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Review articles Imprinting evolution and the price of silence Susan K. Murphy and Randy L. Jirtle* Summary conditions whether the direction of the modifications effected In contrast to the biallelic expression of most genes, shall be upward or downward.'' expression of genes subject to genomic imprinting is Thomas Henry Huxley monoallelic and based on the sex of the transmitting parent. Possession of only a single active allele can lead English Biologist/Evolutionist to deleterious health consequences in humans. Aberrant expression of imprinted genes, through either genetic Mammalian species whose genomes contain imprinted or epigenetic alterations, can result in developmental genes are paying a large expense for an obscure genomic failures, neurodevelopmental and neurobehavioral dis- orders and cancer. The evolutionary emergence of im- modification rooted approximately 150 millions years ago printing occurred in a common ancestor to viviparous when imprinted genes first arose in ancestral mammals.(1,2) In mammals after divergence from the egg-laying mono- the case of imprinting, the cost of evolutionary change is tremes. Current evidence indicates that imprinting re- associated with the normal silencing of one parental copy of gulation in metatherian mammals differs from that in eutherian mammals. This suggests that imprinting each gene, which depends on the sex of the parent from which mechanisms are evolving from those that were estab- it was inherited. This induction of functional haploidy has lished 150 million years ago. Therefore, comparing markedly increased vulnerability to cancer, neurodevelop- genomic sequence of imprinted domains from marsu- mental and neurobehavioral disorders, and has implications pials and eutherians with those of orthologous regions in for the outcomes of assisted reproduction technologies. monotremes offers a potentially powerful bioinformatics approach for identifying novel imprinted genes and Imprinting is present in angiosperm plants and eutherian their regulatory elements. Such comparative studies and metatherian mammals. In this review, we will restrict will also further our understanding of the molecular our focus to autosomal imprinting in mammals. We will evolution and phylogenetic distribution of imprinted discuss the current understanding of the molecular founda- genes. BioEssays 25:577–588, 2003. tions of imprinting, disorders associated with imprinted genes, ß 2003 Wiley Periodicals, Inc. the evolutionary origin of imprinting and the power of phylogenetic comparisons to elucidate imprinted genes and ''It is an error to imagine that evolution signifies a constant the regulatory elements that determine their unusual pattern tendency to increased perfection. That process undoubtedly of expression. involves a constant remodeling of the organism in adaptation to new conditions; but it depends on the nature of those Genomic imprinting We all normally inherit a complete set of chromosomes from each parent, such that for every gene, there is one copy from Department of Radiation Oncology, Duke University Medical Center, our mother and one from our father. One correlate of Gregor Durham. Mendel's principles of inheritance is that for any given gene Funding agencies: This work was supported by grants F32CA94668 and its phenotypic outcome, the sex of the contributing parent (SKM) and R01CA25951 and R01ES08823 (RLJ). is irrelevant. In stark contrast, an estimated 100 to 200 genes *Correspondence to: Randy L. Jirtle, Ph.D., Box 3433, Duke University Medical Center, Durham, NC 27710. E-mail: firstname.lastname@example.com within our genomes are subject to genomic imprinting whereby DOI 10.1002/bies.10277 the expression of RNA (coding or non-coding) and protein is a Published online in Wiley InterScience (www.interscience.wiley.com). direct consequence of the providing parent's sex. In this case, one parent's copy of the gene is expressed, while the other is silent. The two parental copies of imprinted genes share nearly identical genetic information, yet silencing of one allele neces- Abbreviations: AS, Angelman Syndrome; BWS, Beckwith-Wiedemann Syndrome; DMR, differentially methylated region; IC, imprint center; sarily predetermines that any functions ascribed to that LOI, loss of imprinting; PGC, primordial germ cell; PWS, Prader-Willi gene are now dependent on the single active copy. Over Syndrome; UPD, Uniparental disomy. 70 imprinted genes have thus far been identified in mammals (Table 1). BioEssays 25:577–588, ß 2003 Wiley Periodicals, Inc. BioEssays 25.6 577 Review articles 578 BioEssays 25.6 Review articles Imprint erasure, establishment istics are referred to as ''differentially methylated regions'', or and maintenance DMRs. Because the parental genomes are each contained in separate physical compartments only in gametes, these cells must contain information that distinguishes the ''sex'' of Erasure and establishment imprinted genes for appropriate expression in the next gene- The selective silencing of imprinted genes is modulated with ration. These marks, which differ within the offspring on each the life cycle of the organism (Fig. 1). Each gamete carries sex- of the two inherited sister chromatids, must be erased in the specific imprint markings that are required for normal devel- germ cells of each generation and then re-established such opment.(7) Upon fertilization, the two epigenetically distinct that the profile of these cells reflects the sex of the individual in pronuclei exist in the same cellular environment, dominated by which they reside. Once the imprints are founded, these oocyte-specific factors. Remarkably, the paternal pronucleus markings must also be maintained in somatic cells throughout is rapidly and actively demethylated within the zygote prior to all subsequent cellular divisions so that the transcription the first cellular division.(8) In contrast, the maternal genome machinery can appropriately interpret the information to effect becomes demethylated in a passive manner during subse- accurate expression. This dynamic process is complex and quent divisions, presumably due to a lack of methyltransferase involves erasure, establishment, maintenance and implemen- activity on the hemimethylated DNA substrate. Erasure of tation of the imprint markings. the paternal methylation profile by the oocyte is a potentially powerful mechanism by which maternal factors modify chro- matin structure to regulate the paternal genome.(9) Demethy- DNA methylation and imprint marks lation at this stage in development is proposed to be required Other than typical sequence polymorphisms that do not for the activation of genes necessary for early embryonic correlate with imprint status, the nucleotide sequence of the growth.(10) However, imprint methylation marks present on two alleles of imprinted genes are identical. Therefore, the both the paternal and maternal genomes are maintained imprint marks that distinguish the two parental alleles must despite this global demethylation event.(8,11) be epigenetic in nature. Epigenetic alterations are defined as Another reprogramming event occurs later, within the modifications that induce heritable changes in gene expres- primordial germ cells (PGCs) of the developing fetus. Between sion without changes in DNA sequence. Chromatin structure is 10.5 and 12.5 days post-coitum in mice, when the developing an example of an epigenetic characteristic, and is regulated by germ cells are entering the gonads, there is an apparently a number of factors including histone methylation, histone complete eradication of DNA methylation (with the exception acetylation and cytosine methylation. Cytosine methylation is of multicopy repeat sequences).(12) After demethylation in the a vital DNA modification(3) that is important for the regulation of PGCs, parental-specific methylation is re-established during many aspects of cellular function including imprinting.(4,5) The gametogenesis (Fig. 1). This occurs in sperm postnatally 5-carbon position of cytosines in the context of CpG dinucleo- within diploid gonocytes prior to meiosis and within oocytes tides throughout the mammalian genome is subject to the arrested at the diplotene stage of meiosis.(13) A testes-specific covalent attachment of a methyl group. Unlike cytosine, 5- zinc finger DNA-binding protein was recently identified that methylcytosine is highly susceptible to spontaneous deamina- may play a pivotal role in this process. BORIS (Brother Of the tion that results in the generation of a TpG dinucleotide. Regulator of Imprinted Sites), a paralog of the CCCTC-binding Consequently, the mammalian genome has become progres- factor protein, CTCF, functions in the implementation of sively depleted of CpG dinucleotides through the course of imprinting (see below) to control parental-specific expres- evolution. However, there are genomic regions ranging from sion.(14) Despite divergent amino and carboxy termini, both several hundred to several thousand base pairs in length that BORIS and CTCF bind to the same DNA sequences by virtue have maintained the expected number of CpGs. Because they of each having identical zinc finger domains, likely the result are normally unmethylated, these ''CpG islands'' have of a gene duplication event. However, their expression is presumably been protected from such spontaneous deamina- mutually exclusive, suggesting that they carry out similar tion. CpG islands are found associated with the promoter functions that are subject to strict spatiotemporal regulation. regions of roughly 40–50% of housekeeping genes. Important BORIS expression in testes is normally limited to the exceptions to the unmethylated status of CpG islands include discrete period of spermatogenesis when methylation pat- those that are associated with imprinted genes, genes subject terns are erased. This has led to the intriguing hypothesis that to X chromosome inactivation and transposable elements. BORIS may facilitate de novo establishment of the methylation Pathological CpG island hypermethylation is well documented imprints, while CTCF functions to maintain and/or read these for many genes involved in cancer.(6) Methylation of the two imprint marks pre- and postnatally.(14,15) Supporting this parental alleles is not equivalent for imprinted genes, and role, a link between CTCF and the protein complexes requir- sequences exhibiting such contrasting epigenetic character- ed for establishing chromatin silencing through histone BioEssays 25.6 579 Review articles Figure 1. Imprinting throughout development. Methylation marks associated with imprinted genes are established at two distinct phases of development. During gametogenesis, the imprint marks present on the maternal (pink) and paternal (blue) chromosomes are erased (grey), followed by establishment of primary imprints which then reflect only the sex of the individual in which they reside. Just after fertilization, a global demethylation event occurs in the zygote, first in the paternal pronucleus (blue), followed by the maternal pronucleus (pink). Imprint marks that were established in the gametes must resist this demethylation process. Remethylation of the diploid genome occurs post-implantation, and includes setting of the secondary imprints, which are maintained throughout the lifespan of the individual. acetylation has been demonstrated.(16) Furthermore, coloca- sequences in close proximity to germline methylation imprint lization of CTCF and BORIS with the histone methylases marks, and these repeats may function in coordinating the Suv39h1 and Suv39h2(15) suggests a connection between methylation profile. For Rasgrf1, tandem repeats upstream these proteins and the prerequisite methylation of histones for from the promoter are necessary for the correct establishment directing de novo DNA methylation.(17) of methylation at an adjacent CpG island in sperm and The DNA features that direct acquisition of methylation subsequent imprinted expression.(18) Although histone acet- imprints are not clear. Consensus CTCF/BORIS binding sites ylation patterns that appear to be specific to parental origin are located at or near the paternal germline methylation have also been identified, less is known about this aspect imprint marks associated with the Igf2/H19 and Dlk1/Meg3 of chromatin structure in the establishment of germline im- regions (see below). Since CTCF/BORIS binding is regulated prints. It is unlikely to mediate heritability of paternal epigenetic in a combinatorial fashion via multiple zinc finger domains, information, however, since sperm chromatin is devoid of there may be other as yet undefined sequence binding motifs histones and instead consists of DNA wrapped together with associated with other imprinted genes. In addition, these protamines.(19) imprinted genes and many others (e.g., M6p/Igf2r, Peg1/Mest, In the oocyte, the Dnmt3 family of methyltransferases Impact and Rasgrf1) are associated with tandem repeat is required to set maternal-specific methylation patterns 580 BioEssays 25.6 Review articles for imprinted genes. The Dnmt3 family member, Dnmt3L is maintenance of imprint marks are all associated with a wide essential, yet lacks methyltransferase activity.(5) Dnmt3L is variety of disorders and diseases in humans. therefore proposed to provide sequence specificity for the de novo methyltransferases, Dnmt3a and Dnmt3b, by directing them to the DNA regions requiring maternal methylation The price of imprinting imprints.(20) These findings in mice have been underscored Parental-specific gene expression has a deleterious conse- by the report of a human female whose fertilized oocytes quence in that functional haploidy eliminates the protection are devoid of all normal maternal methylation marks at that diploidy normally provides against recessive mutations. imprinted loci. This condition, which suggests a deficit in Moreover, the complex epigenetic mechanisms that regulate DNMT3-mediated imprint marking, leads to post-implantation monoallelic expression of imprinted genes are susceptible to lethality.(21) In addition to their role in establishing methyla- dysregulation at multiple levels. Accordingly, imprinted regions tion patterns in oocytes, Dnmt3a, Dnmt3b and Dnmt3L can of the genome are associated with a number of diverse recruit histone deacetylases, which are thought to synergize developmental disorders and diseases that result from impair- with Dnmts in the initiation of gene silencing.(22) Dnmt3 pro- ed regulation, altered dosage or mutation of these domains. teins are also required for spermatogenesis,(20) but it is not Since imprinted genes often occur in clusters coordinately known whether they provide the de novo methylation required regulated by imprint control centers, single genetic or to establish a paternal imprint mark. epigenetic alterations in these key regions can lead to dis- ruption of many genes resulting in the formation of multiple Maintenance disorders. Once specific patterns of CpG methylation are re-established in the somatic cells within the developing embryo, they must be faithfully maintained throughout many rounds of DNA replica- Imprinting and uniparental disomy tion during growth and development. This is accomplished Uniparental disomies (UPDs) result when an individual through the actions of maintenance methyltransferases such inherits two copies of the same chromosome or subchromo- as DNMT1, which recognizes hemimethylated CpG sites at somal region from only one parent. The karyotype of these replication foci and adds methyl groups to cytosines on the individuals appears normal since they are diploid, but because nascent DNA strand to replicate the methylation pattern of the of the uniparental chromosome inheritance, they can exhibit parent strand. DNA replication is intimately associated with problems not only with non-imprinted genes when recessive establishment of chromatin structure, which involves conco- mutations are exposed, but also for any imprinted genes within mitant incorporation of histone proteins to form nucleosomes. the disomic region due to doubling or absence of expression. Newly synthesized histones arrive at replication foci in an This has led to the identification of multiple syndromes that acetylated form; typically, acetylated histones are associated result from global disruption of imprinting on the disomic with transcriptionally active (euchromatic) genomic regions. chromosomes. For example, paternal UPD for chromosome 6 To perpetuate transcriptionally inactive (heterochromatic) [upd(6)pat] is associated with transient neonatal diabetes, regions, such as pericentric chromosomal regions and the upd(7)mat with Silver-Russell syndrome, upd(14)mat with silenced allele of an imprinted gene, histones must be enzy- intrauterine growth retardation and precocious puberty, and matically deacetylated. DNMT1 has been shown to associate upd(14)pat with growth retardation, small thorax and mental with histone deacetylases at replication foci,(23) combining retardation. Prader-Willi and Angelman syndrome result DNA synthesis with the replication of chromatin structure to from maternal and paternal chromosome 15 duplications, produce a genetically and epigenetically identical daughter respectively, and Beckwith-Wiedemann syndrome occurs with cell. Whereas histone acetylation apparently does not function upd(11)pat (see below). Other UPD-related phenotypes as a germline imprint mark (see above), this chromatin suspected of having an imprinting effect include growth failure modification may have an important role in the maintenance and bronchopulmonary dysplasia found in individuals with of proper imprinted gene expression. Imprinted genes in upd(2)mat and low birth weight and congenital malformations somatic tissues indeed exhibit parent-of-origin-specific his- in individuals with upd(16)mat.(26) tone acetylation patterns that presumably contribute to Interestingly, no individuals with UPD have been reported regulating the transcriptional activity of the maternally and for chromosomes 18 and 19,(26) each of which has at least one paternally derived alleles.(24,25) Therefore, maintenance of the imprinted gene (Fig. 2). This may indicate that aberrant chromatin conformation associated with imprinted genes likely imprinted gene expression from these chromosomes results in requires DNA methylation coupled with histone acetylation. a nonviable pregnancy. This is supported by the observation The importance of the proper regulation of imprinting that mice with maternal duplication in the region of mouse throughout the lifespan of an individual is evidenced by the chromosome 7 syntenic with the imprinted region on human observation that defects in erasure, establishment and chromosome 19q do not survive.(27) BioEssays 25.6 581 Review articles Figure 2. The known human imprinted gene clusters; not to scale. For detailed information see http://www.otago.ac.nz/IGC, http://www.mgu.har. mrc.ac.uk/imprinting/imprinting.html and http:// www.geneimprint.com. The chromosomal location is indicated along with the approximate size of each region. Pink, maternally expressed gene; blue, paternally expressed gene; hatched, imprint status not yet examined in humans; purple, maternal and paternal transcripts distinctly expressed depend- ing on promoter usage or alternative splicing; white, biallelic expression. Blue circles, paternally methylated imprint center (IC); red circles, mater- nally methylated IC. Direction of transcription is indicated. Arrows point toward the telomere for each domain. Imprinting and cancer and methylation changes in the germline DMR (IC2) upstream The role of imprinted genes in growth-related pathways is of H19 (Fig. 2). LOI is correlated with hypermethylation of consistent with the observation that genomic imprinting is the CTCF-binding sites (and consequent loss of boundary involved in many types of cancers. Numerous imprinted genes function) within IC2 on the maternal chromosome in colorectal show abnormal changes in gene expression and alterations in cancer and Wilms' tumor.(30,31) the normal pattern of methylation in cancer. One of the most Tumor cells appear to utilize multiple mechanisms to main- common findings has been a ''loss of imprinting'' (LOI), which tain elevated levels of IGF2. The M6P/IGF2R is a multi- refers to epigenetic modifications that result in either the functional receptor that binds to IGF2 and transports it to the activation of gene expression on the normally silenced allele or lysosomes where it is degraded, therefore playing a critical the loss of expression from the normally active allele. An role in restraining IGF2-mediated cell growth. Not surprisingly, intensively studied example is IGF2, which exhibits LOI in the M6P/IGF2R is subject to loss of heterozygosity in a number numerous childhood and adult cancers (for reviews, see Refs. of tumors(32–36) which is frequently accompanied by inactiva- 28,29). IGF2 imprinting abnormalities are implicated in the tion of the remaining allele by mutations in the IGF2-binding pathogenesis of cancers associated with Beckwith-Wiede- domain.(32) The growth advantage obtained by M6P/IGF2R mann syndrome, including Wilms' tumor, hepatoblastomas, inactivation would be further promoted in tumors that also have rhabdomyosarcoma, and adrenal carcinoma. The mechan- increased IGF2 expression because of LOI; however, this isms underlying biallelic expression of IGF2 are diverse, but potential combination of pathological alterations has not yet can involve alternate promoter usage for IGF2, alterations in been examined. the KvLQT1-AS imprinting center (IC1; the heritable gametic Several other imprinted genes are implicated in tumor imprint mark) in the centromeric part of the imprinted domain, formation by virtue of loss or gain of expression. These include 582 BioEssays 25.6 Review articles genes with proposed tumor suppressive functions (ARH1, genetic alteration with its presence within the affected popu- breast and ovarian cancer; PEG3, gliomas; p57 KIP2, Wilms' lation may be diluted by the contribution of other involved tumor; ZAC, breast cancer; and NNAT, acute myeloid genes. Alternatively, the lack of genetic alterations in candidate leukemia) and those with tumor-promoting functions (PEG1/ genes residing in the genomic regions with linkage associa- MEST, invasive breast cancer, uterine leiomyoma and lung tion may indicate the involvement of epigenetic, rather than cancer; and DLK1, uterine leiomyoma, neuroendocrine genetic, alterations. The breadth of known epigenetic altera- tumors). Unlike genetic mutations, the reversible nature of tions in imprinted genes for BWS, AS and PWS as well as epigenetic lesions may render them amenable to therapeutic cancer underscores the need to evaluate these other devel- intervention. For example, it may be possible to use inhibitory opmental and cognitive disorders in this same light. drugs that target the enzymes responsible for DNA methyla- For example, it is plausible that an individual's epigenetic tion and histone deacetylation such as the DNMT inhibitors, profile, which is heritable and tissue-specific, is composed of 5-azacytidine and 5-aza-20 -deoxycytidine and the histone ''epihaplotypes'' (see also Ref. 41) just as the DNA sequence deacetylase inhibitor, trichostatin A. DNMT inhibitors in fact itself is organized into haplotypes based on genetic variation. are efficacious in treating cancer.(37) This exciting potential for We define epihaplotypes as the aggregation of epigenetic treatment underscores the critical need for further studies on characteristics (e.g., CpG methylation and histone acetylation the mechanisms by which imprinted genes contribute to tumor patterns) for a closely linked group of alleles on the same development. chromosome. We postulate that epigenetic and genetic varia- bility combine to contribute to many of the neurogenetic disorders with parent-of-origin effects. Imprinting and neurobehavioral and How might these epigenetic alterations be induced? One developmental disorders possibility is the positioning of normally methylated transpo- Several neurogenetic and developmental disorders, includ- sable elements in the vicinity of unmethylated CpG islands that ing Angelman, Prader-Willi, and Beckwith-Wiedemann syn- regulate gene expression. This type of genetic fingerprint has dromes are associated with specific imprinted regions and already been shown to dramatically influence phenotype in the genes.(26,38) Prader-Willi syndrome (PWS) is associated with mouse Agouti gene, due to a retroviral element insertion that loss of paternal gene expression at chromosome 15q11-q13. promotes epigenetic instability and ectopic expression, de- Affected infants present with hypotonicity and failure to thrive. pending on the degree of methylation.(42) Given the transpo- Later in development, they exhibit hyperphagia and can son load carried in the human genome combined with the become severely obese. These individuals also have short exclusion of some of these elements from imprinted domains stature, mild mental retardation and obsessive compulsive in primates and rodents,(43,44) the potential for detrimental disorder. Angelman syndrome (AS) results either from loss of effects as a result of transposon-gene adjacency requires maternal gene expression of the same region of chromosome further investigation. 15 or from mutations in UBE3A. Affected individuals exhibit Importantly, epihaplotypes represent the consequence ataxia, severe motor and mental retardation, lack of speech, of a direct interface between environmental influences and epilepsy and hypotonia (see Refs. 38,39 for recent reviews). gene regulation. Methylation (and presumably histone acet- Beckwith-Wiedemann syndrome (BWS) is characterized by ylation) can be altered depending on environmental condi- a generalized disruption of imprinting at chromosome 11p15.5 tions at the cellular and organismic levels, especially during with exomphalos, macroglossia, somatic overgrowth, and pre- early development when the methylation patterns are estab- disposition to multiple types of cancer.(40) In all three of these lished.(45–47) For example, epigenetic alterations are evident imprinting disorders, epigenetic alterations have an important from in vitro culture of embryonic stem cells(48) and pre- contributory or causative role. implantation embryos.(49) Indeed, the majority of cloned A possible etiologic contribution of imprinted genes is sug- embryos exhibit abnormal methylation patterns,(50) and gested by other disorders that exhibit parent-of-origin effects. demethylation of DMR2 in the M6P/IGF2R, with subsequent These include Silver-Russell syndrome (maternal; chromo- loss of gene function, is highly associated with ''large offspring some 7); bipolar disorder (paternal; chromosome 18), schizo- syndrome''.(51) phrenia (paternal; chromosome 22), Tourette's syndrome Another possible explanation for the inability to correlate (maternal; location unknown) and autism (maternal, chromo- neurogenetic disorders having parent-of-origin effects with a some 15; and paternal, chromosome 7). Evidence of a specific specific gene mutation is that the causative mutation may gene's involvement in most of these disorders has not been reside in a previously overlooked chromosomal location. For forthcoming, however, even with extensive mutational ana- example, introns and intergenic regions may contain se- lyses of genes present within the areas of linkage association. quences that are pertinent to regulating a gene(s) whose This may be due to the proposed multigenic nature of these dysregulation ultimately gives rise to the abnormal phenotype. complex disorders in that the ability to correlate a distinct In this regard, comparative genomic approaches will be BioEssays 25.6 583 Review articles invaluable in defining evolutionarily conserved and thus ation. This is a critical time for the proper establish- biologically important regions to evaluate for genetic or ment of the epigenetic characteristics that define chromatin epigenetic alterations. structure (Fig. 1), and the egg and the zygote may there- fore be especially vulnerable to perturbations due to non- Imprinting and behavior physiological environmental conditions encountered in vitro. Several imprinted genes are suggested to contribute to the Such epigenetic defects have already been observed in other ability of female mice to nurture their pups, one of the most species.(51,59,60) fundamental of mammalian behaviors. For the paternally Several recent reports have documented a troubling expressed genes Peg1/Mest (52) and Peg3,(53) possession of a increased incidence of imprinting abnormalities in children nonfunctional paternal allele in parturient females results in conceived through assisted reproductive technology. These apathy toward nest building, lack of pup retrieval, inefficient include reports of three children with AS with rare sporadic pup nursing and, unlike the majority of eutherian mammals, imprinting defects on chromosome 15q11-q13(61,62) and lack of afterbirth ingestion (for review, see Ref. 54). Most pups eleven children with BWS.(63,64) This represents an approx- born to mutant mothers do not survive; those that do survive imate 3- to 6-fold increased risk for BWS in individuals con- are runted. The maternal deficit in nurturing behavior is of ceived through assisted reproduction techniques compared to direct consequence to offspring of a daughter who receives the incidence of BWS in the general population. the defective allele from her father. Studies in rats have shown These reports strongly suggest a need for intensive investi- that Peg3 and Peg5/Nnat are both highly expressed in the gation into the aspects of assisted reproductive technologies vasopressin-positive magnocellular neurons during lactation; that contribute to these types of imprinting defects. Whether Peg3 is also expressed in the oxytocin-positive magnocellular other types of imprinting abnormalities are associated with neurons.(55) In parturient female mice with a defective paternal in vitro fertilizations in humans is currently unknown. Given copy of Peg3, there is also a marked decrease in oxytocin- that this technique has been successfully performed only positive neurons and impaired milk ejection, indicative of an since 1978, it may be too early to determine if individuals that important role for this imprinted genes in lactation.(53) The have been conceived in this manner are at heightened risk as potential contribution of Peg5/Nnat to maternal nurturing adults for cancers associated with imprinting abnormalities. behavior has not yet been examined. In addition to the maternal effects on pup growth and Evolution of imprinting survival, offspring directly inheriting a defective paternal copy Imprinted genes have thus far been identified in metatherian of Peg1/Mest or Peg3 are also growth impaired.(54) For both (marsupial) and eutherian (placental) mammals(1,65) but not in Peg1/Mest and Peg3þ/À offspring derived from crosses of prototherian (monotreme) mammals.(1) Multiple theories have þ/À males to þ/þ females, a 20% decrease in size at birth was been proposed to explain the origins of imprinting early in observed that increased to 35% by one week postpartum for mammalian evolution [e.g., see Refs. 9,66]. According to the Peg1/Mest mutants and four weeks postpartum for Peg3 most debated of these theories, the ''conflict hypothesis'',(67) mutants. This indicates a significant defect in the ability of the genetic contention between the male and female genomes mutant pups to extract adequate resources from their wild- is predicted to occur at the materno-fetal interface. This portion type mother both pre- and postnatally as a consequence of a of development positions the offspring to extract nourishment defective paternally expressed gene. directly from the mother; during this time the father's genes PEG1/MEST, PEG3, and PEG5/NNAT are also imprinted have the opportunity to influence the growth and competitive in humans(56–58) with 90% nucleotide identity between mouse fitness of his offspring within the uterine environment. The and human for PEG1/MEST, 83% identity for PEG3, and 89% mother's genes are also capable of regulating energy dis- identity between rats and humans for PEG5/NNAT. This tribution through the placenta to the offspring. By this rationale, strong level of nucleotide conservation is indicative of a shared imprinting of genes would only be relevant to mammalian biological function. It remains unknown, however, whether the species with intrauterine gestation, and should be absent behavioral effects of Peg1/Mest and Peg3 extend to non- in egg-laying species. For several genes examined so far, rodent mammals. imprinting is indeed absent in both monotremes (egg-laying mammals) and avian species, including the chicken.(65,68) Imprinting and assisted reproduction Imprinting is present in the marsupial opossum,(1,65) which has Attention has recently been focused on the role of imprinting in an 11–13 day intrauterine gestation. Although non-invasive, the outcome of in vitro fertilizations, including those assisted the opossum placenta functions during a short period (ap- by intracytoplasmic sperm injection. Concern has been raised proximately three days) in which there is nutrient transfer via over potential problems associated with abnormalities in the maternal circulation to the developing embryos.(69,70) epigenetic reprogramming that can occur during in vitro oocyte Marsupial fetal development is carried out ex utero, so im- maturation, fertilization and zygote culture prior to implant- printing may also be evident for genes that influence lactation 584 BioEssays 25.6 Review articles (e.g., Peg3 and Peg5/Nnat, Refs. 53,55) and postnatal imprinting of the M6P/IGF2R remain unclear. The lack of DMR2 nurturing behavior (e.g., Peg1/Mest, Ref. 52 and Peg3, in this species, combined with the loss of imprinted M6P/IGF2R Ref. 53) in addition to placental nutrient transfer. It will be expression in humans, is indicative of selective forces that first interesting to determine if genes that influence such postnatal established the imprint but then caused it to be lost. behaviors are imprinted in monotremes. Theoretically, the consequence of loss of imprinted status for M6P/IGF2R in primates and their nearest non-primate relatives combined with retention of IGF2 imprinting is a Evolution of IGF2 and M6P/IGF2R imprinting doubling of the expression ratio of growth-suppressing M6P/ Many imprinted genes have essential roles in fetal growth and IGF2R to growth promoting IGF2. The simplest explanation for development. The best characterized of these were among the the evolutionary alteration in M6P/IGF2R imprint status would first imprinted genes identified: the maternally expressed be a selective pressure that favored the ability to further de- mannose-6-phosphate/insulin-like growth factor 2 receptor crease biological function of IGF2. Since IGF2 serves a (M6p/Igf2r) and paternally expressed insulin-like growth factor mitogenic function, restoration of biallelic expression for the 2 (Igf2). The cation-independent mannose 6-phosphate M6P/IGF2R may have helped to effectively reduce the con- receptor (M6pr) encodes a receptor molecule involved in centration of circulating IGF2 leading to a reduction in overall lysosomal trafficking of proteins with mannose 6-phosphate size. Indeed, mice expressing both M6p/Igf2r alleles exhibit a moieties. This receptor also acquired the ability to bind Igf2 20% decrease in body size late in development that persists sometime between the evolutionary divergence of the therian into adulthood.(76) from prototherian mammals.(1) Interestingly, the M6p/Igf2r Evolutionary studies indicate a rapid shift toward increas- also acquired its imprinted status at this time, indicating that ing brain size in species with increasingly complex social the ability to bind Igf2 perhaps contributed a selective force for structures.(54) Difficulty in parturition and maternal perinatal the father to inactivate his copy of this gene.(71) Igf2 is a highly morbidity and mortality might have initially accompanied this conserved, potent mitogen that stimulates placental and fetal anatomical modification. Therefore, one possibility for the growth in utero.(72) Igf2 serves as a ligand for the Igf1 receptor recent loss of M6P/IGF2R imprinting in an ancestor of the and the insulin type A receptor. This binding functions to initiate near-primates and primates might be that this allowed for and propagate growth-inducing signals and block apoptosis. increased maternal survival during birthing by effectively In contrast, binding of Igf2 to the M6p/Igf2r neutralizes this reducing body and/or brain size. Alternatively, the imprinted growth factor by trafficking it into the lysosomes for degrada- status of the M6p/Igf2r may predispose to tumor formation,(77) tion. The reciprocal imprinting and common biological pathway and acquisition of biallelic expression may have provided an for Igf2 and the M6p/Igf2r has led to much speculation about evolutionary advantage by reducing cancer incidence in in- the origin, evolution, and biologic rationale behind imprinting of dividuals through reproductive age. It remains unclear why a these two genes. loss of imprinting for M6P/IGF2R was favored in this instance Because Igf2 is imprinted in marsupials but not in mono- as opposed to an increase in expression from the maternal tremes, imprinting of this gene must have originated in allele. ancestors of the Therian mammals approximately 150 million years ago. In humans and mice, the heritable imprint methyla- Future focus: comparative sequencing tion mark for Igf2 is carried on the paternal chromosome, just The analysis of genomic sequence information from different upstream from the maternally expressed H19. It is unclear mammalian species has enabled revealing comparisons of whether the imprint machinery visible in species today is the imprinted domains. Included in these studies are comparisons same as that used to initially imprint ancestral genes. Ex- of the chromosome 11 imprinted domain in human, mouse and perimental evidence demonstrates that the mechanism used pig(78–81) and the DLK1/MEG3 imprinted domain in human, for M6p/Igf2r imprinting in the opossum is different from that in mouse and sheep.(82) In addition to determining that imprinted mice.(1) Mice have two DMRs associated with the M6p/Igf2r : gene structure and sequence are conserved, these studies DMR1 spans the promoter region and is paternally methylated also identified a number of novel genes.(79,82,83) Non-exonic and DMR2 is located within intron 2 of the M6p/Igf2r and sequence elements and tandem repeats(82) were also con- carries a heritable maternal methylation imprint.(73) Both served, and similarities and differences between the size, DMRs are required for proper imprinted expression of M6p/ distribution and density of CpG islands were observed.(84) The Igf2r in mice.(74) Interestingly, humans share the same DMR2 tremendous power in such comparisons is highlighted by the methylation profile for M6p/Igf2r yet exhibit biallelic expression recent demonstration that they can reveal elusive cis-acting of this locus in all tissues and developmental stages tested control elements associated with establishment, maintenance thus far.(75) Furthermore, M6P/IGF2R is imprinted in the or regulation of imprinting. marsupial opossum despite its complete lack of a DMR2.(1) For example, phylogenetic comparisons were instrumental The details of the mechanisms used by the opossum to regulate in the recent characterization of the mutation causing the BioEssays 25.6 585 Review articles ''callipyge'' phenotype in sheep.(85) Callipyge sheep are vation such that critical imprinting elements are difficult to characterized by fast-twitch muscular hypertrophy with con- distinguish from the sequences conserved due to the relatively comitant decrease in adiposity in the affected muscles close phylogenetic relationships between these species. In accompanied by a 30% increase in feed efficiency. The contrast, the increased evolutionary distance between the phenotype is most apparent in longissimus dorsi muscle; this monotremes, marsupials and primates coupled with the led to the provocative description of the affected sheep as divergent imprinting between these groups should significantly ''callipyge'' (Greek: calli-, beautiful; -pyge, buttocks). A role for improve the ability to refine extraction of the biologically imprinting was evident from the inheritance pattern of the relevant regions involved in imprinting. phenotype; callipyge animals result only when the trait is Other more global questions about the origins of imprinting inherited from their sire and the dam contributes a nonaffected mechanisms may have answers that will be revealed through allele. Interestingly, homozygous callipyge offspring are of examination of the monotremes. For instance, if further studies normal phenotype, which led to the description of this unusual substantiate that the monotremes lack genomic imprinting inheritance pattern as ''polar overdominance''.(86) entirely, it will be of interest to determine whether the The callipyge mutation was mapped to ovine chromosome monotremes also lack the machinery involved in the establish- 18 in a region orthologous to chromosome 14 in humans and ment of imprinting, such as the testis-specific BORIS protein chromosome 12 in mice that contained the imprinted genes and the DNMTs involved in imprint establishment. Alterna- DLK1 and MEG3.(87,88) Sequence analysis of these and other tively, perhaps sequence characteristics, such as an abun- candidate genes in the linkage interval failed to identify muta- dance of repetitive elements in the vicinity of the tions. Further sequencing of the entire linkage region using relevant genes, has hindered the ability to establish differential sheep identical-by-descent to the founder animal revealed a chromatin structure in the monotremes in spite of the presence single transition mutation that perfectly correlated with the of the essential machinery. Further examination of the mono- callipyge phenotype.(85) This mutation was not in a previously treme and marsupial genomes should provide great insight recognized gene or regulatory region. However, comparison of into this unique phenomenon. human, mouse, bovine, and ovine sequence showed that a 144 bp region encompassing the mutation was highly Conclusions conserved. Further investigation led to the identification of a This review has focused on the mechanisms underlying novel transcript produced from the region containing the genomic imprinting, its deleterious consequences, and the mutation, lending credence to the power of comparative evolution of this unique form of gene regulation. The ''price of genomics to identify otherwise elusive regulatory elements silence'' due to imprinting takes a tremendous toll not only in and genes. terms of societal costs of treating imprinting disorders, but Based on studies of IGF2 and M6P/IGF2R,(1,2,75) the more importantly in human health and the well being of divergence in imprinting among extant prototherian, metather- affected individuals and their families. We are now only ian and eutherian mammals may offer an unprecedented beginning to understand the contribution of imprinted genes to opportunity to identify important regulatory and/or genomic human morbidity and mortality with many more imprinted features of imprinted domains. Comparisons of these regions genes yet to be identified. A more comprehensive under- in the nonimprinted monotremes with those in the imprinted standing of these genes and their regulation will undoubtedly marsupials is expected to reveal the acquisition by marsupials unfold in the coming years. This will not only further our of genomic or epigenetic features relevant to the establish- knowledge of the fundamental roles imprinted genes play in ment of imprinting, such as CpG islands, tandem repeat ele- mammalian development, but also will likely lead to the dis- ments, noncoding RNAs, and perhaps other as yet undefined covery of novel therapeutic approaches to treat the many features. Further comparisons of the marsupial genome to the disorders of imprinting. genomes of rodents and primates should also reveal specific features of imprinted domains that either have maintained the status quo throughout evolution or have evolved further due to Acknowledgments ongoing selective processes. We apologize to the many authors whose work could not be Such differences have already been demonstrated for the cited due to space limitations. We thank Robert Waterland, imprinted opossum M6P/IGF2R, which lacks the DMR2 Kay Nolan, Trang Huynh, Heather Evans and Jennifer Weid- known to be required in all other imprinted species examined man for helpful discussions and critical reading of the to date.(75) We postulate that comparative genomic studies of manuscript, Shirley Libed for insight into early mammalian imprinted domains within the three mammalian subclasses will evolution, and Diana Hanson for graphical assistance. Further prove to be a powerful approach to mapping imprinted information on genomic imprinting can be found at http:// domains. Previous comparisons between rodents, primates, www.geneimprint.com, http://www.otago.ac.nz/IGC and http:// and artiodactyls have revealed extensive areas of conser- www.mgu.har.mrc.ac.uk/imprinting/imprinting.html. 586 BioEssays 25.6 Review articles References 27. Beechey CV, Cattanach BM, Blake A. MRC Mammalian Genetics Unit, 1. Killian JK, Byrd JC, Jirtle JV, Munday BL, Stoskopf MK, MacDonald RG, Harwell, Oxfordshire. World Wide Web Site: Genetic and Physical Im- Jirtle RL. M6P/IGF2R imprinting evolution in mammals. Mol Cell 2000;5: printing Map of the Mouse (http://www.mgu.har.mrc.ac.uk/imprinting/ 707–716. imprinting.html). 2001. 2. Killian JK, Nolan CM, Stewart N, Munday BL, Andersen NA, Nicol S, Jirtle 28. Falls JG, Pulford DJ, Wylie AA, Jirtle RL. Genomic imprinting: implica- RL. Monotreme IGF2 expression and ancestral origin of genomic im- tions for human disease. Am J Pathol 1999;154:635–647. printing. J Exp Zool 2001;291:205–212. 29. Feinberg AP, Cui H, Ohlsson R. DNA methylation and genomic 3. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyl- imprinting: insights from cancer into epigenetic mechanisms. Seminars transferase gene results in embryonic lethality. Cell 1992;69:915–926. in Cancer Biology 2002;12:389–398. 4. Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic im- 30. Cui H, Niemitz EL, Ravenel JD, Onyango P, Brandenburg SA, printing. Nature 1993;366:362–365. Lobanenkov VV, Feinberg AP. Loss of imprinting of insulin-like growth 5. Bourc'his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the factor-II in Wilms' tumor commonly involves altered methylation but not establishment of maternal genomic imprints. Science 2001;294:2536– mutations of CTCF or its binding site. Cancer Res 2001;61:4947–4950. 2539. 31. Nakagawa H, Chadwick RB, Peltomaki P, Plass C, Nakamura Y, de La 6. Murphy SK, Jirtle RL. Non-genotoxic causes of cancer. In: Alison MR, Chapelle A. Loss of imprinting of the insulin-like growth factor II gene Gooderham, NG, eds. The Cancer Handbook. London: Nature Publish- occurs by biallelic methylation in a core region of H19-associated CTCF- ing Group Reference, 2001:317–333. binding sites in colorectal cancer. Proc Natl Acad Sci USA 2001;98:591–596. 7. McGrath J, Solter D. Completion of mouse embryogenesis requires both 32. Kong FM, Anscher MS, Washington MK, Killian JK, Jirtle RL. M6P/IGF2R the maternal and paternal genomes. Cell 1984;37:179–183. is mutated in squamous cell carcinoma of the lung. Oncogene 2000;19: 8. Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the 1572–1578. zygotic paternal genome. Nature 2000;403:501–502. 33. Hankins GR, De Souza AT, Bentley RC, Patel MR, Marks JR, Iglehart JD, 9. Reik W, Walter J. 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Yoon BJ, Herman H, Sikora A, Smith LT, Plass C, Soloway PD. Regulation 41. Rakyan VK, Blewitt ME, Druker R, Preis JI, Whitelaw E. Metastable of DNA methylation of Rasgrf1. Nat Genet 2002;30:92–96. epialleles in mammals. Trends Genet 2002;18:348–351. 19. Steger K. Transcriptional and translational regulation of gene expression 42. Morgan HD, Sutherland HG, Martin DI, Whitelaw E. Epigenetic inheri- in haploid spermatids. Anat Embryol (Berl) 1999;199:471–487. tance at the agouti locus in the mouse. Nat Genet 1999;23:314–318. 20. Hata K, Okano M, Lei H, Li E. Dnmt3L cooperates with the Dnmt3 family 43. Greally JM. Short interspersed transposable elements (SINEs) are ex- of de novo DNA methyltransferases to establish maternal imprints in cluded from imprinted regions in the human genome. Proc Natl Acad Sci mice. Development 2002;129:1983–1993. USA 2002;99:327–332. 21. Judson H, Hayward BE, Sheridan E, Bonthron DT. A global disorder of 44. Ke X, Thomas S, Robinson DO, Collins A. The distinguishing sequence imprinting in the human female germ line. Nature 2002;416:539–542. characteristics of mouse imprinted genes. Mamm Genome 2002;13: 22. Aapola U, Liiv I, Peterson P. Imprinting regulator DNMT3L is a transcrip- 639–645. tional repressor associated with histone deacetylase activity. Nucleic 45. Waterland RA, Jirtle RL. Transposable elements: targets for early nutri- Acids Res 2002;30:3602–3608. tional effects on epigenetic gene regulation. (Submitted). 23. Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new 46. Roemer I, Reik W, Dean W, Klose J. Epigenetic inheritance in the mouse. co-repressor, DMAP1, to form a complex at replication foci. Nat Genet Curr Biol 1997;7:277–280. 2000;25:269–277. 47. Petronis A. Human morbid genetics revisited: relevance of epigenetics. 24. Saitoh S, Wada T. Parent-of-origin specific histone acetylation and Trends Genet 2001;17:142–146. reactivation of a key imprinted gene locus in Prader-Willi syndrome. Am J 48. Dean W, Bowden L, Aitchison A, Klose J, Moore T, Meneses JJ, Reik W, Hum Genet 2000;66:1958–1962. Feil R. Altered imprinted gene methylation and expression in completely 25. Hu JF, Pham J, Dey I, Li T, Vu TH, Hoffman AR. Allele-specific histone ES cell-derived mouse fetuses: association with aberrant phenotypes. acetylation accompanies genomic imprinting of the insulin-like growth Development 1998;125:2273–2282. factor II receptor gene. Endocrinology 2000;141:4428–4435. 49. Niemann H, Wrenzycki C. Alterations of expression of developmentally 26. Engel E, Antonarakis SE. ''Old'' and ''new'' syndromes with uniparental important genes in preimplantation bovine embryos by in vitro culture disomy. In: Genomic imprinting and uniparental disomy in medicine. conditions: implications for subsequent development. Theriogenology New York: Wiley-Liss, Inc., 2002:133–162. 2000;53:21–34. BioEssays 25.6 587 Review articles 50. Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, Reik 71. Wilkins JF, Haig D. Genomic imprinting of two antagonistic loci. Proc R W. Conservation of methylation reprogramming in mammalian develop- Soc Lond B Biol Sci 2001;268:1861–1867. ment: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci 72. Constancia M, et al. Placental-specific IGF-II is a major modulator of USA 2001;98:13734–13738. placental and fetal growth. Nature 2002;417:945–948. 51. Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, 73. Stoger R, Kubicka P, Liu CG, Kafri T, Razin A, Cedar H, Barlow DP. Carolan C, Broadbent PJ, Robinson JJ, Wilmut I, Sinclair KD. Epigenetic Maternal-specific methylation of the imprinted mouse Igf2r locus identi- change in IGF2R is associated with fetal overgrowth after sheep embryo fies the expressed locus as carrying the imprinting signal. Cell 1993;73: culture. Nat Genet 2001;27:153–154. 61–71. 52. Lefebvre L, Viville S, Barton SC, Ishino F, Keverne EB, Surani MA. 74. 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Identification of the single base change causing the IGF2 in marsupials and birds. Dev Genes Evol 2000;210:18–20. callipyge muscle hypertrophy phenotype, the only known example of polar 66. Sleutels F, Barlow DP. The origins of genomic imprinting in mammals. overdominance in mammals. Genome Research 2002;12:1496–1506. Adv Genet 2002;46:119–163. 86. Cockett NE, Jackson SP, Shay TL, Farnir F, Berghmans S, Snowder GD, 67. Moore T, Haig D. Genomic imprinting in mammalian development: a Nielsen DM, Georges M. Polar overdominance at the ovine callipyge parental tug-of-war. Trends Genet 1991;7:45–49. locus. Science 1996;273:236–238. 68. Nolan CM, Killian JK, Petitte JN, Jirtle RL. Imprint status of M6P/IGF2R 87. Fahrenkrug SC, Freking BA, Rexroad CE 3rd, Leymaster KA, Kappes and IGF2 in chickens. Dev Genes Evol 2001;211:179–183. SM, Smith TP. Comparative mapping of the ovine clpg locus. Mamm 69. Harder J, Stonebrook M, Pondy J. Gestation and placentation in two Genome 2000;11:871–876. New World opossums: Didelphis virginiana and Monodelphis domestica. 88. Berghmans S, Segers K, Shay T, Georges M, Cockett N, Charlier C. J Exp Zool 1993;266:463–479. Breakpoint mapping positions the callipyge gene within a 450-kilobase 70. Krause W, Cutts J. Placentation in the opossum, Didelphis virginiana. chromosome segment containing the DLK1 and GTL2 genes. Mamm Acta Anat 1985;123:156–171. Genome 2001;12:183–185. 588 BioEssays 25.6
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I updated my 10.1 2014 yesterday through kies but lost my root.... Is it possible for me to root again and if so how do I go about doing it?
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Raspberry marzipan with sugar of the violet flower
Escalivada - Roasted Eggplant, Peppers and Onions in Olive Oil One of the most popular dishes of the Catalonian kitchen. This classic has an amazing background - eggplant from Almeria and Murcia, onions and peppers from Navarra and olive oil from Andalucia. The peppers are harvested by hand, roasted over firewood and peeled without water to preserve as much flavor as possible. Escalivada means 'cooked over embers' the method Spanish mountain shepherds first used to prepare the dish on cold nights while keeping watch over their sheep. Now you can enjoy this rustic treat at home without having to worry about bringing out the grill! Serve alongside your favorite meats, tossed in a salad or with warm toast. By Conservas Artesanas Rosara.
Galician style mussels - Crafted by my friends at Los Peperestes, true artisans of the seafood of Galicia, these mussels are prepared all naturally, using only the most pristine, plump shellfish. Served in the traditional style of Galicia with a simple sauce of olive oil, red pepper, onion and tomato, this is a favorite treat for my family. Cooked in small batches to maintain their flavor, you can warm them in their sauce and serve with crusty bread or as an easy meal with pasta, rice or potatoes
Imported whole extra grade AA Porcini are the more rare red cap bouchon variety. Plump, medium sized mushroom with nutty, slightly meaty texture.
Sushi Party Soy Wrappers- Original, 20 sheets
Iberian hogs, that feed naturally on acorns in the Dehesa forest. Cured and aged for at least 2 years. This breed is known for producing meat with exceptional marbling, a deep red color, and a slight nutty flavor. Boneless Ham Iberico, Aged 24-28 Months. Approximately 13 lbs.
Extra virgin olive oil from our organic olive groves is mainly made up of the Hojiblanca and Picual varieties resulting in a complex and balance sensory profile.
White Tuna in Olive Oil - Fishing is the lifeblood of the port of Bermeo in northern Spain, where tuna from the crisp waters of the Cantabrian Sea is known as Bonito del Norte (literally 'Northern Beauty'). Fished in season using traditional methods and cooked immediately, this meaty loin is packed in olive oil to seal in flavor and heart-healthy nutrients so you can enjoy it any time. I love it mixed with my homemade mayo for a salad, on a piece of crusty bread or with sliced cucumber. By La Pescadora.
A larger, plumper version of the classic Tortellone shape filled with Porcini, mixed regional mushrooms, Ricotta, & Grana Padano cheeses, parsley & shallots.
Possessing the greatest amount of heat, these are widely revered and enjoyed as the world's greatest swizzle sticks for Bloody Mary's, the same as are used by leading white table cloth restaurants and clubs across the country!
Santa Chiara is a delicate, very fragrant oil and as characteristic of taggiasca olives, has no bitterness. Thanks to its fruity taste and light spicy overtones, Santa Chiara is an ideal finishing oil, exalting every preparation without obscuring its original taste. Recommended for finishing salads, pasta sauces, fish and grilled meat. 500ml / 17oz gold foil bottle.
Made with Extra Virgin Olive Oil and black truffle shavings. Produced in Italy.
Made with Extra Virgin Olive Oil and white truffle shavings. Produced in Italy.
Exceptional thickening & stabilizing abilities ñ keeps ingredients suspended uniformly in salad dressings while providing pour ability w/o flavor masking.
Stabilizes high-fat ingredients which can be transformed into powders. Fats should be liquefied, chilled, & mixed w/ a starting ratio of 60% fat to 40% powder.
A pasta purse filled with grana padana DOP, tellegio DOP, robiola & ricotta cheeses and crisp pieces of fresh pear. approx. 240 pieces / 25 entree portions per case. 6.6lb / 3kg - bag in box.
The ultimate penne, the classic screw-cut pasta is perfect for enhancing any penne recipe or pasta salad.
Cold pressed from choice selected Australian Macadamia Nuts, this oil is cholesterol free and a rich source of monounsaturates. This versatile oil makes a delectable salad dressing or dipping oil and can be used for baking with very high smoke point of 210C/410F.


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Connecticut Transit Bus Line
Bus line connections to/from New Haven along the West Haven shore to downtown Milford.
J7 - Kimberly Ave Line for schedule info
Recent fair: $1.25
Web site link below has lots of transport links and phone numbers
If you are in the down town area, it is quite easy to walk around, but Milford is a small town and most of the stuff you'd do will require you to drive.
Ferni and I like walking around the down town area near the Milford Landing, its a nice little area to walk in.
Metro North
There is a Metro North Station here in Milford although I will be honest I have never personally taken it to Milford.
The train leaves Grand Central Station and takes about 1.5 hours to reach the station stop here in Milford.
I have included the link to the Metro North for anyone looking to get here from NYC
- Trains
Ferni and I always drive to Milford as it is the easiest and most covenient way for us to get around. We usually stop in Milford for a movie, shopping or something to eat and driving allows us to get in the car and stop wherever we want.
- Road Trip
Milford is one of those places that's close enough to spend the day and far enough from home that you feel as if you have escaped. We often hop on I-95 and in a few minutes we are crossing the border of New York into Connecticut and on our way North for a shopping excursion to Milford , a road trip or to visit some friends.
We have also taken the Merritt Parkway to get to Milford since it is usually less congested traffic wise compared to I-95 as well as being picturesque.
- Road Trip
For me the easiest way of getting to and around Milford was to drive. Milford is a short hop off of interstates 84 and/or 95. Just be sure to give yourself extra travel time, because as I have learned, the best laid plans of mice and men, always get screwed up by construction and accidents along interstate 84 and 95.
During one of my trips in summer 2006, I agreed to pick up my travel partner at the railroad station. This was easier than me traveling down to NYC to pick him up since I was coming from Hyde Park. The only thing that made this a pain in the ass, was the one way skinny streets and the construction that was going on at the time.
- Road Trip
From NYC to Milford
Metronorth trains run about every 1/2 hr to Milford from Grand Central and usually cost around 10-11 one way. From the train station, you can walk down past Bank Of America and pop into Cafe Atlantique for a quick bite and a latte ( they also have great beer such as Boddingtons).
- Trains
- Budget Travel
Milford Transit District
Milford Transit District
Sorry no web link, but this bus line runs only around Milford. Link with CT Transit bus to New Haven in downtown Milford. I do believe you can also get buses to Bridgeport CT from downtown, but I have not taken them.
Train service
MTA Metro North trains are easy to get from downtown Milford. To/from Grand Central Station in NYC with peak fares about $16 one way. Commuter time travel has frequent service, then hourly otherwise. BUY YOUR TICKET BEFORE YOU BOARD! Save $3.
AMTRAK service does not stop in Milford, but you can go the short ride to either Bridgeport or New Haven for that. That enables travel from Boston-Washington DC and more.
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"The room was spotless, the bed was comfortable, hot water was always available and the view of the Mediterranean was beautiful. They came to fix a phone with a soft ring right away. A huge, varied and delicious real Israeli breakfast was included in the price and they were happy to make eggs the ..."
A Traveller, Dec 2013, US


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On Her Own: Kaitlyn Gilliland has launched the second chapter of her career
Give Me a Break: Some dancers face years of "nos" before they get a big "yes"
7 Things Your Agent Wishes You Knew: Insider advice that can help you land more contracts
Choreography Knocks: Opportunities for choreographers at all levels
A Growing List of Living Female Choreographers
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We've received word from Oklahoma City Ballet that Robyn (Stewart) Rojas, a former member of the company who both trained and taught at OCB's school, has lost her home in the Moore tornado. Recently married, she is expecting her first child, a boy, in July. A GoFundMe account has been set up by a co-worker. Please consider donating and sharing their story.
If you know of other members of the dance community that have been affected by the recent tornadoes, please email us.
Photo of Rojas in her dancing days, at right, courtesy OCB.


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Useyourlocal.com - We love Pubs!Homepage
Trying to find pubs in Carmarthen that have a real fire? You've come to the right place. We've got every pub and bar in the UK listed. Whether you're looking for a pub or bar to play darts, somewhere to play pool or just want to have a look at a menu before you go, we can help.
0.21 miles
Coracle Tavern in Carmarthen has a beer garden. The pub has a car park, it has disabled facilities. Coracle Tavern is a family friendly pub. The pub serves food, it has an outside area. Coracle Tavern has a real fire.
7.82 miles
Traditional country pub serving home cooked bar meals with Sunday carvey available. Also with 2 newly refurbished En Suite bed and breakfast accommodation.


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Jessica Kegel
Women Win V8 Petite Final, Men Win Third Final
The LMU rowing teams competed at the San Diego Crew Classic this weekend.
Lions Edged for Berg Cup, Win 2V8, V4 Races
The LMU rowing team narrowly missed defending the Berg Cup on Saturday.
Lions Wrap Up Weekend Competition with UCLA, at Parker Cup
LMU women's rowing competed against UCLA at Ballona Creek, while the men hit the water at the Parker Cup in Irvine.
Lions Edged in Varsity 8, Win Second Varsity 8
LMU defeated San Diego State in the second varsity eight, but fell by a second in the varsity eight on Sunday.
Lions Tabbed Fifth in Preseason Poll
LMU was picked to finish fifth in the annual WCC rowing preseason poll.
2014 Iggy Awards
2014 Iggy Awards from the Los Angeles Marriott on May 1, 2014
We Are LMU Lions
A campaign in support of the Lions Athletic Fund, benefiting the LMU Student-Athlete
At LMU...
2012-13...competed in the Varsity 8+ lineup...rowed in the Open 4+ at the Newport Autumn Rowing Festival, helping the squad to a fourth-place finish and its first victory over USD in six years...helped the Varsity 8+ to a victory over WCC-Champion Gonzaga in a head-to-head on March 15 (7:21.84)...helped defend the Berg Cup with a victory in the Varsity 8+ race...guided the Varsity 8+ to a fourth-place finish in the Grand Final at the San Diego Crew Classic...guided the Varsity 8+ to a fourth-place finish at the WCC Championships, helping LMU to a third-place overall finish in the standings...
2011-12...competed in both the Varsity 8+ and Second Varsity 8+ lineups during her freshman season in the spring of 2012... helped the Lions to a fourth-place finish in the V8+ at the 2012 WCC Championships... sixth-place in the V8+ Grand Final at the 2012 Western Intercollegiate Rowing Association (WIRA) Championships... first-place in the V8+ at the 2012 Berg Cup...
High School...


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EA has put out a video showing off its own exercise game carrying the branding EA Sports Active, though that may not be the actual title.
Clearly hoping to cash in as an alternative to Wii Fit, it will retail for around half the price at $60. The exercises involve strapping the Wiimote or Nunchuk to your leg, two of these special straps will be packed in. There's also a "resistance band" which is used in some activities.
You're given an on-screen personal trainer to guide you through your program (and perhaps yell generic slogans at you). A variety of settings are available, but the game recommends at least 20 minutes of usage per day.
Exclusive to the Nintendo Wii, you can expect to see this on shelves in March.
3,022 news items
Peter said:
Cool, looks good but I already got Wii Fit so if you ask me this has come too late. I won't get it but I don't know why people who don't have Wii Fit wouldn't.
Diddy Kong Lover said:
Another bad clone of a good Big N franchise, if EA is going to make something for Wii it should be SSX, seriously Blur sucked.
WiiStud said:
I agree Diddy Kong Lover, Blur did suck, but this does not look too bad to me. Too bad I won't get it. Seems to be geared for chix.
SON OF A WII 1-4 said:
This seems more realistic than Wii Fit. If it costs only $60 I am selling my Wii Fit and getting this.
Master Foot said:
EA Sports Active! Now with miniscule annual updates.
Project Ukulele - Possible Hint at Characters
Could it be?
Free to Play Pokemon Shuffle now available on 3DS eShop
Nintendo offers another micro-transaction based title
Gold Mario Amiibo is a Wal-Mart Exclusive
Get ready to shell out your coins
Area Effect Announces Izle for Wii U
Part action, part adventure, all cel-shaded
Nintendo Confirmed As Sponsor For APEX 2015
Another positive step for the Smash competitive scene
Majora's Mask 3D Collector's Edition Contains Skull Kid Figurine
Pre-orders are now open!
Ackk Studios Rings in the New Year with New Y2K footage
Starting the New Year with a bang!
Is Nintendo Making A New Wii U Gamepad?
Recent commercial may have leaked new GamePad model
Zeldathon Relief Announced
Pot smashing for a great cause
Why the Inclusion of The Duck Hunt Dog in Super Smash Bros. Was Brilliant
How the the most hated character in video games rose to the top of the Smash heap
Michael Jordan said:
No they're making it from the movie ... Speed Racer: The Video Game
daveoneal said:
Thanks Rob. I had no idea this one was published. I'm very happy about it! Yes, Jonathan, it is hard as all get-out, ... Street Fighter 2010: The Final Fight review
daveoneal said:
This was a very well-written review. Apparently Super Hexagon was developed by Terry Cavanagh (who made vvvvvv), so ... Cosmophony review
dirk said:
:p p ... TMNT: Smash Up
dirk said:
cool :p ... TMNT: Smash Up
Wii's World is not officially affiliated with Nintendo! (but they wish we were).


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We found 1 site within 18 miles of Carlisle with 1 bookable
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Dr. Jeffrey S. Nicholl, MD
- 275 Lasalle St Ste HC57
New Orleans, LA 70112
- 3 Locations
- (504) 988-5030
Dr. Jeffrey S. Nicholl graduated from the Georgetown University School of Medicine in 1974. He works in New Orleans, LA and 2 other locations and specializes in Neurology and Epileptology. Dr. Nicholl is affiliated with Tulane University Hospital & Clinic. He speaks English and Spanish.
Gender: Male
Languages Spoken: English, Spanish
Neurology, Epileptology
Medical School: Georgetown University School of Medicine; Graduated 1974
*This physician has multiple office locations. See Office Information for details.


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Caption: Aaron Hale smiles as a car honks in support of medical marijuana. Marijuana advocates protested outside of the federal courthouse in Spokane on Thursday. The group says they would like the city to reduce marijuana enforcement to its lowest priority.
Photo by Colin Mulvany
Published on March 25, 2011
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Due to a SNAFU with my account, the old Relic Radio Facebook page is off limits to me. So, we've got a new one.
Hope you'll join me there! I'll try not to get locked out of this one.
Due to a SNAFU with my account, the old Relic Radio Facebook page is off limits to me. So, we've got a new one.
Hope you'll join me there! I'll try not to get locked out of this one.
An early west-coast offshoot of The Hermit's Cave, The Devil's Scrapbook aired from November 1938, to July 1939. We'll hear what appears to be the only surviving episode of the series, The Conquest Of David Rugg, which aired November 28, 1938.
Opening music: Constancy Part 2 by Kevin MacLeod (incompetech.com)
This week, from the files of David Harding, Counterspy, we'll hear The Case Of The High Class Hijacker, an episode from October 13, 1949.
On today's episode of The Horror, Dark Fantasy presents its story from May 29, 1942 titled, Rendezvous With Satan.
It's The Henry Morgan Show on Legacy Of Laughs this week. We'll hear the March 26, 1947 episode titled, Dedicated To American Landlords.
On this episode of Into The West, we hear from Frontier Gentleman with an episode titled, Big Sam For Governor. This one originally aired March 16, 1958.
Two classic gumshoe's on the show this week, beginning with The Adventures Of Philip Marlowe and an episode titled, The Birds On The Wing. That episode aired November 26, 1949. Then, Pat Novak For Hire brings us his episode from April 23, 1949 titled, Sam Toliver.
Minstrels Of The Mist from Author's Playhouse starts us off this week. That episode first aired August 4, 1944. Then, Believe It Or Not shares a Strange Decision. We close with the Philip Morris Playhouse, and their episode from July 29, 1949 titled, The Iron Man.
This week on Relic Radio Science Fiction we hear from X Minus One. Here's The Snowball Effect, first heard August 14, 1956.
This episode of Strange Tales features a story from The Whistler titled, Mind Over Matter. It first aired January 24, 1943.


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Magnetic Swimmers Cultured
December 22, 2011|
For the first time, researchers culture a bacteria that uses a magnetic sulfide compound to navigate.
December 22, 2011|
For the first time, researchers culture a bacteria that uses a magnetic sulfide compound to navigate.
December 21, 2011|
The Scientist recounts the year's top science prize winners and top-notch scientists that passed away.
December 19, 2011|
A coauthor of the key paper that led to one of this year's Nobel Prizes for Physiology or Medicine claims the recipient wasn't involved in the research.
December 16, 2011|
Scientists near the Fukushima plant are equipping wild monkeys with radiation collars to get better sense of their exposure in the wild.
December 15, 2011|
For nematode worms, a bigger stress response means a healthier, longer life, but fewer babies.
December 14, 2011|
More than one quarter of US researchers studying human embryonic stem cells say they've had trouble acquiring cell lines of interest.
December 13, 2011|
Researchers explain the luminous quality of yellow buttercups.
December 13, 2011|
The age at which BRCA carriers are diagnosed with breast cancer may depend on which parent contributed the mutation.
December 9, 2011|
Researchers found that stress eating can blunt the body's stress response.
December 9, 2011|
Some of the highlights from this year's American Society for Cell Biology meeting, held earlier this week


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Phyllis Schneck, McAfee's chief technology officer and vice president of the security software company's global sector, will be named undersecretary of cybersecurity at the Department of Homeland Security, say sources familiar with DHS cybersecurity operations.
Although DHS and McAfee wouldn't confirm the move, industry sources have said Schneck will be named undersecretary after Acting Undersecretary Bruce McConnell departs. McConnell said in July that he planned to leave in August to return to the private sector.
With her deep background in high-performance computing and cryptography, Schneck emerged as strong candidate for the job in July, as the department searched for a permanent replacement after McConnell's departure. She serves as chairman of the board of directors of the National Cyberforensics and Training Alliance, a partnership among government, law enforcement and the private sector that provides information analytics that have been used to prosecute cyber criminals worldwide.
At McAfee, Schneck is responsible for public sector security and global threat intelligence applications, strategic thought leadership around technology and policy in cybersecurity, and initiatives in adaptive security and intelligence in networks for critical infrastructure protection and cross-sector cyber security.
McConnell became acting undersecretary in April after Mark Weatherford left DHS for the Chertoff Group consultancy after about a year-and-a-half on the job. McConnell had been DHS's senior counselor for cybersecurity before taking on the role of acting undersecretary.
Schneck will take over at a challenging time for DHS cybersecurity, which is in the midst of two major tasks: Carrying out the president's cybersecurity executive order and expanding its internal cybersecurity efforts.
Posted by Mark Rockwell on Aug 06, 2013 at 12:58 PM
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Unisys Federal Systems' Venkatapathi Puvvada, this year's Eagle award winner for industry, stresses the importance of community.
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The latest version of the Cybersecurity Information Sharing Act purportedly protects companies from liability and individuals from government intrusion.
Popular CIO is headed for the private sector.
The Census Bureau hasn't established a time frame for its cloud computing plans, including testing for scalability, security, and privacy protection, as well as determining a budget for cloud services.
President Obama's proposal to boost government coordination with the private sector got a warm welcome in the House Homeland Security Committee.
The spy agency wants to better integrate cybersecurity into its traditional human intelligence operations.
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Fremont 3rd Grade Teacher Accused Of Slapping Student Allowed Back In Classroom, Parents Protest
The family of a Fremont third grade student who was allegedly slapped in the face by a teacher last week is planning a protest after learning the teacher has been cleared to return to work Friday.
#ThankATeacher Trending Before National Teacher Day On May 6th
#ThankATeacher: If you can write an essay, thank a teacher. If you learned a second language, thank a teacher. If you know how to spell "receive," thank a teacher. Those are just some of the memes and quotes you'll see as we approach National Teacher Day on Tuesday, May 6th, 2014.
Benicia Teacher Pleads No Contest To Furnishing Marijuana At After-Hours Potluck
A Benicia school teacher has pleaded no contest to a misdemeanor count of furnishing marijuana at an after-hours employee potluck dinner in November.
San Leandro Teacher Arrested For Alleged Sexual Relationship With Teen
A teacher at San Leandro High School has been arrested and charged for allegedly having a sexual relationship with a teenager, police said Thursday.
Teacher Fired For Refusing To 'Unfriend' Students On Facebook
A 79-year-old teacher was friends with about 250 current students on her Facebook page but that's against school policy…
Former Easy Bay Teacher Faces New Lawsuits In Child Molestation Case
Joseph Martin, a former teacher at Woodside Elementary in Concord, is facing 125 felony molestation charges.
Hundreds Of SF Teachers Skip School Ahead Of Thanksgiving Holiday
The San Francisco Unified School District was forced to scramble for substitutes Tuesday as 432 teachers either called in sick or took a personal day ahead of the Thanksgiving holiday.
Girls, Parents Found Liable For Branding South Bay Teacher As 'Perv'
A Santa Clara jury that found three schoolgirls and their parents liable for defaming a Catholic school teacher they branded as a "perv," is about to decide how much one of the students should pay in punitive damages.
Fremont Teacher Arrested For Allegedly Molesting Girl On High School Campus
Police arrested a teacher on Friday for allegedly molesting a student at John F. Kennedy High School in Fremont.
Bill Eases Path for Firing Teachers for Misconduct
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