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High-throughput and high-density (HD) genetic marker genotyping systems are critical to optimize the efficiency of oil palm breeding and improvement programmes. This study reports the development of the 78 K Infinium® HD customized SNP array, which was used to genotype a thousand palms of a commercial Deli dura x AVROS pisifera family. A total 64,108 (∼82.0%) polymorphic SNP markers were identified of which, 57, 465 (89.6%) were mapped onto the genetic map that has the largest number of markers published so far in oil palm and holds 14,781 SNPs on 2,363 orphan scaffolds (whose chromosomal locations were unknown), which will improve the existing oil palm reference genome (EG5.1). The SNPs were highly informative based on the parent-to-progeny allelic inheritance analysis. The data demonstrated that 4.3% of the progeny resulted from unintentional self-fertilization of the dura female parent. These unintended ‘selfs’ are highly inbred, which will affect their yield. This study also for the first time, describes the homozygosity in the Deli dura and AVROS pisifera, two important parental lines widely used in commercial seed production. As expected, both the parental palms were highly homozygous, having 138 Mb homozygous regions in common, with 70.3% identical alleles. Such a detailed genetic analysis of the individual palm has been made possible with this customized HD SNP array, which will be a valuable tool for routine application in oil palm improvement programmes. The strategy used to design and apply the array will also be of interest for wider scientific research.

Optimal oil palm (Elaeis guineensis) crop yields rely on the purity of the tenera fruit form. The high yielding hybrid tenera fruit form is the consequence of heterozygosity for one of nine genetic variants within the SHELL gene. High-throughput genotyping allows cost-efficient screening prior to planting to decrease unintentional non-tenera palm cultivation. We present a paradigm for dramatically reducing non-tenera cultivation by SHELL genotyping a ~ 10% sampling of seeds per seed production fruit bunch. Identification and seed supply chain removal of bunches above a predetermined non-tenera threshold represent a new paradigm for applying SHELL genetic testing in the industry. In a demonstration involving 121,896 embryos from 1304 independent dura x pisifera controlled crosses from two independent seed production units, we found that 38.4% of bunches achieved a 100% pure tenera prediction rate. The remaining bunches (61.6%) had predicted non-tenera contamination ranging from 1.0 to 89.6%, with an overall average of 3.32% seeds per bunch. SHELL genotyping of expected tenera embryos identified rare aneuploid embryos, confirmed by whole genome sequencing-based heterozygosity and copy number analyses.

Oil palm breeding involves crossing dura and pisifera palms to produce tenera progeny with greatly improved oil yield. Oil yield is controlled by variant alleles of a type II MADS-box gene, SHELL, that impact the presence and thickness of the endocarp, or shell, surrounding the fruit kernel. We identified six novel SHELL alleles in noncommercial African germplasm populations from the Malaysian Palm Oil Board. These populations provide extensive diversity to harness genetic, mechanistic and phenotypic variation associated with oil yield in a globally critical crop. We investigated phenotypes in heteroallelic combinations, as well as SHELL heterodimerization and subcellular localization by yeast two-hybrid, bimolecular fluorescence complementation and gene expression analyses. Four novel SHELL alleles were associated with fruit form phenotype. Candidate heterodimerization partners were identified, and interactions with EgSEP3 and subcellular localization were SHELL allele-specific. Our findings reveal allele-specific mechanisms by which variant SHELL alleles impact yield, as well as speculative insights into the potential role of SHELL in single-gene oil yield heterosis. Future field trials for combinability and introgression may further optimize yield and improve sustainability.

The genome sequence of the African oil palm, the main source of oil production, is used to predict at least 34,802 genes, including oil biosynthesis genes; comparison with the draft sequence of the South American oil palm reveals that the two species may have diverged in the New World and that segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. Two papers published in this issue of Nature deal with the genetics of two variants of one of the most important crops in use today — the African oil palm Elaeis guineensis and its South American cousin Elaeis oleifera. Palm oil accounts for almost half the edible oil consumed worldwide and is also a biofuel, although not without controversy, as in many areas palm oil monoculture has replaced valuable natural forest. Analyses of the 1.8-gigabase genome sequence of E. guineensis and draft sequence of E. oleifera provide insights into oil biosynthesis genes and their regulators, and a record of genome evolution. A key event in the domestication and breeding of the oil palm was loss of the thick, coconut-like shell. The second of the two papers identifies mutations the SHELL gene that specify the different fruit forms found in the oil palm and shows that SHELL gene mutations that originated in pre-colonial Africa are responsible for the single gene hybrid vigour and high yields attained by the oil palm. Oil palm is the most productive oil-bearing crop. Although it is planted on only 5% of the total world vegetable oil acreage, palm oil accounts for 33% of vegetable oil and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8-gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. A total of 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators1, which are highly expressed in the kernel. We also report the draft sequence of the South American oil palm Elaeis oleifera, which has the same number of chromosomes (2n = 32) and produces fertile interspecific hybrids with E. guineensis2 but seems to have diverged in the New World. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations that restrict the use of clones in commercial plantings3, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.

Genetic mapping and whole-genome sequencing studies identify the SHELL gene (a homologue of Arabidopsis SEEDSTICK) as responsible for the three different fruit forms in oil palm (Elaeis guineesis); this has important economic implications for modulating SHELL activity to breed desired fruit forms and enhance oil yields. Two papers published in this issue of Nature deal with the genetics of two variants of one of the most important crops in use today — the African oil palm Elaeis guineensis and its South American cousin Elaeis oleifera. Palm oil accounts for almost half the edible oil consumed worldwide and is also a biofuel, although not without controversy, as in many areas palm oil monoculture has replaced valuable natural forest. Analyses of the 1.8-gigabase genome sequence of E. guineensis and draft sequence of E. oleifera provide insights into oil biosynthesis genes and their regulators, and a record of genome evolution. A key event in the domestication and breeding of the oil palm was loss of the thick, coconut-like shell. The second of the two papers identifies mutations the SHELL gene that specify the different fruit forms found in the oil palm and shows that SHELL gene mutations that originated in pre-colonial Africa are responsible for the single gene hybrid vigour and high yields attained by the oil palm. A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera1,2,3,4. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia5. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation6.

Oil palm (Elaeis guineensis) is the most productive oil bearing crop worldwide. It has three fruit forms, namely dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), which are controlled by the SHELL gene. The fruit forms exhibit monogenic co-dominant inheritance, where tenera is a hybrid obtained by crossing maternal dura and paternal pisifera palms. Commercial palm oil production is based on planting thin-shelled tenera palms, which typically yield 30% more oil than dura palms, while pisifera palms are female-sterile and have little to no palm oil yield. It is clear that tenera hybrids produce more oil than either parent due to single gene heterosis. The unintentional planting of dura or pisifera palms reduces overall yield and impacts land utilization that would otherwise be devoted to more productive tenera palms. Here, we identify three additional novel mutant alleles of the SHELL gene, which encode a type II MADS-box transcription factor, and determine oil yield via control of shell fruit form phenotype in a manner similar to two previously identified mutant SHELL alleles. Assays encompassing all five mutations account for all dura and pisifera palms analyzed. By assaying for these variants in 10,224 mature palms or seedlings, we report the first large scale accurate genotype-based determination of the fruit forms in independent oil palm planting sites and in the nurseries that supply them throughout Malaysia. The measured non-tenera contamination rate (10.9% overall on a weighted average basis) underscores the importance of SHELL genetic testing of seedlings prior to planting in production fields. By eliminating non-tenera contamination, comprehensive SHELL genetic testing can improve sustainability by increasing yield on existing planted lands. In addition, economic modeling demonstrates that SHELL gene testing will confer substantial annual economic gains to the oil palm industry, to Malaysian gross national income and to Malaysian government tax receipts.

The importance of epigenetics in normal development and tissue-specific gene expression, as well as in diseases such as cancer, is well established. DNA methylation is a primary epigenetic modification that is directly linked to the genome itself. Here, we review evidence supporting the promise of DNA methylation-based biomarkers in personalized medicine, discuss standard and emerging technologies for profiling DNA methylation on a genome-wide scale, and forecast how these approaches will be used in parallel to better understand the epigenetics of health and disease and apply that knowledge to advance the field of personalized medicine.

Cytosine-methylation changes are stable and thought to be among the earliest events in tumorigenesis. Theoretically, DNA carrying tumor-specifying methylation patterns escape the tumors and may be found circulating in the sera from cancer patients, thus providing the basis for development of noninvasive clinical tests for early cancer detection. Indeed, using methylation-specific PCR-based techniques, several groups reported the detection of tumor-associated methylated DNA in the sera from cancer patients with varying clinical success. However, by design, such analytical approaches allow assessment of the presence of molecules with only one methylation pattern, leaving the bigger picture unexplored. The limited knowledge about circulating DNA methylation patterns hinders the efficient development of clinical methylation tests and testing platforms. Here, we report the results of a comprehensive methylation pattern analysis from breast cancer clinical tissues and sera obtained using massively parallel bisulphite pyrosequencing. The four loci studied were recently discovered by our group, and demonstrated to be powerful epigenetic biomarkers of breast cancer. The detailed analysis of more than 700,000 DNA fragments derived from more than 50 individuals (cancer and cancer-free) revealed an unappreciated complexity of genomic cytosine-methylation patterns in both tissue derived and circulating DNAs. Both tumor and cancer-free tissues (as well as sera) contained molecules with nearly every conceivable cytosine-methylation pattern at each locus. Tumor samples displayed more variation in methylation level than normal samples. Importantly, by establishing the methylation landscape within circulating DNA, this study has better defined the development challenges facing DNA methylation-based cancer-detection tests.

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer. Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5′ region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.

Somaclonal variation arises in plants and animals when differentiated somatic cells are induced into a pluripotent state, but the resulting clones differ from each other and from their parents. In agriculture, somaclonal variation has hindered the micropropagation of elite hybrids and genetically modified crops, but the mechanism responsible remains unknown. The oil palm fruit 'mantled' abnormality is a somaclonal variant arising from tissue culture that drastically reduces yield, and has largely halted efforts to clone elite hybrids for oil production. Widely regarded as an epigenetic phenomenon, 'mantling' has defied explanation, but here we identify the MANTLED locus using epigenome-wide association studies of the African oil palm Elaeis guineensis. DNA hypomethylation of a LINE retrotransposon related to rice Karma, in the intron of the homeotic gene DEFICIENS, is common to all mantled clones and is associated with alternative splicing and premature termination. Dense methylation near the Karma splice site (termed the Good Karma epiallele) predicts normal fruit set, whereas hypomethylation (the Bad Karma epiallele) predicts homeotic transformation, parthenocarpy and marked loss of yield. Loss of Karma methylation and of small RNA in tissue culture contributes to the origin of mantled, while restoration in spontaneous revertants accounts for non-Mendelian inheritance. The ability to predict and cull mantling at the plantlet stage will facilitate the introduction of higher performing clones and optimize environmentally sensitive land resources.

Fos and Ras function in both dependent and independent signal transduction pathways, and sustained activity of either oncogene is sufficient to induce cell transformation and tumorigenesis. Increased DNA (cytosine-5) methyltransferse (Dnmt1) activity is involved in the mechanism of transformation by both oncogenes, suggesting that inappropriate epigenetic transcription regulation may be a common route of oncogenesis, and that cell transformation may model aspects of the epigenetic deregulation that often occurs in tumors. Here, we have taken a microarray-based gene expression approach to identify differentially expressed genes in cells transformed by c-fos, v-fos, ras or Dnmt1. The cohort of genes differentially expressed in all four transformation systems includes an over-representation of repressed genes, many of which have been functionally implicated in the suppression of transformation or tumorigenesis. Furthermore, we identified four potential tumor suppressor genes subject to epigenetic transcriptional repression in transformed cells. The results emphasize the role of transcription repression in oncogenesis, and they provide insights into the potential common epigenetic mechanisms impacting cell transformation.

The DNA binding activity of the AP-1 transcription factor complex is subject to reduction-oxidation (redox) regulation. This regulation involves posttranslational modification of a conserved cysteine residue in the DNA binding domains of Fos and Jun (2). The critical cysteine residue is highly conserved, and it is flanked by one or two basic amino acids in the four fos family members, the three jun family members, and at least four members of the ATF-CREB family of transcription factors. Chemical oxidation of the cysteines inhibits the DNA binding activity of Fos and Jun, and this inhibition is alleviated by reduction of the cysteines by thiol compounds or by a cellular protein, Ref-1 (43). Loss of redox regulation of AP-1 family members has biological effects. For example, in the oncogenic v-Jun protein, the cysteine has been replaced with a serine (26). Furthermore, serine substitution of the cysteine in Fos results in loss of redox regulation and enhancement of DNA binding and transforming potential (27). Ref-1 is also implicated in redox regulation of DNA binding of several other transcription factors, including NF-κB (43), Egr-1 (16), HIF-1α (7, 15, 25), HLF (7, 22), Pax-5 and Pax-8 (34-36), p53 (10, 18), and others. Ref-1 (also known as HAP1, Ape1, and APEX1) is a bifunctional enzyme. It was independently identified by its AP-1 redox activity (41, 42) and by its sequence identity to class II hydrolytic apurinic/apyrimidinic (AP) DNA repair endonucleases (4, 30, 31). These enzymes function in base excision repair of abasic DNA lesions generated by spontaneous hydrolysis or by exposure to reactive oxygen radicals (6). The DNA repair and redox regulatory activities of Ref-1 are carried out by physically separable domains of the protein (44). The dual activities of Ref-1 suggest that the repair of DNA lesions induced by reactive oxygen species and the regulation of the transcriptional response to an oxidizing cellular environment are tightly coupled. Immunodepletion studies using affinity-purified antibodies specific for Ref-1 demonstrated that Ref-1 is the major redox regulator of AP-1 DNA binding in HeLa cells (43). Furthermore, the essential role of Ref-1 in mammalian development was demonstrated by genetic inactivation of Ref-1 in mice (45). Embryos lacking a functional Ref-1 gene fail to develop beyond embryonic day 6. Death occurs following blastocyst formation, shortly after implantation. These embryos lack both the DNA repair and redox regulatory activities of Ref-1. Therefore, it is not possible to determine whether Ref-1 DNA repair activity, redox regulatory activity, or both are essential for viability. Ref-1 contains two cysteine residues within the redox-active domain (cysteines 64/65 and 93). Previous in vitro studies using recombinant human Ref-1 proteins suggested that cysteine 65 (cysteine 64 of mouse Ref-1) is the redox-active site of Ref-1 (39). These results suggested a sulfhydryl switch mechanism in which cysteine 64/65 of Ref-1 interacts with the conserved cysteines within the DNA binding domains of Fos and Jun and serves as a reductant. Here we report the first genetic analysis of the role of cysteine 64 in Ref-1 redox activity in vivo. Our findings demonstrate that Ref-1 cysteine 64 is not required for Ref-1 redox regulation of AP-1.

Oil palm, a plantation crop of major economic importance in Southeast Asia, is the predominant source of edible oil worldwide. We report the identification of the VIRESCENS (VIR) gene, which controls fruit exocarp colour and is an indicator of ripeness. VIR is a R2R3-MYB transcription factor with homology to Lilium LhMYB12 and similarity to Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1). We identify five independent mutant alleles of VIR in over 400 accessions from sub-Saharan Africa that account for the dominant-negative virescens phenotype. Each mutation results in premature termination of the carboxy-terminal domain of VIR, resembling McClintock’s C1-I allele in maize. The abundance of alleles likely reflects cultural practices, by which fruits were venerated for magical and medicinal properties. The identification of VIR will allow selection of the trait at the seed or early-nursery stage, 3-6 years before fruits are produced, greatly advancing introgression into elite breeding material., Fruit colour is a trait that affects the harvesting, and therefore oil yield, of the economically important oil palm. Here, the authors identify a gene that may control fruit colour in the oil palm and suggest that selection for this gene during early development could advance the breeding potential of this important crop.

This study was originally conceived to test in a rigorous way the specificity of three major approaches to high-throughput array-based DNA methylation analysis: (1) MeDIP, or methylated DNA immunoprecipitation, an example of antibody-mediated methyl-specific fractionation; (2) HELP, or HpaII tiny fragment enrichment by ligation-mediated PCR, an example of differential amplification of methylated DNA; and (3) fractionation by McrBC, an enzyme that cuts most methylated DNA. These results were validated using 1466 Illumina methylation probes on the GoldenGate methylation assay and further resolved discrepancies among the methods through quantitative methylation pyrosequencing analysis. While all three methods provide useful information, there were significant limitations to each, specifically bias toward CpG islands in MeDIP, relatively incomplete coverage in HELP, and location imprecision in McrBC. However, we found that with an original array design strategy using tiling arrays and statistical procedures that average information from neighboring genomic locations, much improved specificity and sensitivity could be achieved, e.g., ∼100% sensitivity at 90% specificity with McrBC. We term this approach “comprehensive high-throughput arrays for relative methylation” (CHARM). While this approach was applied to McrBC analysis, the array design and computational algorithms are fractionation method-independent and make this a simple, general, relatively inexpensive tool suitable for genome-wide analysis, and in which individual samples can be assayed reliably at very high density, allowing locus-level genome-wide epigenetic discrimination of individuals, not just groups of samples. Furthermore, unlike the other approaches, CHARM is highly quantitative, a substantial advantage in application to the study of human disease.

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

Understanding the phenotypic contribution of epigenetic components is making DNA methylation pattern analysis more important in higher eukaryotic genomes as well as human disease. Bisulfite sequencing protocols report DNA methylation occupancy information as a positive assay output that allows methylation patterns to be elucidated from particular devel-opmental or disease states. Reported here is a new method for bisulfite sequencing project management, data analysis, and site-specific methylation test development that is designed for integration in high-throughput genomic and bioinformatics analyses.The phenotypic consequences of epigenetic alterations are being appreciated in nearly every biological system. In higher eukaryotes, DNA methylation patterns serve as a second genomic information code, an easily monitored proximate marker reflecting epigenetic cellular decisions. Most notably, in human cancer, abnormal cellular DNA methylation patterns can directly contribute to the mechanisms of tumorigenesis, most often through the induction of erroneous gene silencing (1). Recent work by many groups has demonstrated that DNA methylation abnormalities may be exploited for the development of powerful cancer diagnostic and prognostic tools (2).One tool in the arsenal of methylation monitoring is bisulfite sequencing (3). Bisulfite treatment of DNA causes the deamination of cytosine and conversion to thymine upon amplification and cloning. However, if the cytosine is methylated, it remains unchanged. In this way, subsequent sequencing of the amplified, mutagenized clones with conventional technology allows one to understand the methylation occupancy at each cytosine.Several groups have released publicly available tools for bisulfite sequencing experimental design or data analysis (4–7). Furthermore, online tools such as methBLAST (medgen.ugent.be/methblast) have become available for in silico bisulfite modification to aid in PCR primer design. However, the utility is limited in that they either only help with experimental design or with data analysis (i.e., pattern elucidation). For those that offer methylation pattern elucidation, they also require the experimenter to employ a locus-by-locus, one-gene-at-a-time approach. Given the revolution in automated sequencing and the capacity available at most genome centers, a combined design and analysis suite that affords investigators the opportunity to take advantage of a high-throughput, low-effort analysis procedure is missing. Most researchers frustratingly address this unmet need by performing each outlined step in the most labor-intensive but readily apparent manner. Typically, this involves a stepwise approach for each target, often aligning trace files by hand and manually performing the occupancy calculations. Moreover, as multiple-locus biomarkers are discovered and employed, validation of methylation patterns in a multi-locus manner from many samples will be necessary. Having an automated analysis capacity will become an even more pressing need.We have created an efficient package of PERL programs called MethylMapper that, when combined with a primer-picking program and BLASTN, simplifies the design and analysis of bisulfite experiments in a high-throughput environment. MethylMapper allows the data to self-organize to minimize mistakes and expedite simultaneous analyses of multiple loci. Furthermore, it makes data quality control as streamlined as possible. The package requires only PERL and NCBI-BLAST (www.ncbi.nlm.nih.gov/blast/download.shtml) and requires very little memory or CPU time. System requirements and CPU usage are dependent only upon the requirements of the BLASTN operation. The package is freely available for download at methylmapper.sourceforge.net.For demonstration purposes, we used MethylMapper to design primers and analyze bisulfite sequencing results for a genomic region spanning the second exon of the SLC4A3 gene on human chromosome 2 (Figure 1). Primer sequences for the analysis shown in Figure 1 are 5′-TGATTTGGGTAAGATTTTGGTTGTGAGTAG-3′ (forward) and 5′-CATCCCTAATAAACAAAACATAAAACT-3′ (reverse). Bisulfite conversion was performed with the EZ DNA Methylation Kit™ (Zymo Research, Orange, CA, USA) under the manufacturer’s recommended conditions. PCR amplification was performed under standard conditions that employed 0.2 pmol of primers, 40 ng of template in a 25-μL volume of water, to which 25 μL of FailSafe™ G 2× premix and 1 U of FailSafe Taq DNA Polymerase (EPICENTRE, Madison, WI, USA) were added. The cycling conditions employed a single 3-min incubation at 95°C, followed by 30 cycles of 95°C for 45 s, 52°C for 15 s, and 72°C for 30 s. Finally, a 10-min chase step was performed by incubating the sample at 72°C. The PCR product was purified from an agarose gel slice and cloned using the pCR2.1-TOPO TA Cloning

Background The c-fos gene was originally identified as the cellular homolog of the oncogene v-fos carried by the Finkel-Biskis-Jenkins and Finkel-Biskis-Reilly murine osteogenic sarcoma retroviruses. Sustained expression of fos is sufficient to induce cellular transformation in vitro and tumorigenesis in vivo. Fos functions as a component of the AP-1 transcription factor complex to regulate gene transcription and several differentially expressed genes have been identified in cells transformed by fos. We have extended these studies by constructing a cellular system for conditional transformation by v-fos. Using Affymetrix-based DNA microarray technology, we analyzed transcriptional changes over the course of transformation and reversion in an inducible v-fos system. Results Microarray analyses of temporal gene expression during the process of v-fos mediated cellular transformation and morphological reversion revealed a remarkably dynamic transcriptome. Of the more than 8000 genes analyzed in this study, 3766 genes were categorized into 18 gene-expression patterns by using self-organizing map analysis. By combining the analysis of gene expression profiles in stably transformed cells with the analysis of sequential expression patterns during conditional transformation, we identified a relatively small cohort of genes implicated in v-fos mediated cellular transformation. Conclusion This approach defines a general conditional cell transformation system that can be used to study the endogenous transcription regulatory mechanisms involved in transformation and tumorigenesis. In addition, this study is the first reported analysis of dynamic changes in gene expression throughout experimentally controlled morphological transformation mediated by v-fos.

We reported previously a model of polyglutamine repeat disorders with insertion of 146 CAG repeats into the murine hypoxanthine phosphoribosyl transferase locus (Hprt(CAG)146; Ordway et al. [1997] Cell 91:753-763), which does not normally contain polyglutamine repeats. These mice develop an adult-onset neurologic phenotype of incoordination, involuntary limb clasping, seizures, and premature death. Histologic analysis demonstrates widespread ubiquinated neuronal intranuclear inclusions (NIIs). We now report characterization of the age of onset of behavioral abnormalities, correlated with the time course of occurrence of NIIs in several brain regions, and the occurrence of NIIs in non-neuronal tissues. Onset of behavioral abnormalities occurred at approximately 22 weeks of age. There was variable time course of expression of NIIs in several brain regions. Assessment of several non-neuronal tissues revealed nuclear inclusions in hepatocytes and choroid plexus epithelium. Gamma-aminobutyric acid (GABA)/benzodiazepine receptors, dopamine D1-like and D2-like receptors, and type 2 vesicular monoamine transporter (VMAT2) binding sites were assayed before and after the onset of behavioral abnormalities. GABA/benzodiazepine receptors were unchanged either before or after the onset of behavioral abnormalities in any region analyzed, whereas striatal D1-like and D2-like receptors were diminished after but not before the onset of symptoms. Dorsal striatal VMAT2 binding sites were decreased before the onset of behavioral changes. Mitochondrial electron transport chain components were assayed with histochemical methods before and after the onset of behavioral changes. There was no change in behaviorally presymptomatic or symptomatic animals. Hprt(CAG)146 mice did not exhibit increased susceptibility to the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Hprt(CAG)146 mice are a useful model for studying polyglutamine repeat disorders.

Several neurological disorders have been attributed to the inheritance of long CAG–polyglutamine repeats. Unlike classical mutations, whose deleterious effects are totally dependent on the context of the gene in which they reside, these translated CAG repeat mutations have been shown to cause neurotoxicity and neuronal intranuclear inclusions when expressed outside their natural gene context. We provide a description of mice with different lengths of repeat in the foreign context of the murine Hprt locus, focusing on aspects of the phenotype that provide an insight into the mechanism by which this unusual mutation might cause toxicity.

The mutations responsible for several human neurodegenerative disorders are expansions of translated CAG repeats beyond a normal size range. To address the role of repeat context, we have introduced a 146-unit CAG repeat into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt). Mutant mice express a form of the HPRT protein that contains a long polyglutamine repeat. These mice develop a phenotype similar to the human translated CAG repeat disorders. Repeat containing mice show a late onset neurological phenotype that progresses to premature death. Neuronal intranuclear inclusions are present in affected mice. Our results show that CAG repeats do not need to be located within one of the classic repeat disorder genes to have a neurotoxic effect.