The necessary and sufficient capabilities of cancer cell have been identified. Strikingly, this list does not include one that would seem to be a key property, namely the ability of cancer cells to kill their "host". This is believed to be a self-evident consequence of the other capabilities (e.g., metastasis), although the available evidence suggests a distinct killer function. Taking into account this unlisted property can significantly affect the current paradigm of carcinogenesis.On the assumption that killer function is a key capability of the cancer cell, it is suggested that cancer has evolved as a mechanism of negative selection of mutant alleles of vitally important genes present in population. Similarly to apoptosis, which is an altruistic suicidal act of a damaged cell, cancer is an altruistic suicidal act of an individual who carries dangerous alleles and presents a hazard for genetic stability of the population. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively.The modern DNA array technology is capable of revealing gene expression profiles responsible for killer function of cancer cell as well as those specific targets in the body that are most strongly affected by the tumor growth.This hypothesis suggests new avenues of cancer research as well as principally new therapeutic strategies. We are now witnessing a post-genomic era of cancer research. Thousands of papers are devoted to discovering molecular mechanisms of this extremely complicated program, the latter term being understood as a prescribed sequence of events with an inevitable result. In almost all studies cancer is considered as a given entity with no attempts made, to my knowledge, to search into its evolutionary origin. However, cancer almost certainly fulfills some evolutionary tasks.First, although cancer is usually mentioned as a representative of a large group of age-related diseases, it principally differs from all the others. Cardiovascular diseases, diabetes, Alzheimer's disease, and many other diseases are manifestations of the "loss-of-function" phenotype due to degeneration and/or death of corresponding cells. Cancer, on the contrary, is an active "gain-of-function" process. Cancer cells acquire numerous new functions, including the unique abilities to adapt to a changing environment and to dodge the blows of the body's protective means, as well as a striking capacity to recruit surrounding normal tissues. The tumor transforms its natural enemies  into unnatural allies, being able to grow only having such a paradoxical support -15. The Second, cancer is an inevitable attribute of the animal world. It hits every species despite the fact that there are huge differences among them in the intracellular machinery, organization of signal pathways and, accordingly, in anticancer defense (or, transformation resistance, see below). In no case, however, is the anticancer defense of a species high enough to exclude completely this illness, despite presumably high cellular plasticity in this respect. This fact would indicate some evolutionary advantages of possessing such a trait. It seems likely that anticancer defense of a species is counterbalanced by opposing forces acting on the population level. In other words, cancer incidence among a species is presumably maintained at certain favorable level, which is coupled in each particular case with an evolutionary adaptation of the intracellular machinery.In conclusion, evolutionary grounds for cancer seem to be evident; it is the explanation of its expediency which presents difficulties. A clue to this problem would be the fact that cancer cell possesses, apart from the well known necessary and sufficient capabilities , a kille14 cells, can not endure a relatively small fraction of cancer cells , this burden often being incompatible with life. Rarely, the immediate cause of death is evident (brain compression, bleeding, perforation of the intestine), but in general it remains obscure. This suggests a deadly influence of cancer upon the body through some unknown mechanisms. Although each of many different forms of cancer has clinical peculiarities, the overall course of the illness and the final result are always the same. So, the notion that the tumor cell, regardless of origin, possesses a special killer function is a statement of an obvious fact. What is surprising in this regard is that no room has been allocated to it in the existing paradigm of carcinogenesis.Carcinogenesis is a multistage process of accumulation of gene defects that determine the characteristic traits of the cancer cell: self-sufficiency in growth signals, insensitivity to anti-growth and pro-apoptosis signals, limitless replicative potential, sustained angiogenesis, tissue invasion, and metastasis . These aParaneoplastic syndromes present evidence that tumors may affect normal tissues remote from the primary site. These syndromes are extremely diverse and affect almost all organs and tissues . The mosparaneoplastic, take place not within the killer function pathway but somewhere aside. Because of this, the term killer function will be used here, without going into its mechanism, as indicating the obvious capability of cancer cells to kill the body.It remains unknown whether the paraneoplastic syndromes are direct manifestations of the killer function itself or they are mere side effects of tumor growth, such as autoimmune diseases. It is noteworthy that cancer is not always accompanied by paraneoplastic syndromes , yet itsde facto only accessory. It seems to be a distinct capability of cancer cells, rather than a derivative of other capabilities, such as uncontrolled proliferation and metastasizing. It is unlikely that active proliferation can by itself exert such a deleterious effect since dozens of billions of cells divide daily in the human body, which is many-fold greater than the proliferation pool of the biggest tumor. Even the metastases, these relatively small foci of ectopic proliferation, can not account by themselves for the inevitable demise. The killer function is also a universal property of the cancer cell since without treatment the lethal outcome is inevitable no matter what is the type of tumor, its ability to develop metastases, recur, induce cachexia, or affect biochemical indices. Finally, this property is specific to the cancer cell, as in normal cell physiology there are no examples of such activity.The killer capability is crucial to the achievement of the final goal, body demise, whereas all the other, promoting proliferation and spread of killer cells are The killer function seems radically different from all other acquired capabilities in that it apparently gives no selective advantage to the cancer cell. On the contrary, for the latter, as a part of the body, acquiring such a function is the same as committing suicide. This changes radically the understanding of the role of cancer cells: they can be regarded not as selfish "cheats" , which pde novo in the same way as the other properties are, namely in the way of numerous step-by-step cycles of mutation-selection. Rather, the cells possess a built-in and ready-for-use program of deadly events, which, just like apoptosis, is launched under certain conditions and then functions automatically. In such a case, mutations of cancer-related genes are not only transformation steps, as generally considered, but also a trigger countdown mechanism that activates the death program directed against the "host". This dual activity leads to appearance of an expanding clone of killer cells progressively strengthening their effect upon the organism . In a certain sense, these are the defects of not the cell  4 to 12 ,26, this14) and large longevity. Indeed, no matter how unlikely is the event by itself it has a real chance to occur under such conditions. If so, one might suggest that animals with a small body weight and short life-span (e.g., rodents) should not suffer from cancer at all, while big animals  should get cancer in their mothers' wombs. Reality, however, does not follow this theory's predictions . All anThe Peto paradox is explained by various transformation resistance of cells from different species ,30. ThisThe Peto paradox can apparently be applied to body tissues as well . Indeed,Most patients develop only one tumor, which presents carcinogenesis as allegedly a local process. Experiments with exposure of animal skin to chemical carcinogens as well as cases of occupational cancer, which demonstrate clearly the link between site of exposure and tumor localization, support this notion. Without questioning the significance of such observations, they seem to have an exceptional and limited character (see below).-4–10-8 per gene per cell division [5 mutations accumulate in the normal stem cell of human colonic crypt [In fact, mutagenesis is intimately related to metabolism and is therefore omnipresent: every day in each cell many thousands of DNA lesions occur due to replication and repair errors, spontaneous depurination, methylcytosine deamination, reactive oxygen species attacks, and telomere shortening. This list should perhaps be extended to include the mutagenic effect of apoptosis resulting from uptake by phagocytosis of DNA from dead cells . Becausedivision ,27; mutadivision ,36. Calcic crypt .TP53, but remaining undetectable by routine diagnostic techniques [When a tumor nodule appears in the body, it seems to be only the tip of an iceberg, maturing in the body for decades and consisting of a multitude of damaged cells at different stages of transformation. This assumption is supported by the clinical experience showing that overt symptoms of the disease are always preceded by precancerous lesions, such as hyperplasia, metaplasia, and dysplasia. This idea finds further development in the concept of "field cancerization", i.e., large (more than 7 cm in diameter) and surrounding the tumor "patches" of damaged cells, recognized on the basis of mutations in chniques .Similar conclusions can be drawn from a notion of mutation as a random and rare event and carcinogenesis as accumulation of genetic defects. Mutagenesis can be described as the process both extensive (measured by number of affected cells) and intensive . Evidently, these parameters are positively correlated with each other: the wider the damaged zone, the deeper the damage of individual cells. The reverse is also true: the deeper the damage of the individual, most "advanced" cells, the wider the lesion area . In other words, appearance of the cell having a complete set of mutations (i.e., cancer cell) is accompanied by formation of a large pool of precancerous cells.The simple model of accumulation of mutant cells (i.e., cells with 1–4 mutations, the latter being the arbitrary transformation threshold) in an exponentially growing cell population is shown in Fig. It follows from the model of multistage carcinogenesis  that in  is the mean number of cells with m mutations after k cell generations and u is the mutation rate. If we assume that the body is composed of 3.5 × 1013 cells (i.e., 245, k = 45), and u is 4 × 10-5 , then aion Fig. . In othetal data  and can tal data ). They mtal data .de facto a triple role: (i) a sensor for general mutagenesis (since its "trigger clock" counts down in accordance with accumulation of mutant cells in the body), (ii) an executioner that unleashes a built-in death program after mutagenesis exceeds some threshold level, and (iii) a founder of a clone of killer cells. Any stem cell in an organism can apparently play such a triple role, thus ensuring reliability of the mechanism.As discussed above, the size of the fraction of pre-malignant cells is dependent on trigger tuning – the more mutations needed for a malignant transformation, the bigger the pool of damaged cells. Hence, the first completely transformed cell plays in vitro [Evolutionary conservation of the death program prompts us to look for an explanation for its appearance. It may consist of the counter-selection of genetically defective individuals. As a matter of fact, if an arising tumor manifests significant whole body mutagenesis, then germ cells are most probably not an exception: a similarity of spontaneous germinal and somatic cell mutation rates was shown for human cells in vitro . High lein vitro . Due to in vitro ,43,44. Bin vitro ,46. In cin vitro , at the While in the case of sporadic tumors the notion that cancer is a local manifestation of generalized mutagenesis is only a more or less validated assumption, it is a truism in the case of hereditary tumors . CarcinoIt is evident that the greater the number of pre-cancerous stages, the more strongly the individual is protected against cancer. A computational model of cancer progression was elaborated recently to show that the appearance of an extra stage and the additional buffering, which arises as a result, reduce the impact of any single hereditary mutation and therefore allow the accumulation of more nonlethal mutations in the population . BecauseThere are many germinal mutations with phenotypic expression but just a few of them  are linA favorable trait is retained only if it manifests itself during the reproductive period. So, the fact that cancer is predominantly a disease of the elderly would seem to be at conflict with a supposition of its evolutionary significance. This is perhaps an apparent conflict: the incidence of cancer among the young may be relatively low exactly because of the efficient selection against the adverse alleles that constantly appear in the population and exist as an inevitable background. As regards the high incidence of cancer at the old age, which seems to have no evolutionary significance, this can be explained from the viewpoint of the evolutionary theory of antagonistic pleiotropy . A genetGerminal mutations in functionally important genes are a strong stimulus for development of a mechanism to prevent their spread in the population. Apart from these highly penetrant rare alleles with a strong hereditary predisposition to cancer, which are merely eradicated from the population, there are many alleles that determine a weak predisposition  .In carcinogenesis, the hypothetical sensor/executor functions under the conditions of continuous interference and noise, which are generated by numerous external and internal locally acting mutagens. From this, many false actions ensue. If one takes the hypothesis that cancer is a means of protection, one must admit that in humans this mechanism is hyperactive and operates beyond the originally set objectives. Both the high cancer incidence in old age and the multitude of cancer cases resulting from local exposure to carcinogens are examples of this hyperactivity that obscure the true evolutionary nature of the phenomenon.Evolution hypotheses that attempt at explaining the appearance of cooperation and altruistic behavior are based on the ideas of kin or group selection ,57. TheiMaintaining DNA integrity is one of the main priorities of living organisms. Depending on the extent of DNA damage, three outcomes are possible: (i) small damage induces repair which restores the initial state; (ii) strong damage launches apoptosis thus preventing cell-to-cell transfer of damaged DNA; (iii) accumulating lesions, when apoptosis is impossible, trigger cancer thus preventing individual-to-individual transfer of damaged alleles of vitally important genes. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively.A striking discovery was made recently that a tumor can survive, propagate, and spread in the body only through the unnatural help coming from normal tissues ,13,59. OBesides, identification of genes responsible for killer function is to be supplemented, using the same technology, with serial analyses of expression profiles of various organs and tissues of tumor-bearing animals at various stages of tumor progression. This can help to identify those specific targets within the body that are most strongly affected by the tumor growth.Recognition of killer function as a crucial capability of cancer cells suggests not only new avenues of cancer research (see above), but also principally new therapeutic strategy. Achievement of better understanding of mechanisms of body death may help to pinpoint new targets for therapy, such as some factors presumably emitted by cancer cells , that exert the deadly effect. These factors are likely to be cancer-specific, so their elimination would not entail severe side effects. The present day cancer therapy is based, without much success, on the imperative to "exterminate the evil" (i.e., the cancer cells). The essence of an alternative strategy which may turn out to be more effective is not to kill the cancer cells, but to neutralize them.The author(s) declare that they have no competing interests.


During the 1990s, health care restructuring in Nova Scotia resulted in downsized hospitals, reduced inpatient length of stay, capped physician incomes and restricted practice locations. Concurrently, the provincial homecare program was redeveloped and out-of-hospital cancer deaths increased from 20% (1992) to 30% (1998). These factors all pointed to a transfer of end-of-life inpatient hospital care to more community-based care. The purpose of this study was to describe the trends in the provision of Family Physician (FP) visits to advanced cancer patients in Nova Scotia (NS) during the years of health care restructuring.Design Secondary multivariate analysis of linked population-based datafiles including the Queen Elizabeth II Health Sciences Centre Oncology Patient Information System , the NS Hospital Admissions/Separations file and the Medical Services Insurance Physician Services database. Setting Nova Scotia, an eastern Canadian province (population: 950,000). Subjects: All patients who died of lung, colorectal, breast or prostate cancer between April 1992 and March 1998 (N = 7,212). Outcome Measures Inpatient and ambulatory FP visits, ambulatory visits by location (office, home, long-term care facility, emergency department), time of day (regular hours, after hours), total length of inpatient hospital stay and number of hospital admissions during the last six months of life.In total, 139,641 visits were provided by family physicians: 15% of visits in the office, 10% in the home, 5% in the emergency department (ED), 5% in a long-term-care centre and 64% to hospital inpatients. There was no change in the rate of FP visits received for office, home and long-term care despite the fact that there were 13% fewer hospital admissions, and length of hospital stay declined by 21%. Age-sex adjusted estimates using negative binomial regression indicate a decline in hospital inpatient FP visits over time compared to 1992–93 levels (for 1997–98, adjusted RR = 0.88, 95%CI = 0.81–0.95) and an increase in FP ED visits (for 1997–98, adjusted RR = 1.18, 95%CI = 1.05–1.34).Despite hospital downsizing and fewer deaths occurring in hospitals, FP ambulatory visits (except for ED visits) did not rise correspondingly. Although such restructuring resulted in more people dying out of hospital, it does not appear FPs responded by providing more medical care to them in the community. Canadians have become increasingly vocal about the need for improved care for the dying. In response, the Special Senate Committee on Euthanasia and Assisted Suicide declaredAs these calls for better care for the dying go out, hospital roles in Canadian communities have been redefined. Hospital restructuring has transferred many aspects of inpatient care to community-based care, including the end-of-life or palliative care of those with cancer. End-of-life care is defined broadly as all care provided to dying persons. Multiple providers are involved in such care and include generalist providers such as family physicians, community nurses and other primary care providers, hospital-based providers, specialists, specialist palliative care providers, volunteers and family. Unless otherwise stated "end-of-life care" reflects any or all of these kinds of care as received by dying patients. When warranted, we will refer to "palliative care program" (PCP) as the comprehensive, organized and specialized program of care for the dying. Such PCPs are often hospital based and include an inpatient palliative care unit, consultant nurses and physicians and may provide consultations and / or care in the community. End-of-life care has been provided in the community in the past, however, this health system restructuring has forced an even greater emphasis on this location for care.Nova Scotia is a small, east coast Canadian province of just under one million people where the percentage of cancer deaths occurring outside of hospitals rose from 19.8% in 1992–93 to 30.2% in 1997–98, an increase of over 50%[While health system restructuring has increased community-based care, restructuring has also affected the context in which family physicians provide this primary medical care. In Nova Scotia, substantial health system restructuring occurred during the study years and initially capped physician incomes, restricted practice locations, downsized hospitals, provided new hospital-in-the-home capacity, initiated redevelopment of a provincial home care program and introduced drug co-payments for seniors receiving government drug benefits.The purpose of this study was to describe the trends in the provision of family physician visits to those dying of the four cancers with the highest mortality in Nova Scotia and to whom, we believe, would be most frequently seen by a family physician, during the years concurrent with this health care restructuring. We hypothesized that, given our previous research showing the trends of advanced cancer patients spending more time out of hospital, and fewer dying in-hospital, family physician community-based services to them would increase.th revision [ICD9-CM]).Study subjects included all Nova Scotians who died due to lung, colorectal, breast or prostate cancer from April 1, 1992 to March 31, 1998 as indicated on the Vital Statistics death certificate  the Queen Elizabeth II Health Sciences Centre Oncology Patient Information System (OPIS) which encompasses the provincial Nova Scotia Cancer Registry (NSCR) and includes provincial Vital Statistics information, (2) the Nova Scotia Medical Services Insurance Physician Services (MSIPS), and (3) the provincial Hospital Admissions and Separates file (HAS). The MSIPS includes data pertaining to all visits provided by physicians in the province who are remunerated via fee-for-service schedules and for physicians who are paid alternatively that provide 'shadow' billing information. More than 96% of FPs in Nova Scotia were fee-for-service at the time of this study.Health services were limited to the 'end-of-life (EOL)', defined in this study as the six months (180 days) prior to the date of death, or from the date of initial cancer diagnosis as recorded in the NSCR to death for persons living less than six months after diagnosis. Service fee codes  and health service identification numbers  were used to identify FP visits.All visits provided by a family physician were counted for each patient during their end-of-life including those in the FP office, patient's home, long-term-care facility (LTC), emergency department (ED), and hospital inpatient settings. Ambulatory visits were defined as all visits, including those to the emergency department, but excluding those made to a patient during a hospital inpatient stay or outpatient procedures. Two 'time of care' categories were created: regular hours (8:01 am to 5 pm), and after hours care (5:01 pm to 8 am, weekends, holidays). Because a common code was not available to identify the time of visit to hospitalized patients, all hospital inpatient visits were considered as occurring during regular hours.In addition to the total length of inpatient hospital stay and the number of hospital admissions, we also examined the total number of specialty visits received by these patients. Demographic and clinical variables included sex, age, fiscal year of death, geographic region, time from diagnosis to death, and tumour site .Initial analyses focused on frequency counts and descriptive measures  of all FP visits, hospital admissions, length of inpatient stay and specialty visits, overall and by fiscal year of death. Each count was expressed as the number of visits provided per 100 "end-of-life (EOL) person-days". Linear temporal trends were assessed using negative binomial regression with a logarithmic link function linking the dependent variable  to fiscal year. Fiscal year was included as a linear predictor adjusting for age and sex, and with log (EOL person-days) as an offset variable. Assessment of a nonlinear trend was made by including fiscal year as both a linear predictor and a qualitative predictor. The Type 3 analysis of year as a qualitative predictor in this model relates to the nonlinear component of trend. Adjusted regression coefficients were exponentiated and reported as rate ratios (RR) with associated 95% confidence intervals (CI). All analyses were conducted using SAS software[In total, 7212 Nova Scotians were identified as having died due to lung, colorectal, breast or prostate cancer over the six-year study period. Males comprised a larger proportion of deaths, as did adults aged 65 years and older, those who died due to lung cancer and survivors of at least 150 days from date of initial cancer diagnosis , were provided by FPs to patients during their end-of-life with 94% of patients receiving at least one FP visit. Variability across fiscal years was minimal, ranging from 92.9% receiving at least one FP visit in 1993–94 to 95.3% in 1997–98.The majority of FP visits were provided to hospital inpatients (64%), followed by the office (15%), home (10%), the emergency department (5%) and long-term care (5%). Of visits provided to hospital inpatients, almost 71% were categorized as a 'subsequent hospital visit', which represents continuing in-hospital care. Other inpatient visits included initial hospital visits (4.7%), visits after four weeks (17.5%), supportive care visits (4.6%) and urgent or emergency care visits (2.5%).Temporal trends associated with ambulatory and inpatient FP visits per 100 EOL person-days are shown in Figure Examination of ambulatory FP visits provided during regular hours (8:01 am-5 pm) showed no significant change over time. However, ambulatory visits provided during 'after' hours (5:01 pm-8 am, weekends) were found to have increased significantly over the study period (p < 0.03). Compared to patients in 1992–93, patients in 1997–98 received 12% more ambulatory visits after hours . Although this increase represented a significant change over time, after hour visits were relatively few compared to the large number of other ambulatory visits and therefore exerted no impact on the overall temporal effect among all ambulatory visits.Significant temporal trends were evident with respect to both the total number of hospital admissions experienced by the patient and the total number of days they spent as a hospital inpatient. A decline in hospital admissions was seen over time, from 1.2 admissions in 1992–93 to 1.1 admissions per 100 EOL person-days in 1997–98. Compared to 1992–93, patients in all subsequent years experienced fewer hospital admissions after accounting for sex and age. By 1997–98 patients experienced 13% fewer hospital admissions than patients in 1992–93 .Over 85% of patients spent at least one day as a hospital inpatient. Patients spent on average a total of 22.7 days in hospital  or 15.6 days per 100 EOL person-days. Total length of hospital inpatient stays declined from 18.6 days per 100 EOL person-days in 1992–93 to 14.8 in 1997–98. Results from the age and sex adjusted regression analysis indicate total length of hospital inpatient stays in 1997–98 were 21% shorter than experienced in 1992–93 .In total, 82,575 visits were made to a medical specialty during the end of life. The number of visits ranged from one to 169, with a median of 11 visits per patient . Age and sex adjusted regression analysis indicate visits to a medical specialty did not change significantly over time.Despite the move to a greater percentage of cancer patients dying out of hospital and despall types of patients in the province from 1992–1999, the total number of office-based, home and long-term-care visits has declined slightly[When we compare these trends to concurrent trends in the province of Nova Scotia for all types of patients, a number of interesting points emerge. For  slightly. This isWe expected to see an increase in home visits for those dying of cancer given the longer period people are spending out of hospital and the greater numbers dying at home. This was not the case. Home and long-term care visits among patients remained stable over time. This trend may have been facilitated by a revamped home care system in 1995 providing more nursing and assisted care visits in the home in the latter part of the decade perhaps reducing the need for family physician visiting.Hospital visits declined quite substantially in the early years of our study and then increased in the final year. Provincial information suggests this trend was true for non-cancer patients as well. Since bThe increase in emergency department visits by family physicians parallels a rise during the study period among the general Nova Scotian population. . This greater emergency department utilization may be a reflection of reduced access to inpatient beds experienced by all patients, including those with advanced cancer.The lack of increased visits made by family physicians in end-of-life care in the community is concerning when one considers the evidence that their participation is associated with a greater likelihood of home death -12 and lIt is important to note that the advantage of using provincial administrative health databases is that we have information for the entire population regarding cancer mortality and physician and hospital utilization. However, there is no clinical information on severity of disease, which would provide a much better understanding of factors influencing health service utilization. In addition, we do not have concurrent home care utilization data or private long-term care facility data to factor into our modeling.Despite health care restructuring of the 1990s which resulted in fewer days in-hospital for those dying with cancer in Nova Scotia, there was no concurrent increase in the family physician visits provided to the dying. This may represent a growing unmet need for community-based medical care. However, further research is needed to examine whether end-of-life-care needs are being met by other health providers such as specialized home palliative care nurses, or general home care nursing services.The author(s) declare that they have no competing interests.FB and BL participated in the conceptualisation and design of the project, the analysis and interpretation of the data, created the first draft of the article, and incorporated co-authors' comments into the final draft. GJ participated in the design of the project, the interpretation of data, and revising the manuscript. GF participated in the analysis and interpretation of data and the revising of drafts. All authors gave approval to the final version.The pre-publication history for this paper can be accessed here:


In yeast, birds and mammals, the SAPK-interacting protein 1 (Sin1) gene product has been implicated as a component of the stress-activated protein kinase (SAPK) signal transduction pathway. Recently, Sin1 has also been shown to interact with the carboxyl terminal end of the cytoplasmic domain of the ovine type I interferon receptor subunit 2 (IFNAR2). However, the function of Sin1 remains unknown. Since SAPK pathways are ancient and the IFN system is confined to vertebrates, the organization of the Sin1 gene and the sequences of the Sin1 protein have been compared across a wide taxonomic range of species.Sin1 is represented, apparently as a single gene, in all metazoan species and fungi but is not detectable in protozoa, prokaryotes, or plants. Sin1 is highly conserved in vertebrates (79–99% identity at amino acid level), which possess an interferon system, suggesting that it has been subjected to powerful evolutionary constraint that has limited its diversification.C. elegans the gene consists of 10 exons and 9 introns organized distinctly from those of vertebrates. In yeast and insects, Sin1 is intronless.Sin1 possesses at least two unique sequences in its IFNAR2-interacting region that are not represented in insects and other invertebrates. Sequence alignment between vertebrates and insects revealed five Sin1 strongly conserved domains (SCDs I-V), but an analysis of any of these domains failed to identify known functional protein motifs. SCD III, which is approximately 129 amino acids in length, is particularly highly conserved and is present in all the species examined, suggesting a conserved function from fungi to mammals. The coding region of the vertebrate Sin1 gene encompasses 11 exon and 10 introns, while in The study reveals the phylogeny of a little studied gene which has recently been implicated in two important signal transduction pathways, one ancient (stress response), one relatively new . Saccharomyces cerevisiae [RAS2Val 19 mutation had elevated levels of cyclic AMP, impaired growth control and were acutely sensitive to heat shock. This phenotype was reversed when the yeast strain was transfected with a cDNA (clone JC310) that encoded a then unknown protein. Although the authors suggested that the inferred interaction between the JC310 product and RAS might be fortuitous, they favored the possibility that that the unknown protein either was a true inhibitor of RAS or that it was a RAS target protein, which when over-expressed, had a protective action. A S. cerevisiae protein encoded by the AVO1 gene showed distant similarity the human JC310 product [Sin1 was originally described as a human protein that modulated Ras function in  clone JC10 that eSchizosaccharomyces pombe cDNA library as a 665 amino acid protein that bound via polypeptide sequences in its C-terminal 244 amino acids to the Sty1/Spc1, stress activated MAP kinase (SAPK) [S. pombe to grow at 37°C, albeit very poorly. Moreover, fusion of the first 486 amino acids of yeast Sin1 (which does not restore growth) with the C-terminal 182 amino acids of the chicken Sin1 sequence protected against heat shock. Together, these data showed that Sin1 functions as a component of the stress-activated Sty1/Spc1 MAP kinase pathway in S. pombe and that a functional homolog of Sin1 exists in vertebrates.Approximately eight years after the identification of JC310 it was again identified, on this occasion in a yeast two-hybrid screen of a No further information concerning Sin1 appeared since the paper of Wilkinson et al.  until ouOvine Sin1, which is 88% identical in sequence to chicken Sin1, can be co-immunoprecipated with the IFN receptor subunit IFNAR2 and shows a similar subcellular distribution to the receptor protein when co-expressed in mammalian cells . AlthougAlthough the SAPK pathway is itself ancient and is found in all the species in which the Sin1 gene exists, the IFN system of receptors and ligands is restricted to vertebrates. We reasoned, therefore, that an analysis of Sin1 gene sequences might not only provide insight into the function of Sin1, but indicate how the protein evolved to interact with IFNAR2. The fact that the Sin1 gene appears to be expressed ubiquitously, that it is highly conserved across a wide range of taxa, and that it is a likely participant in several important signaling pathways, makes it an intriguing candidate for a functional/evolutionary analysis.Schizosaccharomyces pombe and Saccharomyces cerevisiae), the red bread mold (Neurospora crassa) and a number of other fungal species (not shown here), Caenorhabditis elegans, a mosquito species (Anopheles gambiae), fruit fly (D. melanogaster), frog (Xenopus laevis), two fish species (Fugu rubripes and Danio rerio), chicken , mouse (Mus musculus), rat (Rattus norvegicus), human (Homo sapiens), sheep (Ovis aries), cattle (Bos taurus), and pig (Sus scrofa)  are much longer than Sin1 from vertebrate and insect species, which are ~520 aa long. The regions of similarity among these three fungal proteins are confined entirely to the carboxyl termini of these molecules, although several gaps have to be introduced to align them. No similarities are detectable in the amino terminal extensions, which, in the case of S. cerevisiae, is 370 aa long. It is the carboxyl regions of the fungal proteins that can also be aligned with the Sin1 sequences from C. elegans, insects, and vertebrate species, including Ovis aries, the sheep  to 91.3% (sheep and rat)  Table . All thet) Table .There is considerable conservation of Sin1 from mammals to birds (~90%), amphibians ~85%), and fish (~80%) Table . The ins%, and ficonserved region in the middle.The region of the greatest identity between these divergent insect and vertebrate sequences is an acidic region placed in conserved SCD III Fig. , 4, 5. IC. elegans retains the highly conserved 36 amino acid SCD I and the 127 amino acid Domain III  immediately following the end of SCD V.When the ovine Sin1 sequence is scanned for functional motifs , the strThere are numerous motifs that are recognized as potential but weak sites for phosphorylation by either casein kinase II (CK2), protein kinase C, or protein kinase A (data not shown). None of the 17 CK2 sites, the 12 protein kinase C, or the 5 protein kinase A sites present in the ovine Sin1 primary sequence are conserved from mammals to fission yeast, although many are retained across all vertebrates. A weak site for myristylation  and exon 5 (SCD III). As noted above and in Figure S. pombe is comprised of only a single exon.The Sin1 gene from S. pombe gene product is longer than that of mammals, with an extension at its N-terminus, human Sin1 can rescue the stress sensitivity noted in the phenotype of a S. pombe strain that expressed a constitutively active form of RAS, indicating that function, as well as structure, has been conserved over hundreds of millions of years.Sin1 is a little studied gene product of unclear function found in species ranging from mammals to fungi. Although the Two facts should be considered when attempting to infer a role for Sin1 in vertebrates. The first, as discussed in the Background, is the known ability of type 1 IFN to activate MAPK/SAPK in mammalian cells. The second is the proven involvement of Sin1 in the yeast SAPK (Sty1/Spc1) pathway and its involvement in controlling transcription of stress-activated genes . The preS. pombe, S. cerevisiae, and N. crassa, emphasizing the evolutionary distance between these three species. The regions of similarity are confined to the ~600 amino acid C-terminal regions of the three sequences (data not shown), and it is this region that is also conserved in insects and vertebrates (see S. cerevisiae, forms a membrane-associated complex with TOR2 and other protein components (AVO2, AVO3 and LST8), which control cell growth in response to nutrients [S. pombe is involved in a stress response signaling pathway by interacting with Sty1 [S. cerevisiae, although certain landmark amino acids are retained (data not shown). Interestingly, Sin1 from insects and vertebrates, despite having only about 35% identity, are of similar length and possess the five regions of high identity. Conceivably, the SCD III domain is functionally essential in all the species, while SCDs I, II, IV, and V have evolved conserved function within the Metazoa. A not unreasonable assumption is that that Sin1 plays an evolutionarily conserved role in SAPK signaling across a broad range of taxa, including all metazoan and fungal species [The Sin1 gene is remarkably divergent in both length and sequence identity within the fungi ates see  &2. ThisAVO2, AVO and LST8utrients . Cells wAVO2, AVO and LST8AVO2, AVO and LST8In vertebrates Sin1 falls into a class of highly conserved gene products. Its conservation is lower than that of two structural proteins, histone H3 and β-actin, but is comparable to that of CDK1 , a little-studied but widely expressed gene product, is encoded by a single gene in fungi, nematodes, insects, and all vertebrates analyzed and shows modest conservation of amino acid sequence that is consistent with some degree of conserved function in stress-activated signal transduction pathways. Sin1 is highly conserved in vertebrates where it has been implicated in linking interferon responses to the SAPK pathway.C. elegans, S. pombe, and S. cerevisiae, were retrieved from at NCBI Genome databases [Fugu rubripes) and zebrafish (Danio rerio) genome databases at the Ensembl site [Saccharomyces cerevisiae) ORF (open reading frame) database [Sin1 genomic sequences from human, mouse, rat, fruit fly, mosquito, atabases . Sin1 cDatabases . For fismbl site  were usedatabase  was usedPairwise global sequence alignment was performed by using either the BESTFIT or the GAP program from GCG (Madison, WI). Multiple global sequence alignment was performed by using either the PILEUP program (GCG, Madison, WI) and GeneDoc  or ClustSchizosaccharomyces pombe) and chicken : The two Sin1 sequences were published by Wilkinson et al. [Fission yeast : The BLASTp program was used to search the budding yeast ORF database for any protein sequence that had significant similarity to the fission yeast Sin1 protein. The obtained budding yeast Sin1 protein sequence had a GenBank link where its cDNA was available. The cDNA sequence was used to analyze its genomic structure at the NCBI yeast genome site.Budding yeast (Neurospora crassa): Sin1 protein was retrieved from the Neurospora crossa protein data base by searching (BLASTp) with the budding yeast Sin1 protein.Red bread mold (Caenorhabditis elegans): The Sin1 protein sequence was obtained from the C. elegans protein database by searching with ovine Sin1 protein. The cDNA sequence was then obtained from the GenBank link and used to determine the structure of the Sin1 gene.Worm (Drosophila melanogaster): The fruit fly Sin1 protein sequence was retrieved from the D. melanogaster protein database as above. The cDNA sequence was obtained from the GenBank link. Unexpectedly, querying the Drosophila genomic sequence with the C. elegans Sin1 sequence and vice-versa failed to yield a match in either case.Fly (Anopheles gambiae): The mosquito Sin1 protein sequence was retrieved from the Anopheles gambiae str. PEST protein database as above. The cDNA sequence was then obtained from the GenBank link.Mosquito (Fugu rubripes) and Zebrafish (Danio rerio): Both Fugu rubripes and Danio rerio genome databases, which are accessible at two websites, NCBI and ENSEMBL, were queried with Sin1 cDNA sequences from sheep, chicken, and frog. For both species, only the Ensembl site provided the complete genomic sequence. Although the Fugu rubripes genome sequence is incomplete, the exons of Sin1 cDNA can be retrieved and successfully assembled into the full length structure by alignment with other Sin1 cDNA and gene sequences. No GenBank entry was available for the Fugu rubripes Sin1 gene. When a similar method was used to retrieve the Zebrafish Sin1 cDNA sequence, the full length sequence could not be obtained because the region (~20 kb) covering one exon (exon 4) was incomplete. Therefore, the fish Sin1 protein sequence used here is from Fugu rubripes.Puffer fish (Xenopus lavis): The full-length cDNA sequence of Sin1 reported here was from African clawed frog, and was obtained by blasting the Xenopus EST database [Frog (database  with theMus musculus): The mouse Sin1 cDNA sequence was obtained by editing several ESTs, after performing a BLASTn search of the Mus Musculus EST database with the ovine Sin1 cDNA sequence. Searching the mouse genome database with the mouse Sin1 cDNA coding region then allowed the gene, down stream of its transcription start site to be located and its structure to be inferred.Mouse (Rattus norvegicus): The rat Sin1 cDNA sequence was retrieved from several overlapping ESTs, which were obtained by searching the Rattus norvegicus EST database with the ovine Sin1 cDNA sequence. The coding region of the rat Sin1 cDNA was then used to search the rat genome database at the NCBI website for the genomic structure of the gene.Rat (Bos taurus): The full length bovine Sin1 cDNA sequence was obtained from overlapping ESTs, which were obtained by searching the NCBI EST database with the ovine Sin1 cDNA sequence.Cattle (Sus scrofa): The swine Sin1 cDNA sequence was obtained as above by searching the NCBI EST database with the ovine Sin1 cDNA sequence.Pig (Homo sapiens): The sequence published by Colicelli et al. [Human : The sheep Sin1 cDNA sequence was cloned from a sheep endometrial cDNA library in a yeast two-hybrid screen [Sheep  is highlighted by a line above the sequence, and appears not so well conserved in the budding yeast as in other species. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). S. cerevisae, Saccharomyces cerevisae (budding yeast. GenBank accession No. NP_014563).Click here for fileAlignment of Sin1 proteins from the fission yeast and the red bread mold. The Bestfit program was used to align the two sequences. Black shading shows identical residues. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). N. crassa, Neurospora crassa (red bread mold. GenBank accession No. XP_322410).Click here for file


Scientists continue to argue the extent that human activities drive global warming, but few would argue that it exists. The International Panel on Climate Change predicts that greenhouse gases will increase global temperatures by 3.6 degrees F by 2100—a rise unprecedented over the past 10,000 years. What might the world look like as we approach that point? Wetlands will disappear. Floods, hurricanes, and droughts will become progressively more severe. Infectious diseases will increase in virulence and range. Montana's famed glaciers may all but disappear within 30 years. A quarter of species may vanish by 2050.While the effects of climate change on species' geographic ranges and population dynamics have been studied to some extent, scientists know little about how species respond to climate change at the genetic level. In this issue, Elizabeth Hadly and colleagues analyze three different dynamic processes—environmental change, population response, and gene diversity fluctuations—and present evidence that climate change influences variation in genetic diversity.Focusing on two mammal species—the Montane vole and the northern pocket gopher—Hadly et al. asked how the two species responded to historical climate-induced habitat alterations in northwestern Wyoming. They gathered fossils from Yellowstone National Park's Lamar Cave, which contains a treasure trove of carbon-dated deposits that mirror the community of mammals in the area today. Comparing genetic material extracted from fossil samples from different time points over the past 3,000 years to genetic data taken from contemporary animals, Hadly's team tracked genetic changes in populations of the two species and used this information  to estimate changes in effective population size over time.  The genetic and demographic data were then combined with environmental records to analyze the relationship between the factors.Studying these populations in space and time—an approach the authors call “phylochronology”—offers an opportunity to analyze the genetic diversity of a species against the backdrop of environmental fluctuation within an evolutionary time frame. It also suggests how microevolutionary forces—factors that affect genetic variation in populations over successive generations—shape genetic responses to climate change. Such evolutionary forces include mutation, genetic drift , and gene flow (changes in the gene frequency of a population caused by migration).The past 3,000 years includes two periods marked by dramatic climate change—the Medieval Warm Period and the Little Ice Age—that had different effects on local mammal populations depending on their habitat preferences. Habitat specialists, the vole and pocket gopher live in the wet mountain regions of western North America. Though both showed population increases during wetter climates and declines during warmer periods, Hadly et al. predicted the gene diversity fluctuations of the two species would differ based on their different ecological behaviors. And that's what they found: genetic response is tied to population size. Pocket gophers have low population densities, stick close to home, and are fiercely territorial, while voles live in high-density populations and range more widely. For the gophers, population declines resulted in reduced gene diversity; for the voles—which have a larger effective population size and greater dispersal between populations—population declines resulted in increased gene diversity. But what forces underlie these differences in genetic variation?A recent study suggests that migration (a primary agent of gene flow) is most common in and between low-density patches in vole populations, which implicates gene flow as the driver of gene diversity patterns. But the authors don't rule out selection as a possibility, and suggest how to go about resolving the question. Hadly et al. show that phylochronology opens a unique window onto the relatively recent evolutionary past and offers “the potential to separate cause from effect.” They also conclude that “differences in species demography can produce differential genetic response to climate change, even when ecological response is similar.” With a 3-degree temperature increase in just the past 50 years in the American West, conservation of biodiversity may well depend on such insights.


The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs. Wuchereria bancrofti, assessment of antigenemia offers the convenience of daytime testing and greater sensitivity than testing for microfilaremia; however, both microfilaremia and antigenemia develop from months to years after exposure, reducing their utility for detection of low levels of infection or recrudescence of transmission [The exponential growth of the lymphatic filariasis elimination program has highlighted the need for tools that can be used to monitor progress toward programmatic endpoints (e.g. when to stop mass treatment) as well as to conduct surveillance to detect any potential resumption of transmission. Measurement of microfilaremia, the recognized gold standard for demonstrating the impact of community-wide interventions, is not an optimal monitoring or surveillance tool because of the requirement for nocturnal blood collection in much of the world and because it is a relatively insensitive test for infection . For Wucsmission -6. Entomsmission . By provAntibody detection has served as the basis for diagnostic assays for filariasis for many decades. The best of these assays are sensitive for infection but are not specific, both because they cannot distinguish current infection from past infection or exposure to the parasite and because there is some degree of cross-reactivity with other helminth infections. On the positive side, prior studies have shown that virtually all residents of filariasis-endemic areas mount antifilarial antibody responses within the first few years of life. Thus, prevalence rates of antifilarial antibodies in children may be a useful index for assessing changes in transmission of the infection ,9.Antibody assays based on crude filarial extracts are limited by cross-reactions with other nematode antigens . RecombiBrugia malayi or Wuchereria bancrofti. Comparable results have been reported with assays for SXP [B. malayi cDNA library by antibody screening [B. malayi or B. timori microfilaremia were tested [Bm14 and WbSXP belong to a family of related genes that encode proteins that are strong immunogens in many parasitic nematode infections . SXP and for SXP ,16,17. Bcreening ,19. ELISe tested -22.The present study was designed to compare objectively the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. We report here the results of this multicenter evaluation.Serum specimens from patients of known infection status  was defined on the basis of agreement among at least 4 of 5 labs. If only 3 of 5 labs obtained the same result or if 3 did and one of the two remaining laboratories did not obtain an interpretable result, this was considered to represent a lack of consensus (recorded as 'NC' or no consensus in Tables W. bancrofti (91%) and Brugia (96%) infections, while the other two tests performed better with specimens from the homologous infections (Table Brugia patients, but displayed only modest sensitivity (45%) in terms of its performance with W. bancrofti samples. Results were comparable for both the BmR1 dipstick and cassette (data not shown). Similarly, the WbSXP assay was positive for 30 of 33 (91%) serum specimens from W. bancrofti patients, but only 39% of Brugia cases.The Bm14 ELISA displayed comparable sensitivity for both Wuchereria and Brugia infections, and there was little reactivity with specimens from persons with O. volvulus, Loa loa or other helminths (e.g. Strongyloides). The Bm14 assay, and to a lesser extent, the WbSXP assay, appeared to function as a 'pan-filaria' assay, showing reactivity with the specimens from persons with W. bancrofti, B. malayi, L. loa and O. volvulus. None of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections  still remains. For mapping exposure ,25.B. malayi infection [Antibody assays almost certainly will find other uses in the context of lymphatic filariasis (LF) elimination programs. For example, antifilarial antibody tests may be sensitive markers of transmission intensity or provide evidence of ongoing exposure to filarial infection long before the development of antigenemia or microfilaremia. Primates develop antibody responses to Bm14 within 4–8 weeks following nfection . In a lonfection . Less isAs LF programs reach their planned end point (5 or more years of > 80% drug coverage in targeted populations), it will be necessary to determine whether or not transmission has been interrupted and whether mass drug administration can be stopped. Parasitologic testing, whether for microfilaremia or antigenemia, will require testing of thousands of persons to demonstrate that infection levels are below 0.1%, the level established by the Global Program as the end point for defining the elimination of LF. Since antibody responses develop in the absence of demonstrable infection, detecting incident antibody responses should provide a more sensitive measure of transmission than microfilaria or antigen detection. Children born following the cessation of transmission should be antibody-negative, while older children and adults may have evidence of residual antibody reactivity ,27. A teOperational use of antibody assays requires far more practical experience with the assays than we now have. Of greatest concern is the specificity of the tests employed. For example, ELISA tests often use a statistical approach to establish cutoff values for positive results. A test that is 99% specific will predictably have some false positive results if large numbers of samples are tested. Further work will be needed to establish rates of antibody positivity that exceed the number that are likely to occur by chance. In addition, it will be necessary to develop and validate algorithms for confirming the presence of infection or ongoing transmission in situations where low antibody prevalence rates are detected. Despite these caveats, we believe that antibody tests based on antigens like Bm14, BmR1, and Wb-SXP will prove to be useful tools that can be used to facilitate decision making by program managers in the context of filariasis elimination programs.GW, RN, and PK have relationships with companies interested in developing commercial applications of the Bm14, BmR1 and WbSXP assays, respectively.GW, RN, PK, and VBL were responsible for the initial development of the assays. All of the authors participated in the planning of this multicenter study. DG was responsible for coordinating specimen shipment and database management. Participating labs included PL and DG from CDC, RN from the Universiti Sains Malaysia, PK and VBL from Anna Center for Biotechnology, CS from NIH and GW from Washington University School of Medicine. PL and EO were responsible for coordinating the study. PL wrote the first draft of the manuscript, but all of the authors participated in the editing of subsequent versions.


Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine. Artificial prions - infectious, misfolded proteins - can be created by fusing the replication element of one prion to aggregation sequences from another Saccharomyces cerevisiae and Podospora anserina, prions are transmitted from cell to cell through mating and cell division, resulting in readily assayed phenotypes with a non-Mendelian pattern of inheritance (The aggregation of misfolded proteins underlies a diverse range of human diseases, including sporadic amyloidoses such as Alzheimer's disease and hereditary neuropathies such as Huntington's disease . Prions eritance .+PSI] (URE3] (+PSI] and [URE3] can be eliminated by transient growth in the presence of guanidine hydrochloride (GuHCl), which “cures” cells of prions by inhibiting Hsp104p, a molecular chaperone needed for prion replication , also promoted the aggregation of other Q/N-rich proteins, emulating the effect of [+PIN] (A surprisingly large number of proteins in nd Ure2p . From amnd Ure2p . When th strains . Aggregaf [PIN+] .Many sequences with Q/N content as high as that of Sup35p and Ure2p, including human polyglutamine expansion disease proteins, form visible aggregates when overexpressed in yeast as GFP fusions . However–psi]) and an aggregated prion state ([+PSI]) with diminished translation termination activity, which can be monitored by nonsense suppression of the mutant ade1–14 allele (–psi] strains form red colonies on yeast extract-peptone-dextrose (YEPD) medium and cannot grow in the absence of adenine, [+PSI] strains suppress the premature stop codon in ade1-14, and thus appear pink or white on YEPD medium and grow on adenine-free medium (a phenotype termed adenine prototrophy, Ade+). The N or prion domain of Sup35p (residues 1-112) is required for [+PSI] formation but is dispensable for the translation termination activity of the carboxy-terminal C domain  supported a [+PSI]-like prion state termed [+NU] (sup35 (new1 mutants (L.Z.O., unpublished data).We had earlier identified New1p as an uncharacterized protein with a Sup35p-like N-terminal domain; when fused to the M and C domains of Sup35p, the first 153 residues of New1p (New1ed [NU+] . Sup35p ed [NU+] . The pri] (sup35  and new11–153 into a [+NU] prion state (induction). Finally, we fused these constructs to the M and C domains of Sup35p (–M-C), introduced them in place of endogenous SUP35, and assessed whether these proteins could adopt stable prion states (maintenance).To understand the sequence requirements for the prion behavior of New1p, we constructed a series of truncated prion domains A and exa+NU]  aggregated and induced [+PSI] at levels comparable to the full prion domain, in agreement with +PSI], albeit with decreased efficiency. Although a construct lacking oligopeptide repeat 2 entirely (residues 1–49) did not induce [+PSI] de novo, this GFP fusion could nonetheless decorate preexisting Sup35p aggregates. Thus, while oligopeptide repeat 2 contributes to the aggregation of Sup35p, the primary determinants of prion induction reside in the amino-terminal Q/N-rich region and oligopeptide repeat 1.We found that the Q/N-rich tract and a small portion of the adjacent oligopeptide repeat are responsible for Sup35p aggregation and de novo [+PSI] aggregates. Constructs that did not form fluorescent foci could not retain [+PSI], suggesting that aggregation is a prerequisite for prion maintenance. However, aggregation is not sufficient for prion inheritance, as Sup35p constructs with deletions spanning oligopeptide repeats 3–5 could not support a prion state despite their ability to form aggregates and efficiently induce [+PSI]. Only the sixth (incomplete) oligopeptide repeat proved dispensable for [+PSI] maintenance, consistent with an earlier report (In contrast, the rest of the oligopeptide repeat region is needed for stable inheritance of [r report .PSI No More) shows strong interference with [+PSI] in certain strain backgrounds through a poorly understood mechanism  to distinguish between these two functions. PNM2-1 (echanism .+PSI] induction. Earlier work indicated that PNM2-1 is capable of seeding [+PSI] in vivo (–psi] [+PIN] cells with a wild-type SUP35 locus led to both focus formation and [+PSI] induction (+PSI] through a defect in seeding (Using both in vivo and in vitro assays, we established that PNM2-1 does not have a defect in aggregation or [ in vivo . Consistnduction A. A prev seeding . We test seeding B and by  seeding C using a+PSI]. When the wild-type SUP35 gene of a [+PSI] strain was replaced with PNM2-1, the strain retained the prion on synthetic defined (SD) yeast medium that selected for [+PSI] (SD-ade medium) but reverted to [–psi] at a high frequency in nonselective YEPD medium, resulting in sectored colonies  propagons per cell, the PNM2-1 strain had dramatically fewer: 41 of 50 cells had no [+PSI] propagons at all (i.e., were [–psi]), and among the remaining nine [+PSI] cells, the median propagon number was six (+PSI] prions, a defect in propagon replication causes mitotic instability, demonstrating the importance of oligopeptide repeat 2 in prion replication or segregation.We next used a recently described assay to measure the number of heritable prion seeds (propagons) in a PNM2-1 strain. Here, prion replication is inhibited by GuHCl treatment. As the cells divide, preexisting propagons are diluted but not destroyed. The number of propagons present in a colony arising from a single cell is then evaluated by removing the GuHCl prion replication block after a large number (10 or more) of cell divisions and counting the total number of [t colony . Whereas was six F. Thus, +F], after transient overexpression of F-M-GFP. As with [+NU], [+F] induction did not require [+PIN] (data not shown). [+F] could be eliminated by GuHCl treatment  or aggregates as a result of the resident prion (pink/white). Consistent with the induction data, F-M-C and New11–153-M-C were not incorporated into [+PSI] aggregates; likewise, Sup35p did not interact with [+F] or [+NU] aggregates. However, [+F] prions recruited New11–153-M-C and, to a lesser extent, [+NU] recruited F-M-C. Thus, F and New1p prion domains can cross-interact during de novo induction and at normal levels of expression, indicating that the NYN repeat is sufficient to specify homotypic interaction between two otherwise distinct prion domains.We next explored the specificity of [e [PSI+] E. Howeveon assay  ; conversely, Sup35p did not enter [+Q] aggregates.Can a simple aggregation-prone sequence such as polyglu-tamine  be turne+] cells B. Transi+] cells C. Severareatment D, and direatment E. We fur] strain F. The Q6A number of epigenetic traits in fungi result from the stable inheritance of self-propagating, infectious protein aggregrates (prions) . Prion iAggregation underlies the growth phase of the prion replication cycle A and occ+NU] but requires an adjacent oligopeptide repeat to form a minimal heritable New1p prion, [+NU]mini. Similarly, in Sup35p, the Q/N-rich amino terminal region mediates aggregation whereas most of the oligopeptide repeats are needed for the inheritance of [+PSI] propagons. Oligopeptide repeats 1 and 2 appear to contribute to both growth and inheritance, consistent with earlier reports that expansion and deletion within this region modulate in vitro polymerization of Sup35p and the appearance of [+PSI] in vivo  within its prion domain has an amino acid content reminiscent of the oligopeptide repeat sequence (i.e., numerous Q, N, S, Y, and G residues)  . Only twesidues) , but it esidues) , it is u+F] prion cross-interacted with New1p but not Sup35p. Like [+NU], [+F] could be induced in the absence of a prion-promoting (PIN) factor. Finally, [+F] could itself act as a PIN factor, as does [+NU] (data not shown). Notably, the NYN repeat of New1p functions as an aggregation module apparently without regard to its position within a protein; this sequence induced prions when overexpressed by itself or with oligopeptide repeats at its N-terminal (in New11–153 and New150–100) or C-terminal regions (in the F chimera). These results suggest that aggregation sequences are portable and functionally separable from the oligopeptide repeat, perhaps constituting a structurally discrete amyloid core. Indeed, a peptide derived from the amino-terminal region of Sup35p forms a self-seeding amyloid in vitro , followed by loss of the maintainer spontaneously or through 5-FOA counterselection. The PNM2-1 strain in +PSI]. HIS3-marked oligopeptide repeat truncations and PNM2-1 maintainers were from –f] strain was generated by “gamma” chromosomal integration of pRS306 F-M-C into the SUP35 locus of YJW 584; excision of the wild-type gene was confirmed by PCR of Ade- colonies arising from subsequent growth on 5-FOA. The [–q] strain was made by “omega” chromosomal gene replacement (SUP35 with a C.glabrata HIS3-marked –M-C variant (with or without oligopeptide repeats) into the SUP35 locus of a diploid [+PSI] [+PIN] strain. After sporulation, gene replacement was confirmed by PCR and by loss of [+PSI] in half of the haploid progeny. Yeast culture methods were according to standard procedures . Unless otherwise noted, cultures displaying 10% or more cells with foci were scored as positive. For induction, dilutions of the above cultures were plated onto SD-ade and YEPD media to determine percentage of Ade+. In qualitative assessments, strains were scored as positive if 5% or more of plated cells grew on SD-ade medium after 5 d. In [+NU] maintenance experiments, strains with indicated maintainers were tested for the ability to support an Ade+ state following New11–153-GFP overexpression. In [+PSI] maintenance experiments, strains that began as [+PSI] were tested for Ade+ after plasmid shuffle gene replacement with the indicated maintainer. For decoration, a [+PSI] [+PIN] strain was transformed with the indicated inducers, grown in selective medium with 50 μM CuSO4, and examined by fluorescence microscopy during midlogarithmic phase. Propagon counts were performed as described in For aggregation, inducers were overexpressed by growth of cells in selective medium with 50 μM CuSOCentrifugation was performed as described in Data S1(30 KB ZIP).Click here for additional data file.The GenBank accession numbers for the proteins discussed in this paper are Hsp104p (NP_013074), New1p (NP_015098), Rnq1p (NP_09902), Sup35p (NP_010457), Ure2p (NC_014170), YDR210W (NP_010496), and YBR016W (NP_010319).