Redundant cis-acting elements control expression of the Drosophila affinidisjuncta Adh gene in the larval fat body.

The alcohol dehydrogenase (Adh) gene in the Hawaiian species of fruit fly, Drosophila affinidisjuncta, like the Adh genes from all Drosophila species analyzed, is expressed at high levels in the larval fat body via a larval-specific promoter. To identify the cis-acting elements involved in this highly conserved aspect of Adh gene expression, deleted D. affinidisjuncta genes were introduced into D. melanogaster by somatic transformation. Unlike previously described methods, this transformation system allows analysis of Adh gene expression specifically in the larval fat body. The arrangement of sequences influencing expression of the proximal promoter of this gene in the larval fat body differs markedly from that described for the Adh gene from the distant relative, D. melanogaster. Multiple redundant elements dispersed 5' and 3' to the gene, only some of which map to regions carrying evolutionarily conserved sequences, affect expression in the fat body. D. affinidisjuncta employs a novel mode of Adh gene regulation in which the proximal promoter is influenced by sequences having roles in expression of the distal promoter. This gene is also unique in that far upstream sequences can compensate for loss of sequences within 200 bp of the proximal RNA start site. Furthermore, expression is influenced in an unusual, context-dependent manner by a naturally-occurring 3' duplication of the proximal promoter--a feature found only in Hawaiian species.     

cis-acting control elements for Krüppel expression in the Drosophila embryo.

Krüppel (Kr), a gap gene of Drosophila, shows complex spatial patterns of expression during the different stages of embryogenesis. In order to identify cis-acting sequences required for normal Kr gene expression, we analysed the expression patterns of fusion gene constructs in transgenic embryos. In these constructs, bacterial lacZ expression was placed under the control of Kr sequences in front of a basal promoter. We identified cis-acting Kr control units which drive beta-galactosidase expression in 10 known locations of Kr expression in early and late embryos. More than one cis-regulatory element drives the expression in the anterior domain at the blastoderm stage, in the nervous system, the midline precursor cells and in the amino-serosa. In addition, two cis-acting elements direct the first zygotic expression of Kr in a striped subpattern within the central region of the blastoderm embryo. Both elements respond to alterations in the activities of maternal organizer genes known to be required for Kr expression in establishing the thoracic and anterior abdominal segments in the wild-type embryo.     

Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.     

Computational identification of cis-acting elements affecting post-transcriptional control of gene expression in Saccharomyces cerevisiae

Understanding the regulation of gene expression requires the identification of cis -acting control elements that modulate gene function. The recent availability of complete genome sequences and profiles of mRNA expression has facilitated the development and utilization of computational methods to identify discrete regulatory elements. We have developed an oligomer counting method that identifies sequences that occur significantly more often in a group of interest relative to other genes in the genome. The use of a second parameter, which measures the frequency of oligomers within the group of interest, allows the detection of false positive signals caused by very infrequent oligomers that would otherwise appear as significant. Applying this method to gene groups that have a common expression pattern or shared function should identify oligomers that comprise cis -acting control elements. As a test of this method, we applied this approach to a set of intron-containing yeast genes, where we easily identified the known splicing signals as control elements. We have used this training set to examine how this method is affected by the length of the oligomer examined, as well as the size and composition of the gene group. These simulations allowed us to identify rules for selecting groups of genes to analyze. Finally, application of this method to nuclear genes encoding proteins targeted to the mitochondria identified a new putative cis -acting sequence in the 3'-untranslated region of this family of genes, which may play a role in mRNA localization or the regulation of mRNA stability or translation.     

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

Extinction of alpha-fetoprotein gene expression in somatic cell hybrids involves cis-acting DNA elements.

alpha-Fetoprotein (AFP), a liver-specific protein, is extinguished in somatic cell hybrids formed by the fusion of mouse hepatoma cells (BWTG3) with rat fibroblast cells (JF1). Our studies show that the extinction of mouse AFP expression in these somatic cell hybrids may involve at least two cis-acting regulatory domains, i.e., the enhancer elements and a tissue-specific promoter region, which are located in the 5'-flanking region of the AFP gene.     

cis-acting regulatory elements abrogate allele-specific HLA class I gene expression in healthy individuals.

Pedigree analysis of a Dutch family revealed the presence of an HLA-A "blanc" allele segregating with the HLA-B8, -Cw7, -DR3, -DR52, -DQ2 haplotype. Precipitation studies, using selected mAb and sera directed against conserved epitopes of HLA class I products, failed to detect the expression of a corresponding HLA-A locus product. cDNA nucleotide sequence analysis of the HLA-A "blanc" specificity showed that the obtained sequence was identical to the authentic HLA-A1 gene revealing no mutations, deletions or recombinations that could influence translation or transport of a putative translation product to the cell surface. Mitogen stimulation, EBV transformation, or treatment with rIFN-gamma and rTNF-alpha did not induce HLA-A1 expression. Furthermore, cell-mediated lympholysis analysis revealed that individuals carrying the nonexpressed HLA-A1 gene could mount a strong anti-HLA-A1 T cell response, indicating that HLA-A1 was not expressed during T cell ontogeny. Therefore, this study describes for the first time the allele-specific down-regulation of the expression of a classical HLA class I gene segregating in healthy individuals.     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.