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Polycomb silencing mechanisms and genomic programming.

Polycomb complexes, best known for their role in the epigenetic silencing of homeotic genes, are now known to regulate a large number of functions in organisms from flies to man. They control transcription activators, pattern-forming genes, maintenance of stem cells and are implicated in cell proliferation and oncogenesis. Our understanding of Polycomb mechanisms derives principally from the study of homeotic genes in Drosophila, where they act in an all-or-none fashion to silence expression in inappropriate parts of the organism. This review summarizes what has been learned from homeotic genes and examines the possible extensions of Polycomb mechanisms to allow for dynamic regulatory behavior and the reprogramming of silenced chromatin states.

Animals↗

Centrosome inheritance after fertilization and nuclear transfer in mammals.

Centrosomes, the main microrubule organizing centers in a cell, are nonmembrane-bound semi-conservative organelles consisting of numerous centrosome proteins that typically surround a pair of perpendicularly oriented cylindrical centrioles. Centrosome matrix is therefore oftentimes referred to as pericentriolar material (PCM). Through their microtubule organizing functions centrosomes are also crucial for transport and distribution of cell organelles such as mitochondria and macromolecular complexes. Centrosomes undergo cell cycle-specific reorganizations and dynamics. Many of the centrosome-associated proteins are transient and cell cycle-specific while others, such as y-tubulin, are permanently associated with centrosome structure. During gametogenesis, the spermatozoon retains its proximal centriole while losing most of the PCM, whereas the oocyte degenerates centrioles while retaining centrosomal proteins. In most mammals including humans, the spermatozoon contributes the proximal centriole during fertilization. Biparental centrosome contributions to the zygote are typical for most species with some exceptions such as the mouse in which centrosomes are maternally inherited and centrioles are assembled de novo during the blastocyst stage. After nuclear transfer in reconstructed embryos, the donor cell centrosome complex is responsible for carrying out functions that are typically fulfilled by the sperm centrosome complex during normal fertilization, including spindle organization, cell cycle progression and development. In rodents, donor cell centrioles are degraded after nuclear transfer, and centrosomal proteins from both donor cell and recipient oocytes contribute to mitotic spindle assembly. However, questions remain about the faithful reprogramming of centrosomes in cloned mammals and its consequences for embryo development. The molecular dynamics of donor cell centrosomes in nuclear transfer eggs need further analysis. The fate and functions of centrosome components in nuclear transfer embryos are being investigated by using molecular imaging of centrosome proteins labeled with specific markers including, but not limited to, green fluorescent protein (GFP).

Animals↗

Nucleolar remodeling in nuclear transfer embryos.

Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synthesizing nucleoli become structurally distinct toward the end of the 4th post-fertilization cell cycle. In embryonic cell nuclear transfer embryos, fully developed nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail to develop fully functional nucleoli. In bovine in vivo developed embryos, a range of important nucleolar proteins (e.g., topoisomerase I, upstream binding factor and RNA polymerase I, fibrillarin, nucleophosmin and nucleolin) become localized to the nucleolar anlage over several cell cycles. This relocation is completed toward the end of the 4th cell cycle. A substantial proportion of bovine embryos produced by nuclear transfer of embryonic or somatic cells to bovine ooplasts display aberrations in protein localization in one or more blastomers. This information is indicative of underlying aberrations in genomic reprogramming and may help to explain the abnormalities observed in a proportion of fetuses and offspring derive from nuclear transfer embryos.

Animals↗

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

Amino Acids↗

Origin and progress of nuclear transfer in nonmammalian animals.

This chapter traces the origin and progress of nuclear transfer that later became the paradigm for cloning animals. Classic studies in cytology, embryology, or genetics spanning more than five centuries that led to nuclear transfers in unicellular animals and to those in oocytes of insects, fish and amphibians are reviewed. The impetus for the development of successful nuclear transfers in amphibian oocytes in 1952 was to determine whether or not differentiated somatic cell nuclei are developmentally equivalent to zygote nuclei. Experiments in amphibians demonstrated several important results: (1) specialized somatic cell nuclei are extensively multipotent; (2) fertile adult amphibians can be cloned from embryonic and larval nuclei; (3) serial cloning expands the number of clones; (4) transplanting nuclei into oocyte cytoplasm induces reprogramming of their gene function; and (5) amphibian cloning became the model for cloning mammals. Subsequent studies in mice, a more technically favorable species, revealed that specialized cell nuclei are equivalent to zygote nuclei.

Amphibians↗

Expression of imprinted genes in cloned mice.

Genomic imprinting is a mammalian specific epigenetic modification of the genome. Assessment of the integrity of the imprinting memory in somatic cell cloned animals is important not only for understanding of the "reprogramming" process during cloning by nuclear transfer, but also for the applications of this technique for therapeutic cloning in the future. In this chapter, we summarize the analytical methods for assessment of monoallelic expression of imprinting genes and expression analysis. From a practical point of view, the authors suggest the use of intersubspecific F1 hybrids between the laboratory mouse (Mus musculus musculus) and the JF1 strain (Mus musculus molossinus). We also list the sequence for PCR primers to detect the polymorphism of imprinted genes between musculus and molossinus.

Animals↗

Culture medium preferences of pre-implantation cloned mouse embryos.

Somatic cloning technology involves the transfer of a somatic cell nucleus into an enucleated oocyte, followed by activation and in vitro culture. Efficiency in terms of live offspring generally remains very low. Little attention has been devoted so far to the impact of culture environment on cloned embryo development. Failure of genomic reprogramming of the donor nucleus in nuclear transfer (NT) experiments could lead to an altered phenotype in these cloned embryos that could be manifested by different medium preferences of the NT embryos. We describe here the application of sequential culture media to support preimplantation development of mouse embryos reconstructed using conventional NT techniques. Embryo-quality analysis was performed on NT blastocysts obtained. Additionally, NT embryos that arrested during development also were analyzed.

Animals↗

Optimization of procedures for cloning by somatic cell nuclear transfer in mice.

Cloning by somatic cell nuclear transfer is a complex procedure that is dependent on correct interactions between oocyte and donor cell genome. These interactions require minimal insult to either the oocyte or the transplanted nucleus. Available data also indicate that reprogramming the donor cell genome may be slow, so that the cloned embryo expresses genes typical of the donor cell, and thus has different characteristics from normal embryos. Procedures that minimize damage to the donor genome and that address the unique characteristics of the cloned construct should enhance the efficacy of the method.

Animals↗

A viral conspiracy: hijacking the chemokine system through virally encoded pirated chemokine receptors.

Several herpesviruses and poxviruses contain genes encoding for G protein-coupled receptor (GPCR) proteins that are expressed on the surface of infected host cells and/or the viral envelope. Most of these membrane-associated proteins display highest homology to the subfamily of chemokine receptors known to play a key role in the immune system. Virally encoded chemokine receptors have been modified through evolutionary selection both in chemokine binding profile and signaling capacity, ultimately resulting in immune evasion and cellular reprogramming in favor of viral survival and replication. Insight in the role of virally encoded GPCRs during the viral lifecycle may reveal their potential as future drug targets.

Animals↗

Endothelial cell- and lymphocyte-based in vitro systems for understanding KSHV biology.

Kaposi sarcoma (KS), the most common AIDS-associated malignancy, is a multifocal tumor characterized by deregulated angiogenesis, proliferation of spindle cells, and extravasation of inflammatory cells and erythrocytes. Kaposi sarcoma-associated herpesvirus (KSHV; also human herpesvirus-8) is implicated in all clinical forms of KS. Endothelial cells (EC) harbor the KSHV genome in vivo, are permissive for virus infection in vitro, and are thought to be the precursors of KS spindle cells. Spindle cells are rare in early patch-stage KS lesions but become the predominant cell type in later plaque- and nodular-stage lesions. Alterations in endothelial/spindle cell physiology that promote proliferation and survival are thus thought to be important in disease progression and may represent potential therapeutic targets. KSHV encodes genes that stimulate cellular proliferation and migration, prevent apoptosis, and counter the host immune response. The combined effect of these genes is thought to drive the proliferation and survival of infected spindle cells and influence the lesional microenvironment. Large-scale gene expression analyses have revealed that KSHV infection also induces dramatic reprogramming of the EC transcriptome. These changes in cellular gene expression likely contribute to the development of the KS lesion. In addition to KS, KSHV is also present in B cell neoplasias including primary effusion lymphoma and multicentric Castleman disease. A combination of virus and virus-induced host factors are similarly thought to contribute to establishment and progression of these malignancies. A number of lymphocyte- and EC-based systems have been developed that afford some insight into the means by which KSHV contributes to malignant transformation of host cells. Whereas KSHV is well maintained in PEL cells cultured in vitro, explanted spindle cells rapidly lose the viral episome. Thus, endothelial cell-based systems for studying KSHV gene expression and function, as well as the effect of infection on host cell physiology, have required in vitro infection of primary or life-extended EC. This chapter includes a review of these in vitro cell culture systems, acknowledging their strengths and weaknesses and putting into perspective how each has contributed to our understanding of the complex KS lesional environment. In addition, we present a model of KS lesion progression based on findings culled from these models as well as recent clinical advances in KS chemotherapy. Thus this unifying model describes our current understanding of KS pathogenesis by drawing together multiple theories of KS progression that by themselves cannot account for the complexities of tumor development.

Animals↗

Alternative splicing: therapeutic target and tool.

Alternative splicing swells the coding capacity of the human genome, expanding the pharmacoproteome, the proteome that provides targets for therapy. Splicing, both constitutive and regulated forms, can itself be targeted by conventional and molecular therapies. This review focuses on splicing as a therapeutic target with a particular emphasis on molecular approaches. The review looks at the use of antisense oligonucleotides, which can be employed to promote skipping of constitutive exons, inhibit inappropriately activated exons, or stimulate exons weakened by mutations. Additionally this manuscript evaluates methods that reprogram RNAs using reactions that recombine RNA molecules in trans. Preliminary, but exciting, results in these areas of investigation suggest that these methods could eventually lead to treatments in heretofore intractable ailments.

Alternative Splicing↗

Subversion of host defense mechanisms by adenoviruses.

Adenoviruses (Ads) cause acute and persistent infections. Alike the much more complex herpesviruses, Ads encode numerous immunomodulatory functions. About a third of the viral genome is devoted to counteract both the innate and the adaptive antiviral immune response. Immediately upon infection, E1A blocks interferon-induced gene expression and the VA-RNA inhibits interferon-induced PKR activity. At the same time, E1A reprograms the cell for DNA synthesis and induces the intrinsic cellular apoptosis program that is interrupted by E1B/19K and E1B/55K proteins, the latter inhibits p53-mediated apoptosis. Most other viral stealth functions are encoded by a separate transcription units, E3. Several E3 products prevent death receptor-mediated apoptosis. E3/14.7K seems to interfere with the cytolytic and pro-inflammatory activities of TNF while E3/10.4K and 14.5K proteins remove Fas and TRAIL receptors from the cell surface by inducing their degradation in lysosomes. These and other functions that may afect granule-mediated cell death might drastically limit lysis by NK cells and cytotoxic T cells (CTL). Moreover, Ads interfere with recognition of infected cell by CTL. The paradigmatic E3/19K protein subverts antigen presentation by MHC class I molecules by inhibiting their transport to the cell surface. In concert, these viral countermeasures ensure prolonged survival in the infected host and, as a consequence, facilitate transmission. Elucidating the molecular mechanisms of Ad-mediated immune evasion has stimulated corresponding research on other viruses. This knowledge will also be instrumental for designing better vectors for gene therapy and vaccination, and may lead to a more rational treatment of life-threatening Ad infections, e.g. in transplantation patients.

Adenoviridae↗

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Culture Techniques↗

Cell sociology and the problem of automation in the development of pluricellular animals.

The principles of automation (automatism and programming) in the unfolding of spatiotemporal patterns during animal development are deduced from experimental data reconsidered from the point of view of cell sociology. The developmental programme in the egg is not part of the genetic information but a part of the cytoplasmic information. Throughout development cells store extra-cellular information released by their neighbours in the form of cytoplasmic information. Successive determinations cannot be considered as successive reprogrammings of cells: each one consists of a selection of one specific programme from the total information previously stored. This programme specifies cell interactions in the determined population as a whole; it is very imprecise and is progressively completed during the course of further differentiation by information released by neighbouring cell populations. Complicated patterns may emerge from only two homogeneous populations involved in distinct differentiation pathways and confronting each other. Consequently the "egg developmental programme" provides gene effectors and specific physico-chemical conditions necessary for the staring of at least two distinct differentation pathways. Experimental data suggest that there are two components in this programme. One is a molecular machinery which starts at fertilization in the whole cytoplasm. It yields two programmes of differentiation, typically first an endodermal and then an ectodermal one. The other component of the egg developmental programme, which does not require specific information, allows the interception of the first (endodermal) programme. The application of informatics to developmental automatism is discussed in the latter part of the paper.

Cell Differentiation↗

Denervated chicken breast muscle displays discoordinate regulation and differential patterns of expression of alpha f and beta tropomyosin genes.

The expression of the alpha fast (alpha f) and beta tropomyosin (TM) genes has been analysed with muscle-specific and common cDNA probes after unilateral nerve section of the pectoralis major muscle (PM) in 4-week-old chickens. The following were observed in denervated muscles. (1) The beta TM mRNA, which was repressed during development, reaccumulates in a biphasic curve with the increase in the beta TM protein lagging behind the changes in its mRNA. Accordingly, no beta TM is seen in products translated in vitro from total and polyA+ RNA obtained 1 week after denervation. No such translation block is seen with RNA obtained from control or muscles denervated for 6 weeks. (2) No changes in the alpha fTM mRNA and corresponding protein are observed. (3) RNA processing of the two genes is not changed. (4) In the contralateral muscles, transitory increases in alpha f and beta TM mRNAs are observed while the corresponding proteins remain unchanged. Our data suggest that muscle fibres display early and long-term responses to the loss of neural input which might result from a combination of changes produced by regenerative processes and reprogramming of existing fibres. Moreover, in contrast to normal development, no reciprocal changes of alpha f and beta TM expression are seen in denervated muscles.

Animals↗

Fibronectin distribution during cell type conversion in newt lens regeneration.

The distribution of fibronectin during the cell type conversion from iris into lens that occurs in newt lens regeneration was studied by immunofluorescence. Newts were lentectomized and irises at different stages of dedifferentiation and redifferentiation were examined using as a probe a rabbit antiserum prepared to Xenopus plasma fibronectin. In the normal iris, fibronectin is predominantly located at the basal surface of the pigmented iris epithelial cells. During activation and early dedifferentiation fibronectin staining is progressively displayed at the basolateral and apical surface of the depigmenting cell, to eventually surround the surface of the dedifferentiated cells. As cells redifferentiate into lens fibers, staining for cell surface fibronectin decreases and is displayed mainly in the nascent lens capsule. Fibronectin deposition may be associated with the formation of intercellular spaces during dedifferentiation. The fibronectin-rich extracellular matrix could be important in cell reprogramming.

Animals↗

[Teratoma and the mammalian embryo].

Teratomas are tumors that occur in the ovary or testis of mammals and are composed of a mixture of differentiated cells from various organs. Experimental investigations on teratomas have shown that a "biological therapy" of a tumor is feasible if these cells participate in normal differentiation and are able to reprogram changes in gene expression.

Animals↗

[Correlation between protein thiols and the doubling time of various rat hepatomas].

The SH content of the soluble proteins (nanomol./mg protein) from five transplantable rat hepatomas and from the DENA-hepatoma were determined with dithionitrobenzoate (Ellman reagent). Both the total number of thiols as well as the number of SH groups that can be blocked by hydroxypentenal (HPE) increase with increasing growth rate of the tumors. In comparison with the protein thiol content of the slowest growing DENA-hepatoma (doubling time 100 days), the total protein thiols of the fastest growing Yoshida hepatoma (doubling time 2,5 days) increase by 100% and the HPE-sensitive protein thiols by 350%. The total protein thiols are significantly correlated with the growth rate (probability of error 5%), the HPE-sensitive thiols are correlated with high significance (probability of error less than 1%). These results are in accordance with the "Molecular Correlation Concept" of G. Weber and might possibly be understood as a consequence of reprogramming of gene expressions.

Animals↗