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PubMed1994

Molecular genetic approaches to understanding brain development and behaviour.

An understanding of brain development and brain function at the level of the genome is developing rapidly, because of the availability of new technologies in molecular and cellular biology. This understanding can be further enhanced by an interactive exchange between the disciplines of behavioural neuroscience and molecular genetics. New genes are being cloned almost daily, but their function remains an enigma. The purpose of this review is to illustrate how reporter genes can be used to map the brain's genetic activity in developmental time and anatomical space. The production of mutants in the homozygous condition may further lead to a morphological or behavioural phenotype. A knowledge of behavioural neuroscience can provide a prescreen of the reporter distribution and thereby make predictions concerning the type of behavioural analysis required. This approach allows selective cloning and sequencing of those genes which have either a morphological or behavioural phenotype but are transcribed at low levels. It is known that genomic imprinting influences brain development, and also that human genetic mutations and deletions influence imprinting in mental retardation as well as certain behavioural disorders. Precisely how such imprinted genes influence brain development and behaviour is being pursued by the use of chimeras. The distribution of maternal or paternal disomy cells in the brain and the way they influence behaviour may reveal the phenotype and how this is brought about.

Alleles↗

PubMed2001

Trap a gene and find out its function: toward functional genomics in Drosophila.

Many declared aims of the genome projects have been achieved. The total genomic sequences of several relatively noncomplex/complex organisms (such as E. coli, yeast, Caenorhabditis, Drosophila) are being determined, and the nucleotide sequencing of the entire human genome will be complete in the near future. However, this achievement is not the end of the road but rather the first step toward the functional understanding of the genome of humans and other organisms. The determined linear nucleotide sequences remain only lists of A, C, G and T, unless they are given functional significance. The coding sequences of genes can be identified in a relatively reliable manner by computational methods, but the exact function of their protein products can rarely be determined without obtaining much additional information, e.g., by biochemical or cell biological methods. Thus, following sequencing, the next step must be to assign functions to the identified genes. The final goal of genome research today may look futuristic, but the knowledge of the function of every single gene and the interactions between them will finally allow us to understand the development and functioning of an organism as a whole. Gene-trapping methodology is a powerful strategy for cloning and identifying functional genes, as it marks a gene with a tag and simultaneously generates a corresponding genetic variation for that particular locus. Therefore, gene trapping is an extremely useful tool for functional genomics, establishing a correlation between the physical and genetic maps of the genome. The relative simplicity of its genome and the availability of huge bodies of genetic and molecular information make Drosophila melanogaster one of the most important model organisms. Its genome will serve as a "reference" for the in-depth analysis of the organization of more complex eukaryotic genomes. Multifaceted approaches to Drosophila functional genomics and the dual-tagging gene trap system newly developed for functional analysis of Drosophila genes are discussed in this review.

Animals↗

PubMed1999

twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development.

The Drosophila Pax-6 gene eyeless (ey) plays a key role in eye development. Here we show tht Drosophila contains a second Pax-6 gene, twin of eyeless (toy), due to a duplication during insect evolution. Toy is more similar to vertebrate Pax-6 proteins than Ey with regard to overall sequence conservation, DNA-binding function, and early expression in the embryo, toy and ey share a similar expression pattern in the developing visual system, and targeted expression of Toy, like Ey, induces the formation of ectopic eyes. Genetic and biochemical evidence indicates, however, that Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey. Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program.

Amino Acid Sequence↗

PubMed2005

GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana.

Lateral root development occurs throughout the life of the plant and is responsible for the plasticity of the root system. In Arabidopsis thaliana, lateral root founder cells originate from pericycle cells adjacent to xylem poles. In order to study the mechanisms of lateral root development, a population of Arabidopsis GAL4-GFP enhancer trap lines were screened and two lines were isolated with GAL4 expression in root xylem-pole pericycle cells (J0121), i.e. in cells competent to become lateral root founder cells, and in young lateral root primordia (J0192). These two enhancer trap lines are very useful tools with which to study the molecular and cellular bases of lateral root development using targeted gene expression. These lines were used for genetic ablation experiments by targeting the expression of a toxin-encoding gene. Moreover, the molecular bases of the enhancer trap expression pattern were characterized. These results suggest that the lateral-root-specific GAL4 expression pattern in J0192 is due to a strong enhancer in the promoter of the LOB-domain protein gene LBD16.

Arabidopsis↗

PubMed2003

An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila.

The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Here we show that two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute.

Alleles↗

PubMed2003

Repopulation of rat liver by fetal hepatoblasts and adult hepatocytes transduced ex vivo with lentiviral vectors.

Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified ( approximately 10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach.

Albumins↗

PubMed2002

A genetically clamped renin transgene for the induction of hypertension.

Experimental analysis of the effects of individual components of complex mammalian systems is frequently impeded by compensatory adjustments that animals make to achieve homeostasis. We here introduce a genetic procedure for eliminating this type of impediment, by using as an example the development and testing of a transgene for "genetically clamping" the expression of renin, the major homeostatically responding component of the renin-angiotensin system, one of the most important regulators of blood pressure. To obtain a renin transgene whose expression is genetically clamped at a constant level, we have used single-copy chosen-site gene targeting to insert into a liver-specific locus a single copy of a modified mouse renin transgene driven by a liver-specific promoter/enhancer. The resulting transgene expresses renin ectopically at a constant high level in the liver and leads to elevated plasma levels of prorenin and active renin. The transgenic mice display high blood pressure, enhanced thirst, high urine output, proteinuria, and kidney damage. Treatment with the angiotensin II type I receptor antagonist, losartan, reduces the hypertension, albuminuria, and kidney damage, but does not affect expression of the transgene. This genetically clamped renin transgene can be used in models in which hypertension and its complications need to be investigated in a high prorenin/renin environment that is not subject to homeostatic compensations by the animal when other factors are changed.

Animals↗

PubMed2004

Conservation and non-conservation of genetic pathways in eye specification.

In this review we highlight two genetic pathways important for eye morphogenesis that are partially conserved between flies and vertebrates. Initially we focus on the ey paradigm and establish which aspects of this genetic hierarchy are conserved in vertebrates. We discuss experiments that evaluate the non-linear relationship amongst the genes of the hierarchy with a concentration on vertebrate functional genetics. We specifically consider the Six genes and their relationship to sine oculis, as tremendous amounts of new data have emerged on this topic. Finally, we highlight similarities between Shh/Hh directed morphogenesis mediated by basic Helix-Loop-Helix factors in vertebrate retinal cell specification and in specification of fly photoreceptors.

Animals↗

PubMed1991

The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding.

FIS protein is involved in several different cellular processes stimulating site-specific recombination in phages Mu and lambda as well as transcription of stable RNA operons in E.coli. We have performed a mutational analysis of fis and provide genetic and biochemical evidence that a truncated version of FIS lacking the N-terminal region is sufficient for specific DNA binding and for stimulating lambda excision. These mutants also retain their ability to autoregulate fis gene expression. Such mutant proteins, however, cannot stimulate the enhancer dependent DNA inversion reaction.

Amino Acid Sequence↗

PubMed2004

Embryonic stem cells form an organized, functional cardiac conduction system in vitro.

A functional pacemaking-conduction system is essential for maintaining normal cardiac function. However, no reproducible model system exists for studying the specialized cardiac pacemaking-conduction system in vitro. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the cells expressing these markers remain unknown. The ability to accurately identify cells that function as cardiac pacemaking cells is crucial for being able to study their molecular phenotype. In differentiating murine embryonic stem cells, we demonstrate the development of an organized cardiac pacemaking-conduction system in vitro using the coexpression of the minK-lacZ transgene and the chicken GATA6 (cGATA6) enhancer. These markers identify clusters of pacemaking "nodes" that are functionally coupled with adjacent contracting regions. cGATA6-positive cell clusters spontaneously depolarize, emitting calcium signals to surrounding contracting regions. Physically separating cGATA6-positive cells from nearby contracting regions reduces the rate of spontaneous contraction or abolishes them altogether. cGATA6/minK copositive cells isolated from embryoid cells display characteristics of specialized pacemaking-conducting cardiac myocytes with regard to morphology, action potential waveform, and expression of a hyperpolarization-activated depolarizing current. Using the cGATA6 enhancer, we have isolated cells that exhibit electrophysiological and genetic properties of cardiac pacemaking myocytes. Using molecular markers, we have generated a novel model system that can be used to study the functional properties of an organized pacemaking-conducting contracting system in vitro. Moreover, we have used a molecular marker to isolate a renewable population of cells that exhibit characteristics of cardiac pacemaking myocytes.

Animals↗

PubMed1995

Genetic analysis of embryonic cis-acting regulatory elements of the Drosophila homeotic gene sex combs reduced.

The homeotic gene Sex combs reduced (Scr) of Drosophila melanogaster is expressed in the labial and prothoracic segments of the ectoderm, in parasegments two and three of the CNS, and in the visceral mesoderm of the anterior and posterior midgut. The mutationally defined function of Scr is to specify the identity of the labial and prothoracic segments and to control the development of the gastric caeca. The Scr locus occupies a chromosomal region of approximately 80 kb within the Antennapedia complex (ANT-C). To understand how Scr's spatiotemporal expression pattern is generated in the embryo, we have mapped its transcriptional regulatory elements using three approaches. First, we examined the expression pattern of Scr in embryos containing chromosomal rearrangements that remove potential Scr regulatory elements. Second, we made and analyzed a set of Scr minigene transformants. Third, we analyzed a set of Scr-lacZ enhancer tester constructs. Using more sensitive anti-SCR antisera, we discovered that Scr is expressed in tissues that were not previously thought to accumulate SCR: a stripe of ectodermal cells in the parasegment 2 region of stage 5 embryos, the embryonic salivary glands, and the dorsal ridge. Four DNA fragments that had previously been shown in an analysis of Scr-lacZ reporter constructs to contain putative Scr enhancer elements were found to have functional enhancers; similarly, another Scr fragment was found to contain a functional repressor. Our results suggest that regulation of Scr in the labial segment and the CNS requires the apparently synergistic action of multiple, widely spaced enhancer elements. Regulation in the prothorax also appears to be controlled by multiple enhancers:one complete pattern element and one subpattern element. In contrast, Scr regulation in the visceral mesoderm is controlled by an enhancer(s) located in only one DNA fragment.

Animals↗

PubMed2001

Polymorphism of the human alpha1 immunoglobulin gene 3' enhancer hs1,2 and its relation to gene expression.

We studied the hs1,2 transcriptional enhancer identified downstream of the human alpha1 gene of the immunoglobulin H (IgH) locus, for which two different allelic configurations (a and b) were previously reported by Southern blotting. By using a polymerase chain reaction (PCR) method we amplified minisatellites within the hs1,2 core enhancer, with variable numbers of tandem repeats (VNTR) defining three 'PCR alleles' alpha1A, alpha1B and alpha1C (including one, two and three repeats, respectively). Five different alpha1 h1,2 genotypes were encountered in a population of 513 donors, representing 13.8, 34.5, 49.7, 1.3 and 0.6% for the AA, BB, AB, AC and BC genotypes, respectively. Luciferase assays showed that increasing the number of minisatellites increased the transcriptional strength of the alpha1 hs1,2 enhancer. Simultaneous determination of Southern blot alleles and VNTR alleles only showed a partial linkage between both types of polymorphism, altogether defining at least six different allelic forms of the 3'alpha1 region. In conclusion, the present study further demonstrates the genetic instability of the 3'alpha region, for which multiple alleles have been generated through inversions and internal deletions and/or duplications. This study also strengthens the hypothesis that the polymorphism at the IgH 3' regulatory region of the alpha1 gene could play a role in the outcome of diseases involving immunoglobulin secretion.

3' Untranslated Regions↗

PubMed1995

The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors.

The HBx protein of hepatitis B virus (HBV) is a transcriptional activator that is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. Recently, we and others have shown that HBx stimulates the Ras-Raf-MAP kinase cascade, which leads to enhanced cell proliferation and the activation of transcription factors AP-1 and NF-kappa B. Other studies have shown that HBx can activate transcription by interacting directly with nuclear components of the transcription machinery. Therefore we examined the basis for the different reported activities of HBx. Here, we show that HBx is a complex protein, displaying independent activities in different intracellular locations. The intracellular distribution of HBx protein was first investigated using scanning confocal laser immunomicroscopy and by genetic studies. Our work has established that HBx expressed in cultured cells is found authentically in both the cytoplasm and the nucleus. HBx is not strongly associated with any intracellular structures, but some preferential accumulation was observed near the cell surface. Next, HBx variants were constructed containing a functional or mutant nuclear localization sequence. We show that when HBx is engineered to relocate exclusively to the nucleus, it no longer activates the Ras-Raf-MAP kinase cascade, nor does it activate transcription factors AP-1 and NF-kappa B. Surprisingly, nuclear HBx fully retains the ability to stimulate HBV enhancer I, which is activated independently of the Ras and protein kinase C pathways. Therefore HBx protein stimulates signal transduction pathways in the cytoplasm and transactivates transcription elements in the nucleus. Furthermore, SV40 T antigen is shown to induce the nuclear sequestration of HBx protein and to block its activation of NF-kappa B, demonstrating that HBx is regulated by proteins that alter its intracellular distribution. The conflicting functions of HBx protein in viral infection and possibly carcinoma may involve the regulation of its differential distribution in the cell.

Amino Acid Sequence↗

PubMed2004

The human SCGB2A2 (mammaglobin-1) promoter/enhancer in a helper-dependent adenovirus vector directs high levels of transgene expression in mammary carcinoma cells but not in normal nonmammary cells.

Expression of secretoglobin family 2A member 2 (SCGB2A2, also known as mammaglobin-1) has been detected in a high percentage of primary and metastatic breast tumors, to a lesser extent in normal breast, but not in other normal tissues. Plasmid transfection studies in our lab and others, however, were unable to identify the genetic elements regulating this specificity. Here we demonstrate that a 25-kb DNA fragment derived from the human SCGB2A2 gene upstream of the protein coding sequence was highly active and preferentially expressed in breast cancer cells when introduced via a helper-dependent adenoviral (HDAd) vector. HDAd delivery was selected for its high cloning capacity, its high efficiency of gene transfer, and the absence of cis-acting viral sequences that can potentially interfere with specificity of the inserted promoters. A series of vectors with deletions in the 25-kb fragment was constructed to identify important regulatory regions of the SCGB2A2 promoter. We have determined that elements controlling the specificity of expression reside within the first 345 bp upstream of the coding sequence. In addition, we identified a strong enhancer several kilobases upstream of this minimal promoter. We suggest that the SCGB2A2 promoter/enhancer should be particularly advantageous for gene therapy protocols involving oncolytic viruses or toxic gene transfer via adenovectors to mammary tumors.

Adenoviridae↗

PubMed2005

A POU factor binding site upstream of the Chx10 homeobox gene is required for Chx10 expression in subsets of retinal progenitor cells and bipolar cells.

Retinal progenitor cells (RPCs) undergo a series of changes over time that affect their competency to produce different cell types at different times in development. The transcriptional machinery that regulates these changes, as well as associated gene expression changes, have not been characterized. An analysis of the regulatory region of the retinal homeodomain transcription factor, Chx10, was carried out using in ovo electroporations in chick and transgenic mice. An RPC enhancer was defined that mediates reporter activity in subsets of RPCs and directs high-level expression in intermediate and late RPCs. Using bioinformatic and biochemical analysis, a key binding site in this enhancer was found and was shown to be bound by the POU domain factors, Brn-2 and Tst-1/SCIP, in retinal extracts. Analysis of the Brn-2 expression pattern shows that it is expressed in intermediate and late RPCs, but not early RPCs, and thus partially overlaps with expression of the reporter activated by the defined Chx10 enhancer. Biochemical analysis also revealed binding of both Chx10 and Brn-2 to an enhancer of the CNS progenitor cell marker, Nestin. Nestin expression in the retina is restricted to intermediate/late RPC subsets, and genetic evidence is presented that demonstrates that Chx10 represses Nestin expression in early RPCs. A bipolar cell enhancer for Chx10 also was defined, and a role for Brn-2 in expression of Chx10 in bipolar cells is predicted. These data identify Brn-2 as a new marker of subsets of RPCs and suggest a mechanism by which a combination of POU factors and Chx10 define RPC gene expression patterns, such as that of Nestin.

Animals↗

PubMed1993

Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog.

We have previously reported the isolation and characterization of a new form (PGHS-2) of prostaglandin G/H synthase (PGHS, cyclooxygenase) from chicken embryo fibroblasts. To further study the regulation and structure of the gene, we have cloned the entire chicken PGHS-2 (previously termed miPGHSch) gene with its 5'-flanking region from a chicken genomic library. A genomic Southern blot showed the existence of a single PGHS-2 gene. The size of the gene was estimated at 8.9 kb through DNA sequencing and polymerase chain reaction analysis. The PGHS-2 gene was found to contain 10 exons, giving it a structure similar to that of the human PGHS-1 and murine PGHS-2 genes. The transcription start site was determined by primer extension, and the nucleotide sequence of 1.6 kb of the 5'-flanking region immediately upstream of the transcription start site was determined. The promoter sequence contained a TATA box and a variety of enhancer elements, including a serum response element, an AP-1, an NF-kappa B, and several SP-1 and AP-2 sites. Chloramphenicol acetyltransferase (CAT) assays showed that the first 158 nucleotides of the promoter efficiently drove transcription of the CAT reporter gene in serum-stimulated cells. Dexamethasone, a potent inhibitor of prostaglandin synthesis, had no effect on CAT activity, although this drug is known to markedly decrease PGHS-2 mRNA in vivo. This suggests that dexamethasone may inhibit PGHS-2 mRNA expression at the post-transcriptional level. Analysis of hamster/mouse somatic cell hybrids with radiolabeled cDNA probes demonstrated that PGHS-1 mapped to chromosome 2 and PGHS-2 mapped to chromosome 1 of the mouse genome.

3T3 Cells↗

PubMed1996

Genetic characterization of the homeodomain-independent activity of the Drosophila fushi tarazu gene product.

The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results.

Animals↗

PubMed1986

Papillomavirus transforming functions.

The bovine papillomavirus type 1 (BPV-1) has served as a model for unravelling the molecular genetics of the papillomaviruses. BPV-1 transformation of rodent cells in tissue culture has provided a means to study the viral functions involved in latent infection of cells and in the induction of cellular proliferation functions. BPV-1 has been shown to encode two independent transforming genes, each of which can induce cellular transformation in susceptible rodent cells. These two genes apparently act synergistically in transforming mouse C127 cells. Deletion mutagenesis studies have shown that the expression of one of these genes (E6) is required for efficient tumorigenesis and anchorage independence. BPV-1 also encodes functions which may act indirectly to affect transformation. BPV-1 contains transcriptional enhancers which can act in a position-independent and orientation-independent manner to increase the transcriptional activity of a heterologous gene. One of these elements, which is located in a non-coding region of the genome, can be trans-activated by a specific viral gene product encoded by the E2 open reading frame. Mutations which eliminate this trans-activation function also have a dramatic effect on transformation and on stable plasmid maintenance.

Animals↗