Demethylation of genes in animal cells

1990 ◽  
Vol 326 (1235) ◽  
pp. 241-251 ◽  
Keyword(s):  
Tissue Culture ◽  
Germ Line ◽  
Animal Cell ◽  
Gene Activation ◽  
Housekeeping Gene ◽  
Cell Types ◽  
Embryonic Cells ◽  
Animal Cells ◽  
Developmentally Regulated ◽  
I Gene

Tissue-specific animal cell genes are usually fully methylated in the germ line and become demethylated in those cell types in which they are expressed. To investigate this process, we inserted a methylated IgG K gene into fibroblasts and lymphocytes at various stages of development. The results show that this gene undergoes demethylation only in the mature lymphocytes and therefore suggest that the ability to demethylate a gene is developmentally regulated. These studies were supported by similar experiments using the rat Insulin I gene, and in this case it appears that the cis -acting elements that control demethylation may be different from those responsible for gene activation. The ability to demethylate the housekeeping gene APRT is also under developmental control, because this occurs only in embryonic cells, both in tissue culture and in transgenic mice.

scholarly journals A Spindle Checkpoint Functions during Mitosis in the Early Caenorhabditis elegans Embryo

2005 ◽  
Vol 16 (3) ◽  
pp. 1056-1070 ◽  
Author(s):  
Sandra E. Encalada ◽  
John Willis ◽  
Rebecca Lyczak ◽  
Bruce Bowerman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Metaphase Plate ◽  
Time Lapse ◽  
Cell Types ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Dividing Cells

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

scholarly journals Stable C. elegans chromatin domains separate broadly expressed and developmentally regulated genes

2016 ◽  
Author(s):  
Kenneth J. Evans ◽  
Ni Huang ◽  
Przemyslaw Stempor ◽  
Michael A. Chesney ◽  
Thomas A. Down ◽  
...  
Keyword(s):  
Domain Structure ◽  
Germ Line ◽  
Cell Types ◽  
Chromatin Domain ◽  
Developmentally Regulated ◽  
Chromatin Domains ◽  
Domain Organization ◽  
C Elegans ◽  
Border Regions ◽  
Early Embryos

AbstractEukaryotic genomes are organized into domains of differing structure and activity. There is evidence that the domain organization of the genome regulates its activity, yet our understanding of domain properties and the factors that influence their formation is poor. Here we use chromatin state analyses in early embryos and L3 larvae to investigate genome domain organization and its regulation in C. elegans. At both stages we find that the genome is organized into extended chromatin domains of high or low gene activity defined by different subsets of states, and enriched for H3K36me3 or H3K27me3 respectively. The border regions between domains contain large intergenic regions and a high density of transcription factor binding, suggesting a role for transcription regulation in separating chromatin domains. Despite the differences in cell types, overall domain organization is remarkably similar in early embryos and L3 larvae, with conservation of 85% of domain border positions. Most genes in high activity domains are expressed in the germ line and broadly across cell types, whereas low activity domains are enriched for genes that are developmentally regulated. We find that domains are regulated by the germ line H3K36 methyltransferase MES-4 and that border regions show striking remodeling of H3K27me1, supporting roles for H3K36 and H3K27 methylation in regulating domain structure. Our analyses of C. elegans chromatin domain structure show that genes are organized by type into domains that have differing modes of regulation.Significance statementGenomes are organized into domains of different structure and activity, yet our understanding of their formation and regulation is poor. We show that C. elegans chromatin domain organization in early embryos and L3 larvae is remarkably similar despite the two developmental stages containing very different cell types. Chromatin domains separate genes into those with stable versus developmentally regulated expression. Analyses of chromatin domain structure suggest that transcription regulation and germ line chromatin regulation play roles in separating chromatin domains. Our results further our understanding of genome domain organization.

scholarly journals Stable Caenorhabditis elegans chromatin domains separate broadly expressed and developmentally regulated genes

2016 ◽  
Vol 113 (45) ◽  
pp. E7020-E7029 ◽  
Author(s):  
Kenneth J. Evans ◽  
Ni Huang ◽  
Przemyslaw Stempor ◽  
Michael A. Chesney ◽  
Thomas A. Down ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Domain Structure ◽  
Germ Line ◽  
Cell Types ◽  
Chromatin Domain ◽  
Developmentally Regulated ◽  
Chromatin Domains ◽  
Domain Organization ◽  
Border Regions ◽  
Early Embryos

Eukaryotic genomes are organized into domains of differing structure and activity. There is evidence that the domain organization of the genome regulates its activity, yet our understanding of domain properties and the factors that influence their formation is poor. Here, we use chromatin state analyses in early embryos and third-larval stage (L3) animals to investigate genome domain organization and its regulation in Caenorhabditis elegans. At both stages we find that the genome is organized into extended chromatin domains of high or low gene activity defined by different subsets of states, and enriched for H3K36me3 or H3K27me3, respectively. The border regions between domains contain large intergenic regions and a high density of transcription factor binding, suggesting a role for transcription regulation in separating chromatin domains. Despite the differences in cell types, overall domain organization is remarkably similar in early embryos and L3 larvae, with conservation of 85% of domain border positions. Most genes in high-activity domains are expressed in the germ line and broadly across cell types, whereas low-activity domains are enriched for genes that are developmentally regulated. We find that domains are regulated by the germ-line H3K36 methyltransferase MES-4 and that border regions show striking remodeling of H3K27me1, supporting roles for H3K36 and H3K27 methylation in regulating domain structure. Our analyses of C. elegans chromatin domain structure show that genes are organized by type into domains that have differing modes of regulation.

Characteristics of five cell types appearing during in vitro culture of embryonic material from Drosophila melanogaster

Development ◽  
1970 ◽  
Vol 23 (1) ◽  
pp. 53-69
Author(s):  
Glen Shields ◽  
James H. Sang
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Culture Medium ◽  
Cell Types ◽  
Embryonic Cell ◽  
Basic Information ◽  
Embryonic Cells ◽  
Continuous Multiplication ◽  
Material Progress

Although attempts have been made over a number of decades to achieve successful tissue culture of Drosophila material, progress has been held back until recently by a lack of basic information on the chemical and physiological characteristics of the haemolymph of the organism necessary for a reasoned formulation of a culture medium. In 1963, Begg & Cruickshank published details of the mineral composition, osmotic pressure and pH of the haemolymph of third instar larvae, and this has provided the basis for a more satisfactory approach, as reflected in an increasing amount of fruitful work since reported. Much of this work has been done with embryonic material. Horikawa & Fox (1964) claimed continuous multiplication of a small type of early embryonic cell; Lesseps (1965) described re-aggregation of dissociated embryonic cells in culture, with possible development of muscle cells, nerve cells and oenocytes in the aggregates.

scholarly journals Applications and analysis of hydrolysates in animal cell culture

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Yin Ying Ho ◽  
Hao Kim Lu ◽  
Zhi Feng Sherman Lim ◽  
Hao Wei Lim ◽  
Ying Swan Ho ◽  
...  
Keyword(s):  
Cell Culture ◽  
Growth Factors ◽  
Culture Media ◽  
Animal Cell ◽  
Cell Types ◽  
Animal Cell Culture ◽  
High Price ◽  
Biological Research ◽  
Animal Cells ◽  
Beneficial Effects

AbstractAnimal cells are used in the manufacturing of complex biotherapeutic products since the 1980s. From its initial uses in biological research to its current importance in the biopharmaceutical industry, many types of culture media were developed: from serum-based media to serum-free to protein-free chemically defined media. The cultivation of animal cells economically has become the ultimate goal in the field of biomanufacturing. Serum serves as a source of amino acids, lipids, proteins and most importantly growth factors and hormones, which are essential for many cell types. However, the use of serum is unfavorable due to its high price tag, increased lot-to-lot variations and potential risk of microbial contamination. Efforts are progressively being made to replace serum with recombinant proteins such as growth factors, cytokines and hormones, as well as supplementation with lipids, vitamins, trace elements and hydrolysates. While hydrolysates are more complex, they provide a diverse source of nutrients to animal cells, with potential beneficial effects beyond the nutritional value. In this review, we discuss the use of hydrolysates in animal cell culture and briefly cover the composition of hydrolysates, mode of action and potential contaminants with some perspectives on its potential role in animal cell culture media formulations in the future.

NUTRITION OF ANIMAL CELLS IN TISSUE CULTURE: VII. USE OF REPLICATE CELL CULTURES IN THE EVALUATION OF SYNTHETIC MEDIA

1954 ◽  
Vol 32 (1) ◽  
pp. 306-318
Author(s):  
Raymond C. Parker ◽  
George M. Healy ◽  
Dorothy C. Fisher
Keyword(s):  
Tissue Culture ◽  
Cell Cultures ◽  
Subsequent Treatment ◽  
Relative Toxicity ◽  
Animal Cells ◽  
Washed Cell ◽  
Mouse Cells ◽  
Synthetic Media ◽  
Simple Screening ◽  
Natural Media

The replicate culture assay procedures of Earle and his associates have been adapted for use in evaluating the effectiveness of synthetic media. For this purpose, use has also been made of Earle's L strain mouse cells. Washed and continuously stirred suspensions of these or similar strains of cells may be dispensed, with reasonable assurance of uniformity, into a series of replicate cultures, the number depending on the volume of the suspension and the capacity and effectiveness of the stirring and dispensing unit. For use with synthetic media, the original procedures for the preparation and care of the replicate cultures and for their subsequent treatment for the counting of isolated, stained nuclei have been modified considerably. This paper describes the procedures that were finally adopted and also describes a relatively simple screening procedure in which washed cell suspensions may be used to advantage in making preliminary assays of synthetic media and in testing the relative toxicity or growth stimulating effects of substances added to, or derived from, natural media.

NUTRITION OF ANIMAL CELLS IN TISSUE CULTURE: VII. USE OF REPLICATE CELL CULTURES IN THE EVALUATION OF SYNTHETIC MEDIA

1954 ◽  
Vol 32 (3) ◽  
pp. 306-318 ◽  
Author(s):  
Raymond C. Parker ◽  
George M. Healy ◽  
Dorothy C. Fisher
Keyword(s):  
Tissue Culture ◽  
Cell Cultures ◽  
Subsequent Treatment ◽  
Relative Toxicity ◽  
Animal Cells ◽  
Washed Cell ◽  
Mouse Cells ◽  
Synthetic Media ◽  
Simple Screening ◽  
Natural Media

The replicate culture assay procedures of Earle and his associates have been adapted for use in evaluating the effectiveness of synthetic media. For this purpose, use has also been made of Earle's L strain mouse cells. Washed and continuously stirred suspensions of these or similar strains of cells may be dispensed, with reasonable assurance of uniformity, into a series of replicate cultures, the number depending on the volume of the suspension and the capacity and effectiveness of the stirring and dispensing unit. For use with synthetic media, the original procedures for the preparation and care of the replicate cultures and for their subsequent treatment for the counting of isolated, stained nuclei have been modified considerably. This paper describes the procedures that were finally adopted and also describes a relatively simple screening procedure in which washed cell suspensions may be used to advantage in making preliminary assays of synthetic media and in testing the relative toxicity or growth stimulating effects of substances added to, or derived from, natural media.

scholarly journals A Developmentally Regulated Gene, ASI2, Is Required for Endocycling in the Macronuclear Anlagen of Tetrahymena

2010 ◽  
Vol 9 (9) ◽  
pp. 1343-1353 ◽  
Author(s):  
Lihui Yin ◽  
Susan T. Gater ◽  
Kathleen M. Karrer
Keyword(s):  
Signal Transduction ◽  
Mitotic Cycle ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Ciliated Protozoa ◽  
Dna Rearrangement ◽  
Developmentally Regulated ◽  
Content Type ◽  
Molecular Events ◽  
Multicellular Organisms

ABSTRACT Ciliated protozoa contain two types of nuclei, germ line micronuclei (Mic) and transcriptionally active macronuclei (Mac). During sexual reproduction, the parental Mac degenerates and a new Mac develops from a mitotic product of the zygotic Mic. Macronuclear development involves extensive endoreplication of the genome. The present study shows that endoreplication of macronuclear DNA in Tetrahymena is an example of endocyling, a variant of the mitotic cycle with alternating S and G phases in the absence of cell division. Thus, endocycling is conserved from ciliates to multicellular organisms. The gene ASI2 in Tetrahymena thermophila encodes a putative signal transduction receptor. ASI2 is nonessential for vegetative growth, but it is upregulated during development of the new Mac. Cells that lack ASI2 in the developing Mac anlagen are arrested in endoreplication of the DNA and die. This study shows that ASI2 is also transcribed in the parental Mac early in conjugation and that transcription of ASI2 in the parental Mac supports endoreplication of the DNA during early stages of development of the Mac anlagen. Other molecular events in Mac anlage development, including developmentally regulated DNA rearrangement, occur normally in matings between ASI2 knockouts, suggesting that ASI2 specifically regulates endocycling in Tetrahymena.

Stable transfer and restricted expression of a cloned class I gene encoding a secreted transplantation-like antigen

1985 ◽  
Vol 5 (6) ◽  
pp. 1295-1300
Author(s):  
Y Barra ◽  
K Tanaka ◽  
K J Isselbacher ◽  
G Khoury ◽  
G Jay
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Class I ◽  
Regulatory Sequences ◽  
Transcriptional Enhancer ◽  
Gene Encoding ◽  
Specific Expression ◽  
Tissue Specific ◽  
Functional Studies ◽  
I Gene

The identification of a unique major histocompatibility complex class I gene, designated Q10, which encodes a secreted rather than a cell surface antigen has led to questions regarding its potential role in regulating immunological functions. Since the Q10 gene is specifically activated only in the liver, we sought to define the molecular mechanisms which control its expression in a tissue-specific fashion. Results obtained by transfection of the cloned Q10 gene, either in the absence or presence of a heterologous transcriptional enhancer, into a variety of cell types of different tissue derivations are consistent with the Q10 gene being regulated at two levels. The first is by a cis-dependent mechanism which appears to involve site-specific DNA methylation. The second is by a trans-acting mechanism which would include the possibility of an enhancer binding factor. The ability to efficiently express the Q10 gene in certain transfected cell lines offers an opportunity to obtain this secreted class I antigen in quantities sufficient for functional studies; this should also make it possible to define regulatory sequences which may be responsible for the tissue-specific expression of Q10.

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