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Results for “Enhancer Elements, Genetic”

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PubMed2005

Genetic screens for clock mutants in Drosophila.

The isolation and analysis of mutant flies (Drosophila melanogaster) with altered circadian rhythms have led to an understanding of circadian rhythms at the molecular level. This molecular mechanism elucidated in fruit flies is similar to the mechanism of the human circadian clock, which confers 24-h rhythmicity to our sleep/wake behavior, as well as to many other aspects of our cellular and organismal physiology. In fruit flies, genes can be mutated to abolish circadian rhythms (i.e., produce arrhythmia) or alter the period of the circadian rhythm; these genes encode key components of the circadian oscillator mechanism. Other mutations have identified components of the input pathways (by which light and temperature synchronize the circadian clock to environmental cycles) or output pathways (which connect the circadian oscillator to the physiological response). Mutations in genes are typically generated by chemical mutagenesis or mutagenesis with transposable elements. Flies with mutagenized chromosomes are processed in a series of genetic crosses, which allow specific chromosomes to be screened for semidominant mutations, recessive mutations, enhancer/suppressor mutations, or genes that can be overexpressed to alter circadian rhythms. Circadian phenotypes, which are assayed to identify mutants, include eclosion (emergence of the adult from the pupal case), locomotor activity (similar to human sleep?wake behavior), and circadian oscillations of gene expression. It is argued that screens for new circadian genes will continue to reveal novel components of the circadian mechanism.

Animals↗

PubMed1995

Identification of a new subclass of Alu DNA repeats which can function as estrogen receptor-dependent transcriptional enhancers.

We have utilized a genetic selection system in yeast to identify novel estrogen-responsive genes within the human genome and to define the sequences in the BRCA-1 gene responsible for its estrogen responsiveness. This approach led to the identification of a new subclass within the Alu family of DNA repeats which have diverged from known Alu sequences and have acquired the ability to function as estrogen receptor-dependent enhancers. Importantly, these new elements confer receptor-dependent estrogen responsiveness to a heterologous promoter when assayed in mammalian cells. This transcriptional activity can be attenuated by the addition of either of three different classes of estrogen receptor antagonists, indicating that these elements function as classical estrogen receptor-dependent enhancers. Furthermore, this enhancer activity is restricted to a specific subset of DNA repeats because consensus Alu elements of four major subfamilies do not respond to the estrogen receptor. Previously, most Alu sequences have been considered to be functionally inert. However, this work provides strong evidence that a significant subset can confer estrogen responsiveness upon a promoter within which they are located. Clearly, Alu sequences must now be considered as important contributors to the regulation of gene transcription in estrogen receptor-containing cells.

Animals↗

PubMed2003

Evidence that the mouse 3' kappa light chain enhancer confers position-independent transgene expression in T- and B-lineage cells.

One of the major obstacles for successful application of murine leukemia virus (MLV) vectors to genetic therapy of lymphocyte disorders is low levels of transgene expression or the eventual loss of expression. To overcome this problem, an improved retroviral vector was constructed utilizing the myeloproliferative sarcoma virus (MPSV) long terminal repeat (LTR), which provided a significantly higher level of transgene expression in human lymphoid cells than did MLV vectors. Nevertheless, transgene expression remained low in a large percentage of transduced cells. To address whether lymphocyte enhancer elements might improve transgene expression mediated by retroviral vectors in lymphocytes, we cloned the mouse immunoglobulin 3' kappa light chain enhancer gene (mE3') into the MPSV vector. We found that the mE3' conferred a higher, more uniform and sustained level of expression in transduced T- and B-cell lines, and in primary T cells, than did the control vector lacking this element. Integration sites were diverse and a single copy of the proviral genome was present in all examined transduced cells. The mE3' failed to enhance transgene expression in most nonlymphoid cells, indicating it is relatively lineage-specific. Taken together, these results provide strong evidence that the mE3' functions as a locus control region (LCR) in conferring enhanced integration-site-independent expression of a retroviral transgene.

Animals↗

PubMed2001

Distant cis-elements regulate imprinted expression of the mouse p57( Kip2) (Cdkn1c) gene: implications for the human disorder, Beckwith--Wiedemann syndrome.

Complex phenotypes and genotypes characterize the human disease, Beckwith--Wiedemann syndrome (BWS). Genetic and epigenetic mutations are found in five different genes which all lie within a 1 Mb imprinted domain on human chromosome 11p15. Only two of these genes, p57(KIP2) (CDKN1C) and IGF2, are likely to be functionally involved in this disease. The presence of the additional mutations therefore suggests a role for the regulation of these two genes by distant cis-elements. The mouse Igf2 gene is regulated by enhancers and imprinting elements which lie >120 kb downstream of its promoter. Here we show that key elements for expression of the mouse p57(Kip2) (Cdkn1c) gene also lie at a distance. Enhancers for expression within skeletal muscle and cartilage lie >25 kb downstream of the gene. In addition, we find no evidence for allele-specific expression of p57(Kip2) (Cdkn1c) from our bacterial artificial chromosome transgenes that span 315 kb around the locus. This suggests that a key imprinting element for p57(Kip2) (Cdkn1c) also lies at a distance. Therefore, BWS in humans may result from disruption of appropriate expression of the p57(KIP2) (CDKN1C) gene through mutations that occur at a substantial distance from the gene.

Animals↗

PubMed2004

High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics.

A library of 29,482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12,707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value < 1.00 e(-05)). To illustrate the in silico reverse genetic approach, identification of 83 T-DNA insertions within genes coding for transcription factors (TF) is presented. Based both on the estimated number of members of several large TF gene families (e.g. Myb, WRKY, HD-ZIP, Zinc-finger) and on the frequency of insertions in chromosome 1 predicted genes, we could extrapolate that 7-10% of the rice gene complement is already tagged by T-DNA insertion in the 6116 independent transformant population. This large resource is of high significance while assisting studies unravelling gene function in rice and cereals, notably through in silico reverse genetics.

Base Sequence↗

PubMed2004

Advances in the molecular biology of tombusviruses: gene expression, genome replication, and recombination.

The tombusviruses are among the most extensively studied messenger-sensed RNA plant viruses. Over the past decade, there have been numerous important advances in our understanding of the molecular biology of members in this genus. Unlike most other RNA viruses, the synthesis of tombusvirus proteins has been found to involve an atypical translational mechanism related to the uncapped and nonpolyadenylated nature of their genomes. Tombusviruses also appear to employ an unusual mechanism for transcription of the sg mRNAs that template translation of a subset of their viral proteins. In addition to these new insights into tombusvirus gene expression, there has also been significant progress made in our understanding of tombusvirus RNA replication. These studies have been facilitated greatly by small genome-derived RNA replicons, referred to as defective interfering RNAs. In addition, the development of an in vitro system to study viral RNA synthesis has allowed for dissection of some of the steps involved in the replication process. Another exciting recent advance has been the creation of yeast-based systems that support amplification of tombusvirus RNA replicons and will allow the identification of host factors involved in viral RNA synthesis. Lastly, the recombinogenic nature of tombusvirus genomes has made them ideal systems for studying RNA-RNA recombination and genetic rearrangements, both in vivo and in vitro. In this review, we compile recent information on each of the aforementioned processes-translation, transcription, replication and recombination-and discuss the significance of the results.

Base Sequence↗

PubMed1995

A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers.

Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial beta-galactosidase (beta-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. beta-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue.

Animals↗

PubMed2005

Genetic analysis of the human papillomavirus type 31 differentiation-dependent late promoter.

Human papillomaviruses infect stratifying squamous epithelia, causing benign and malignant lesions. Upon differentiation of the host keratinocyte, the virus undergoes a dramatic increase in both DNA replication and transcription from the late promoter, leading to expression of late genes and virion morphogenesis. In human papillomavirus type 31 (HPV31), the late promoter is designated p742 and includes multiple start sites embedded within the E7 gene. In this report, we mapped viral DNA elements that control transcriptional activity from p742. Enhancer elements in the viral upstream regulatory region positively regulate this promoter. The region containing the transcriptional start sites is dispensable for activity, and at least two separate elements in the E6/E7 region are capable of supporting transcription. Of these, we mapped one to a 150-bp region of the E7 open reading frame and designate it the core p742 promoter. Using GF109203X, an inhibitor of protein kinase C signaling, we show that p742 activation is independent of viral genome amplification. Finally, we mapped elements in the region of p742 that confer responsiveness to differentiation and show that the upstream regulatory region does not contribute to the differentiation response of p742. These studies are an important step toward understanding the functioning and regulation of this multiple-start promoter.

Cells, Cultured↗

PubMed1999

Main features of DNA-based immunization vectors.

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Enhancer Elements, Genetic↗

PubMed2003

Phylogenetic analysis of the mammalian Hoxc8 non-coding region.

The non-coding intergenic regions of Hox genes are remarkably conserved among mammals. To determine the usefulness of this sequence for phylogenetic comparisons, we sequenced an 800-bp fragment of the Hoxc9-Hoxc8 intergenic region from several species belonging to different mammalian clades. Results obtained from the phylogenetic analysis are congruent with currently accepted mammalian phylogeny. Additionally, we found a TC mini satellite repeat polymorphism unique to felines. This polymorphism may serve as a useful marker to differentiate between mammalian species or as a genetic marker in feline matings. This study demonstrates usefulness of a comparative approach employing non-coding regions of Hox gene complexes.

Animals↗

PubMed1989

Regulation of larval cuticle protein gene expression in Drosophila melanogaster.

Genes that encode 3rd instar larval cuticle proteins (LCP's) of Drosophila melanogaster are located in at least two chromosomal sites. The genes encoding four of the five predominant LCP's are located in a cluster at the chromosomal region 44D. They are organized in pairs that are transcribed divergently, and expressed with different timing during the third larval instar. Towards understanding the basis of gene regulation within the 44D cluster, we have analyzed genetic variants, including the 2-3 variant, which has an insertion of a copia-like transposable element, H.M.S. Beagle, within the 44D cluster. The Beagle element appears to inactivate the LCP-3 gene by inserting into its TATA box, but also may cause the precocious expression of two other LCP genes, LCP-1 and LCP-f2, in the cluster. The long terminal repeat (LTR) of the Beagle element apparently contains a sequence, perhaps an enhancer-like element, which causes altered expression of these genes. We have also investigated the cis-regulatory elements involved in expression of the LCP-2 gene in wild-type larvae. We have identified two upstream regions that may contain separate cis-regulatory elements. The region between -252 bp and -515 bp may be essential for any expression of LCP-2. Additionally, the region between -515 bp and -795 bp appears to be required for the normal level of expression of the LCP-2 gene.

Animals↗

PubMed2003

Retrovirus silencer blocking by the cHS4 insulator is CTCF independent.

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.

Animals↗

PubMed1994

Regulated expression of a dominant negative form of Rev improves resistance to HIV replication in T cells.

Infection by the human immunodeficiency virus (HIV) has remained refractory to treatment, and molecular genetic interventions have been developed for the treatment of the acquired immunodeficiency syndrome (AIDS). Previous studies have focused on the development of gene products which inhibit productive HIV replication, including transdominant proteins, RNA decoys and ribozymes. In this report, we show that appropriate expression vectors which optimize production and regulated synthesis of a transdominant mutant form of Rev improve its antiviral effect. The combination of a strong constitutive enhancer, a Tat activation response (TAR) regulatory element and transdominant Rev take advantage of three aspects of early viral gene expression to confer increased resistance to HIV replication. This vector may be useful, alone or in combination with other antiviral genes, to provide gene therapy for AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex.

The central complex is an important center for higher-order brain function in insects. It is an intricate neuropil composed of four substructures. Each substructure contains repeated neuronal elements which are connected by processes such that topography is maintained. Although the neuronal architecture has been described in several insects and the behavioral role investigated in various experiments, the exact function of this neuropil has proven elusive. To describe the architecture of the central complex, we study 15 enhancer-trap lines that label various ellipsoid body neuron types. We find evidence for restriction of gene expression that is correlated with specific neuronal types: such correlations suggest functional classifications as well. We show that some enhancer-trap patterns reveal a single ellipsoid body neuron type, while others label multiple types. We describe the development of the ellipsoid body neuropil in wild-type animals and propose developmental mechanisms based on animals displaying structural mutations of this neuropil. The experiments performed here demonstrate the degree of resolution possible from the analysis of enhancer-trap lines and form a useful library of tools for future structure/function studies of the ellipsoid body.

Animal Structures↗

PubMed2004

Neurogenic phenotypes induced by RNA interference with bHLH genes of the Enhancer of split complex of Drosophila melanogaster.

The Enhancer of split gene complex [E(spl)-C] of Drosophila melanogaster harbors seven highly related genes encoding transcriptional regulators with a basic helix-loop-helix (bHLH) domain. They are activated by the Notch signaling pathway in order to inhibit proneural gene activity, for example, during neurogenesis in the developing embryo. The E(spl) proteins are at least partly redundant, despite some remarkable differences in their expression patterns. We attempted to address the degree of redundancy by means of RNA interference. We find a quantitative correlation between the degree of a neurogenic phenotype and the number of genes affected. Surprisingly, interference with m3 results in a high rate of mortality which cannot be reproduced by genetic mutation. Most likely, m3 dsRNA interferes with unrelated genes involved in other aspects of embryonic development.

Animals↗

PubMed2005

Influence of combined methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase enhancer region (TSER) polymorphisms to plasma homocysteine levels in Korean patients with recurrent spontaneous abortion.

OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR) mutations known to be associated with hyperhomocysteinemia may be a risk factor for recurrent spontaneous abortion. Recently 28-bp tandem repeat polymorphism in thymidylate synthase enhancer region (TSER) was reported to affect plasma homocysteine level. We investigated the association between plasma homocysteine level and MTHFR and TSER genotypes. METHODS: Plasma homocysteine level was measured by fluorescent polarizing immunoassay. MTHFR mutations (C677T and A1298C) were identified by PCR-restriction fragment length polymorphism assay. TSER mutation was analyzed by PCR method. RESULTS: Average homocysteine level was significantly higher in MTHFR 677TT genotype (9.80+/-3.87 micromol/L) than in MTHFR 677CT (7.04+/-1.99 micromol/L) in MTHFR 677CC genotype (8.14+/-1.74 micromol/L) in Korean patients with unexplained recurrent spontaneous abortion (p=0.0143). While MTHFR 1298AA showed the highest level, plasma homocysteine levels were not significantly different among MTHFR 1298AA (8.42+/-2.65 micromol/L), 1298AC (6.98+/-2.44 micromol/L) and 1298CC (6.09+/-0.32 micromol/L) (p=0.2058). There was no significant difference among TSER genotypes (2R2R, 8.61+/-1.68 micromol/L; 2R3R, 7.84+/-2.16 micromol/L; 3R3R, 8.05+/-2.81 micromol/L; p=0.9319). Among the combined genotypes of MTHFR C677T and TSER, 677TT-3R3R genotype had the highest homocysteine level (11.47+/-4.66 micromol/L). 1298AA-3R3R had the highest level (8.54+/-3.05 micromol/L) among the combined genotypes of MTHFR A1298C and TSER. CONCLUSION: Although there was no significant difference found among combined genotypes, 3R3R showed elevated homocysteine levels in MTHFR 677TT and 1298AA in Korean patients with unexplained recurrent spontaneous abortion. Thus TSER polymorphism may be a genetic determinant of plasma homocysteine level in Korean patients as well as MTHFR C677T polymorphism.

Abortion, Habitual↗

PubMed2003

An enhancer-titration effect induces digit-specific regulatory alleles of the HoxD cluster.

Mice carrying transgenes targeted upstream the HoxD cluster display abnormal digits, with alterations resembling those obtained with loss of functions of Hoxd genes. Because the HoxD cluster remained entirely untouched by the insertional events, we investigated whether these phenotypes were induced by regulatory modifications at a distance. We report here that these targeted relocations behaved as hypomorphic alleles of the distantly located gene Hoxd13 and showed that posterior Hoxd genes located in cis with the integration site were down-regulated. Genetic analyses suggested that this down-regulation resulted from the titration of the activity of a remote located enhancer sequence. These results indicate that the transcriptional efficiency of Hoxd genes in digits could be modulated by the presence of other, unrelated, promoters, within the regulatory landscape of this enhancer. Modifications in these latter transcription units may thus impact upon digit morphology, through misregulation of Hoxd genes, thus illustrating the "buffering effect" that such a global regulatory element can exert upon a short genomic interval.

Animals↗

PubMed1988

A transcriptionally defective long terminal repeat within an endogenous copy of mouse mammary tumor virus proviral DNA.

Mouse mammary tumor virus proviral DNA is endogenous to most inbred strains of mice but in many strains is not transcriptionally active. This inactivity may be due to defects in the proviruses themselves or to position effects mediated by DNA sequences flanking the proviral units. The transcriptional competence of long terminal repeats (LTRs) derived from endogenous proviral DNA at genetic loci Mtv-8, Mtv-9, and Mtv-17 of the C57BL/6 mouse strain was examined with a transient transfection assay in which gene expression was monitored by expression of chloramphenicol acetyltransferase. LTRs from Mtv-8 and Mtv-9 were able to direct glucocorticoid-induced chloramphenicol acetyltransferase expression in this assay, while the LTR from Mtv-17 was only about 5% as effective. Analysis of chimeric LTRs indicated that the glucocorticoid-inducible transcriptional enhancer element within the Mtv-17 LTR is active when linked to a functional promoter from Mtv-8, whereas the promoter from Mtv-17 is defective in directing hormone-induced gene expression, even when linked to the Mtv-8 glucocorticoid-responsive enhancer. The DNA sequence of transcriptional control regions of the LTRs of all three endogenous proviral units was determined; this analysis revealed that the source of the defect in Mtv-17 is a single G-to-A transition at position-75 with respect to the site of transcription initiation that resides within the previously defined binding site for the transcription factor nuclear factor 1. Competition experiments with a gel electrophoresis mobility shift assay indicated that the affinity of nuclear factor 1 for DNA derived from Mtv-17 is significantly less than for comparable sequences derived from Mtv-8.

Animals↗