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At least 109 records · Page 6Linked to original sources
PubMed1993

The regulation of human globin gene switching.

This paper describes the mechanism of regulation of the human beta-globin on the basis of a number of natural mutations and experiments in transgenic mice. From these data we conclude that this multigene locus is regulated at a number of different levels involving specific interactions between the Locus Control Region (LCR) and the individual genes. Most important is the action of stage specific transcription factors acting on sequences immediately flanking the genes. In addition, specificity is obtained through specific interaction of the genes with the LCR and through competition of the genes for interaction with the LCR.

Animals↗
PubMed2003

In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart.

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.

Adenoviridae↗
PubMed2005

The alc-GR system: a modified alc gene switch designed for use in plant tissue culture.

The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.

Animals↗
PubMed2002

Globin gene switching in primates.

Evolutionary approaches to the identification of DNA sequences required for transcription of the genes of the beta-globin cluster are reviewed. Sequence alignments of non-coding regions from widely divergent species revealed many conserved motifs (phylogenetic footprints) that were putative transcription factor binding sites and candidate binding proteins were identified. The differential timing of the prosimian and simian gamma-globin genes was analyzed by identifying base changes in the vicinity of the phylogenetic footprints. These differential phylogenetic footprints were shown to bind different nuclear factors, and the behavior of constructs with human or galago gamma-promoters in transgenic mice indicated that DNA motifs near the gamma-globin genes are sufficient to determine the developmental stage of expression. Locus control region alignments have identified many conserved sequence differences outside of the hypersensitive sites. Globin protein and mRNA expression profiles during embryological development in a series of catarrhine (Old World monkeys and apes) and platyrrhine (New World monkeys) primates have been determined. While all catarrhines examined to date have globin expression patterns that are highly similar to the well-established human switching behavior, platyrrhines have inactivated their gamma 1 genes by a variety of mechanisms, and have an earlier gamma-beta switch.

Animals↗
PubMed2002

Hexon gene switch strategy for the generation of chimeric recombinant adenovirus.

The usefulness of adenovirus as a vehicle for transgene delivery is limited greatly by the induction of neutralizing anti-adenoviral immunity following the initial administration, thereby resulting in shorter-term and reduced levels of transgene expression. In this paper, we outline a strategy for the generation of recombinant Ad5-based adenovectors that have undergone a complete hexon exchange in an effort to circumvent pre-existing anti-vector humoral immunity. Eighteen different chimeric adenoviral vectors (from subgroups A, B, C, D, and E) have been constructed using a combination of direct cloning and bacterial homologous recombination methods. However, only chimeric Ad5-based constructs in which the hexons from Ad1, Ad2, Ad6, and Ad12 are incorporated in place of the Ad5 hexon were successfully rescued into viruses. Despite several attempts, the remaining fourteen chimeric adenovectors were not rescuable. In vivo rodent studies using transgenes for human immunodeficiency virus type 1 (HIV-1) gag and secreted human alkaline phosphatase (SEAP) suggest that the Ad5/Ad6-gag chimera (wherein Ad5 hexon was replaced with that of Ad6) is able to evade neutralizing antibodies generated against Ad5 vector efficiently. However, it appears that cross-reactive cytotoxic T lymphocytes (CTL) may also play a role in controlling in vivo infectivity of Ad5/Ad6-gag chimera. The Ad5/Ad12 chimera was found to be extremely ineffective in the i.m. delivery and expression of HIV-1 gag in mice compared to the Ad5/Ad6 construct. Implications of these results will be discussed.

Adenoviruses, Human↗
PubMed1992

Genetics of the white-opaque transition in Candida albicans: demonstration of switching recessivity and mapping of switching genes.

Spheroplast fusion has been used to analyze the genetics of the reversible phenotypic transition, white-opaque, in Candida albicans WO-1. This transition involves changes in cell shape, permeability, and colony morphology. Fusion of switching with nonswitching cells gave nonswitching fusants, suggesting that the white-opaque phenotype is recessive. Chromosome loss induced by heat shock gave segregants of the fusants which were able to undergo the transition, indicating that the repressor function is genetically defined and probably limited to one or two chromosomes. Chromosomes R, 1, 3, 4, and 7 were eliminated as unique sites for the repressor, leaving 2, 5, and 6 as possible locations. When a ura3 (chromosome 3) nonswitching strain was fused with a switching strain, all ura3 segregants induced by heat shock were incapable of the phenotypic transition. Therefore, some or all of the genes (called SWI genes) essential for the transition are located on chromosome 3. UV irradiation-induced recombination did give rise to Ura- switching progeny, showing that the failure to switch was not due to a side effect of the pyrimidine requirement. The failure to isolate normally switching ura3 progeny generated by UV irradiation suggests a close linkage between the two genes.

Candida albicans↗
PubMed1990

Autonomous developmental control of human embryonic globin gene switching in transgenic mice.

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.

Animals↗
PubMed1996

A C. elegans Hox gene switches on, off, on and off again to regulate proliferation, differentiation and morphogenesis.

Hox genes establish body pattern throughout the animal kingdom, but the role these genes play at the cellular level to modify and shape parts of the body remains a mystery. We find that the C. elegans Antennapedia homolog, mab-5, sequentially programs many independent events within individual cell lineages. In one body region, mab-5 first switches ON in a lineage to stimulate proliferation, then OFF to specify epidermal structures, then ON in just one branch of the lineage to promote neuroblast formation, and finally OFF to permit proper sense organ morphology. In a neighboring lineage, continuous mab-5 expression leads to a different pattern of development. Thus, this Hox gene achieves much of its power to diversify the anteroposterior axis through fine spatiotemporal differences in expression coupled with a changing pattern of cellular response.

Alleles↗
PubMed2004

The role of p22 NF-E4 in human globin gene switching.

The human stage selector protein, a complex containing the ubiquitous transcription factor CP2 and the erythroid-specific factor p22 NF-E4, facilitates the interaction of the gamma-globin genes with the locus control region in fetal erythroid cells. Enforced expression of p22 NF-E4 in K562 cells and human cord blood progenitors increases fetal globin gene expression, and in progenitors, reduces beta-globin expression. To examine the role of NF-E4 in an in vivo model of hemoglobin switching, we enforced the expression of p22 NF-E4 in transgenic mice carrying the human beta-globin locus yeast artificial chromosome. Although murine erythropoiesis and globin gene expression is unaffected in these mice, the expression profile of the human globin genes is altered. All three transgenic lines displayed an increased gamma:beta-globin ratio in E12.5-14.5 fetal liver, resulting in a delay in the fetal/adult switch. At E12.5, this is primarily due to a reduction of beta-gene expression, whereas at E14.5, the increased gamma:beta ratio is due to enhanced gamma-gene expression. Despite this, the switch in globin subtype is fully completed in the adult bone marrow. These findings indicate that p22 NF-E4 is capable of influencing human globin gene expression in vivo but is incapable of overriding the intrinsic mechanisms governing gamma-gene silencing in this context.

Animals↗
PubMed1981

Alpha-Globin-gene switching during the development of chicken embryos: expression and chromosome structure.

We have analyzed the chromosome structure and expression of the alpha-gene cluster in developing chicken embryos. Using recombinant lambda clones (Dodgson and Engel, 1980), we show a striking relationship between chromosomal regions that are unmethylated, regions that are DNAase I-sensitive and regions that are transcribed. Adjacent regions at both the 5' and 3' sides of the active alpha genes are methylated and relatively insensitive to DNAase I. The active alpha subdomain defined by these assays begins right at the 5' side of the first alpha gene (alpha D) in the active cluster in definitive red cells and extends through a 1.5 kb spacer, into the second alpha gene (alpha A), and 1.5 kb beyond the 3' side of that gene. The sharp boundaries of this subdomain suggest that specific DNA sequences may establish its borders. The extension of the active chromosomal domain beyond the most stable nuclear transcript suggests that transcription may proceed beyond the 3' ends of both alpha A and alpha D. This has been verified by in vitro runoff nuclear transcription. Presumably, poly(A) addition occurs before transcription is terminated. During the switch from the primitive to definitive lineage of erythroblasts, the so-called U gene becomes inactive. This inactivity is reflected in its assembly into a more DNAase-resistant structure. The associated DNA also becomes methylated, and no transcription is detectable by endogenous RNA polymerases. A DNAase I-hypersensitive region at the gene becomes inactivated after the switch to the definitive lineage.

Animals↗
PubMed1992

Histone acetylation and globin gene switching.

An affinity-purified antibody that recognises the epitope epsilon-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic beta rho gene (active at 5 days) and from the adult beta A gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription.

Acetylation↗
PubMed1996

Globin gene switching. In vivo protein-DNA interactions of the human beta-globin locus in erythroid cells expressing the fetal or the adult globin gene program.

To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene.

Adult↗
PubMed1998

Mating-type gene switching in Saccharomyces cerevisiae.

Saccharomyces cerevisiae can change its mating type as often as every generation by a highly choreographed, site-specific recombination event that replaces one MAT allele with different DNA sequences encoding the opposite allele. The study of this process has yielded important insights into the control of cell lineage, the silencing of gene expression, and the formation of heterochromatin, as well as the molecular events of double-strand break-induced recombination. In addition, MAT switching provides a remarkable example of a small locus control region--the Recombination Enhancer--that controls recombination along an entire chromosome arm.

Animals↗
PubMed1999

Cell-surface area codes: mobile-element related gene switches generate precise and heritable cell-surface displays of address molecules that are used for constructing embryos.

We present an updated area code hypothesis supporting the proposal that cell surface display of seven-transmembrane olfactory receptors, protocadherins and other cell surface receptors provide codes that enable cells to find their correct partners as they sculpture embryos. The genetic mechanisms that program the expression of such displays have been largely unknown until very recently. However, increasing evidence now suggests that precise developmental control of the expression of these genes during embryogenesis is achieved in part by permanent and heritable changes in DNA. Using the developing immune system as a model, we discuss two different types of developmentally programmed genetic switches, each of which relies on recombination mechanisms related to mobile elements. We review new evidence suggesting the involvement of mobile element related switch mechanisms in the generation of protocadherin molecules, and their possible involvement in the control of expressions of olfactory receptors. As both recombinase and reverse transcriptase mechanisms play a role in the switching of the immunoglobulin genes, we searched the databases of expressed sequence tags (dbEST) for expression of related genes in other tissues. We present data revealing that transposases and reverse transcriptases are widely expressed in most tissues. We also searched these databases for expression of env (envelope) gene products, stimulated by provocative results suggesting that these molecules might function as cellular address receptors. We found that env genes are also expressed in large numbers in normal human tissues. One must assume that these three different types of mobile-element-related messenger RNA molecules (transposases, reverse transcriptases, and env proteins) are expressed for use in functions of value in the various tissues and have been preserved in the genome because of their selective advantages. We conclude that it is possible that many specific cell lineage decisions are made and remembered by means of genetic switches similar to those that control the immunoglobulin and protocadherin and, probably, the seven transmembrane/olfactory gene families. We also conclude that complex genetic programs utilizing mobile-element-related genes program these events.

Cell Membrane↗
PubMed2005

Novel method of generating prostate-specific Cre-LoxP gene switching via intraductal delivery of adenovirus.

BACKGROUND: In order to facilitate elucidation of oncogene or tumor suppressor gene function on initiation and progression of prostate cancer, it would be advantageous to develop an effective method to generate spatially and temporally controlled gene modification in murine prostates. METHODS: Adenovirus expressing Cre-recombinase (Adeno-Cre) was intraductally injected into the prostate of ROSA26 reporter mice. Immmunohistochemical and X-gal staining were performed on prostate tissue sections harvested from mice at various time points following viral injection to confirm expression and activity of Cre-recombinase, respectively. RESULTS: Adenovirus was intraductally delivered to the anterior lobe of the mouse prostate. Using this method of intraductal injection, we were able to precisely obtain Adeno-Cre infection to a majority of epithelial cells but not in the stromal cells or other organs. We further demonstrated that Adeno-Cre infected epithelial cells not only expressed Cre-recombinase enzyme but more importantly, Cre-recombinase activity was revealed through positive X-gal staining in Rosa26 reporter mice, thus, confirming epithelial-specific Cre-loxP recombination in Adeno-Cre infected prostate tissue sections. CONCLUSIONS: This novel method of direct genetic delivery into adult murine prostates could provide an alternative to the more expensive and time-consuming transgenic/knockout approaches. The latter also have other limitations such as the availability of cell-type specific or temporally-regulated promoters, and the complication of genetic background differences, which can potentially be complemented by the technology we describe here.

Adenoviridae↗
PubMed1998

An ethanol inducible gene switch for plants used to manipulate carbon metabolism.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Alcohol Dehydrogenase↗