regions characterized by an open structure, a hallmark of actively transcribed genes. Target site selection might be influenced by several factors, including the function of cellular proteins that interact with integrase, the viral protein that catalyzes the integration reaction. Interestingly, a common functional feature that unifies these cellular co-factors is that, to a different extent, they are all involved in the regulation of chromatin structure or transcription. Inappropriate retroviral integration might lead to insertional mutagenesis and cellular transformation, as recently observed in a gene therapy clinical trial exploiting retroviral vectors for gene transfer into hematopoietic progenitors. Thus, the deeper understanding of the molecular mechanisms regulating integration site selection is also essential for the design of safer and more effective gene transfer vectors.

Chromatin↗
PubMed2005

[Detection of integration status of human papillomavirus 16 in cervical precancerous lesions].

OBJECTIVE: To investigate the prevalence of integration of human papillomavirus 16 (HPV16) DNA into the host genome in cervical squamous intraepithelial lesions (SIL). METHODS: Multiplex PCR was used to detect the HPV/HPV16 infection and integration status of HPV16 in the surplus cells from liquid-based cytological samples from 108 patients with cervical cancer precursor lesions. Consensus primers GP5+/GP6+ were used to amplify a 150 bp long fragment in the conserved region of the HPV L1 gene so as to detect the presence pf HPV. Scion Image 4.0 electrophoresis image analysis soft was used to calculate the E2/E6 ratio so as to evaluate the episomal and integrated status of HPV16 infection: in episomal form, both targets should be equivalent, and in integrated form, E2 gene would be absent, while in mixed form of episomal/integrated mixed form, the copy number of E2 would be less than that of E6. RESULTS: Sixty-two out of the 108 patients (57.41%) had HPV infection. HPV16 were found in 32 of the 108 samples (29.63%). Among the 32 cases HPV16 DNA was exclusively episomal in 15 cases (46.88%), concomitant in 13 cases (40.62%), and integrated in 4 cases (12.50%). The prevalence of integrated and/or concomitant forms of HPV-16 DNA increased with progression of cervical disease. The prevalence of integrated form was 54.55% in the patients of CIN3 type, 50.00% in CIN2 type, 28.07% in CIN1 type, and 11.54% in the inflammatory type with significant differences between any 2 groups (all P < 0.01). CONCLUSIONS: HPV16 integration into the host genome is already present in some of CIN lesions. The multiplex PCR estimation of the HPV L1, HPV16 E2, E6 genes and E2/E6 ratio could be a simple method for detecting HPV/HPV16 infection and its integration status in liquid-based residual samples. It would be a helpful complementary tool for cytological screening to identify those patients at high risk of developing high-grade squamous intraepithelial lesions and cervical cancer.

Adult↗
PubMed1995

Integration is the art of decomposition.

A growing demand for integrating stand-alone information systems with an HIS can be observed. Different forms of integration can be distinguished: technical integration, user interface integration, and integration of the information service. The integration of the information service appears to be especially difficult. An integrated information service can only be achieved when the applications use each other's information services. We will discuss three approaches for letting applications belonging to another system utilize the necessary information service of a host system. The first approach makes use of the user interface, and the necessary information service has to be extracted from the service to a user. Another, often used, approach is the extension of the applications with coupling modules. The coupling modules communicate messages and translate them into the context of the application. The applications are modified scarcely and the coupling modules have to supply the necessary information services. When the coupling problem is more complex (e.g., a larger set of data is to be exchanged interactively), a growing effort will be needed for these adapting modules. A better approach is to split the application into building blocks. Applications are composed of building blocks. The application, which should deliver the integrated information service, is also composed of such building blocks. The building blocks are designed to offer information services that can be used by other systems as well. If we succeed in decomposing the applications into suitable building blocks, an integrated information service could be more easily achieved.

Hospital Information Systems↗
PubMed1996

Expression of HPV16 E6 or E7 increases integration of foreign DNA.

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Cinnamates↗
PubMed1996

Particulate versus integrated evolution of the upper body in late pleistocene humans: a test of two models.

Evolutionary biologists are largely polarized in their approaches to integrating microevolutionary and macroevolutionary processes. Neo-Darwinians typically seek to identify population-level selective and genetic processes that culminate in macroevolutionary events. Epigeneticists and structuralists, on the other hand, emphasize developmental constraints on the action of natural selection, and highlight the role of epigenetic shifts in producing evolutionary change in morphology. Accordingly, the ways in which these paradigms view and address morphological contrasts between classes of related organisms differ. These paradigms, although seldomly explicitly stated, emerge in paleoanthropology as well. Considerations of postcranial morphological contrasts between archaic and modern humans typically fall into one of two broad interpretive models. The first derives from the neo-Darwinian perspective and holds that evolution in the postcranial skeleton was largely mosaic (operating in a particulate manner), and that temporal change in specific traits informs us about behavioral shifts or genetic evolution affecting isolated anatomical regions (i.e., adaptive behavioral inferences can be made from comparative studies of individual trait complexes). The alternative model follows from the epigeneticist paradigm and sees change in specific postcranial traits as correlated responses to change in overall body form (involving shifts in regulation of skeletal growth, or selective and developmental responses to broad adaptive shifts). By this view, integration of functional systems both constrains and directs evolution of various traits, and morphological contrasts inform us about overall change in body form related to change in such things as overall growth patterns, climatic adaptation, and technological dependency. These models were tested by confirmatory factor analysis using measures of upper body form and upper limb morphological traits in Eurasian Neandertal and early modern fossils and recent human samples. Results indicate (1) a model of morphological integration fits the data better than a model of no integration, but (2) this integration accounts for less than half of the variance in upper limb traits, suggesting a high degree of tolerance for particulate evolution in the context of an integrated upper body plan. Significant relationships were detected between joint shapes and body size, between humeral shaft shape and body size and chest shape, and between measures of biomechanical efficiency and robusticity. The observed morphological differences between late archaic and early modern humans reflect particulate evolution in the context of constraints imposed by genetic and morphological integration. While particulate approaches to interpreting the fossil record appear to be justified, attention must also be paid to delineating the nature and extent of morphological integration and its role in both constraining and producing observed patterns of variation between groups. Confirmatory factor analysis provides a means of examining trait covariance matrices, and serves as a useful method of identifying patterns of integration in morphology.

Biological Evolution↗
PubMed2006

Patterns of craniofacial integration in extant Homo, Pan, and Gorilla.

Brain size increased greatly during Pleistocene human evolution, while overall facial and dentognathic size decreased markedly. This mosaic pattern is due to either selective forces that acted uniquely on each functional unit in a modularized, developmentally uncoupled craniofacial complex, or alternatively, selection that acted primarily on one unit, with the other responding passively as part of a coevolved set of ontogenetically and evolutionarily integrated structures. Using conditional independence modeling on homologous linear measurements of the height, breadth, and depth of the cranium in Pan (n = 95), Gorilla (n = 102), and recent Homo (n = 120), we reject the null hypothesis of equal levels of overall cranial integration. While all three groups share the pattern of greater neurocranial integration with distinct separation between the face and neurocranium (modularization), family differences do exist. The apes are more integrated in their entire crania, but display a particularly strong pattern of integration within the facial complex related to prognathism. Modern humans display virtually no facial integration, a pattern which is likely related to their markedly decreased facial projection. Modern humans also differ from their great ape counterparts in being more integrated within the breadth dimension of the cranial vault, likely tied to the increase in brain size and eventual globularity seen in human evolution. That the modern human integration pattern differs from the ancestral African great ape pattern along the inverse neurocranial-facial trend seen in human evolution indicates that this shift in the pattern of integration is evolutionarily significant, and may help to clarify aspects of the current debate over defining modern humans.

Animals↗
PubMed2006

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin-1beta.

OBJECTIVE: Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin-1beta (IL-1beta), and combinations thereof. METHODS: Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17beta-estradiol, and dehydroepiandrosterone (DHEA), with or without IL-1beta. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix. RESULTS: Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 microg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration-dependent stimulation of integration (with maximum values observed with DHEA at 3 x 10(-5)M, testosterone at 10(-8)M, and 17beta-estradiol at 10(-11)M). For testosterone and 17beta-estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL-1beta at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL-1beta and sex hormones, integration of articular cartilage was reduced. CONCLUSION: This is the first study to demonstrate that steroid hormones such as 17beta-estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL-1beta. In the absence of IL-1beta or after neutralization of IL-1beta, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.

Animals↗
PubMed2006

Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines.

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-). METHODS: A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone. RESULTS: Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation. CONCLUSIONS: This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

Bacteriophages↗
PubMed1990

Viral integration, fragile sites, and proto-oncogenes in human neoplasia.

To evaluate the trend of viral integration in the human genome, chromosomal localization of five DNA-containing viruses compiled from literature data was compared to the location of fragile sites and proto-oncogenes. A total of 35 regionally mapped viral integration sites from tumors and transformed cells were distributed over 19 chromosomes. Of the 35 integration sites 23 (66%) were at the bands of fragile sites, and 7 were one band away (20%). This statistically defines the correlation as highly significant (P = 0.0000183, Fisher's F-test). Five integration sites did not correspond to the location of a fragile site. Thirteen integration sites and proto-oncogenes mapped at the same bands (37%), 6 (17%) were one band apart, and at 16 integration sites (46%) no proto-oncogenes were localized (P = 0.00491). Eighteen viral integration sites, fragile sites, and proto-oncogenes (51%) were localized at the same bands or one band distant. This clustering of viral integration sites, fragile sites, and proto-oncogenes is statistically highly significant (P = 0.0000118), and indicates nonrandom viral integration in the human genome.

Chromosome Fragile Sites↗
PubMed1990

Influence of cellular sequences on instability of plasmid integration sites in human cells.

To learn more about mechanisms of genome instability in human cells, I investigated DNA sequences that promote high rates of recombination by analyzing rare unstable plasmid integration sites in simian virus 40-transformed human fibroblasts. Previous studies had hypothesized that rearrangement or loss of integrated sequences could be attributed to adjacent cellular DNA. Consistent with this interpretation, a cloned fragment containing both the integrated plasmid and 2.0 kb of adjacent cell DNA from one such unstable integration site in the cell line LM205 demonstrated a much higher incidence of rearrangements when integrated into other chromosome locations than did the original plasmid. To further test this hypothesis, portions of cellular DNA from this region were integrated in duplicate in other locations to determine their ability to promote restriction-fragment-length polymorphism, an indicator of high rates of homologous recombination. Although two types of instability were observed, neither could be attributed solely to the cell sequences being tested in the plasmid. The first type of instability was a transient deletion or amplification of the plasmid DNA soon after integration, which appeared to be a general phenomenon often associated with any type of newly integrated sequence. A second type of instability continued indefinitely for many cell generations, as did that observed in cell line LM205. Because this was rare (one of 78 clones tested), it could not be attributed solely to cell sequences contained within the plasmid. However, the rearrangements in this cell clone occurred exclusively within the cell DNA adjacent to the integration site, again suggesting a role for cis-acting cell sequences in this process. The inability to identify specific cell sequences responsible for instability may therefore indicate that a complex combination of sequences is involved, possibly within both the plasmid and cell DNA.

Base Sequence↗
PubMed1979

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.

Animals↗