Ultrabithorax function in butterfly wings and the evolution of insect wing patterns.
BACKGROUND: . The morphological and functional evolution of appendages has played a critical role in animal evolution, but the developmental genetic mechanisms underlying appendage diversity are not understood. Given that homologous appendage development is controlled by the same Hox gene in different organisms, and that Hox genes are transcription factors, diversity may evolve from changes in the regulation of Hox target genes. Two impediments to understanding the role of Hox genes in morphological evolution have been the limited number of organisms in which Hox gene function can be studied and the paucity of known Hox-regulated target genes. We have therefore analyzed a butterfly homeotic mutant 'Hindsight', in which portions of the ventral hindwing pattern are transformed to ventral forewing identity, and we have compared the regulation of target genes by the Ultrabithorax (Ubx) gene product in Lepidopteran and Dipteran hindwings. RESULTS: . We show that Ubx gene expression is lost from patches of cells in developing Hindsight hindwings, correlating with changes in wing pigmentation, color pattern elements, and scale morphology. We use this mutant to study how regulation of target genes by Ubx protein differs between species. We find that several Ubx-regulated genes in the Drosophila haltere are not repressed by Ubx in butterfly hindwings, but that Distal-less (Dll) expression is regulated by Ubx in a unique manner in butterflies. CONCLUSIONS: . The morphological diversification of insect hindwings has involved the acquisition of different sets of target genes by Ubx in different lineages. Changes in Hox-regulated target gene sets are, in general, likely to underlie the morphological divergence of homologous structures between animals. (+info)
Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation.
The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity. (+info)
Analysis of two cosmid clones from chromosome 4 of Drosophila melanogaster reveals two new genes amid an unusual arrangement of repeated sequences.
Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing approximately 5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome. (+info)
The mouse Aire gene: comparative genomic sequencing, gene organization, and expression.
Mutations in the human AIRE gene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; OMIM 240300). Previously, we have cloned hAIRE and shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization of Aire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of the AIRE gene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT-PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product. (+info)
ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4.
To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5'-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5'-ATGACGAGGAGGA-3', restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system. (+info)
Cloning and functional characterization of the 5'-flanking region of the human bone morphogenetic protein-2 gene.
Bone morphogenetic protein-2 (BMP-2) is involved in bone formation, organogenesis or pattern formation during development. The expression of BMP-2 is regulated accurately and coordinately with that of other transforming growth factor-beta (TGF-beta) superfamily members. To elucidate the mechanism underlying the regulation of BMP-2 expression, a 6.7 kb SpeI-SalI fragment, from the P1 phage library, encompassing the 5'-flanking region of the human BMP-2 gene, was isolated and sequenced. Transcription start sites were mapped by the 5'-rapid amplification of cDNA ends (RACE) method. It has been found that the human BMP-2 gene contains, largely, two promoter regions surrounded by GC-rich sequences with several Sp1 consensus motifs. The proximal promoter possesses a single start site, whereas several start sites are clustered in the distal promoter region. Neither TATA nor CAAT consensus sequences are found in the proximity of the start sites for either promoter. Interestingly, in no case is the transcription-initiation site common between the human and mouse BMP-2 genes, although the sequence of the BMP-2 gene is well conserved in the promoter region between two species. Transient transfection experiments with the reporter fused with various lengths of the BMP-2 promoter sequence demonstrated that there exist enhancer elements in an 1.1 kb GC-rich fragment covering both promoter regions. It is noteworthy that the enhancer elements are 5'-flanked by a 790 bp strong repressor element that is characterized by numerous AT stretches. This intriguing organization may be amenable to the tight control of the expression of BMP-2 that is essential for development or bone morphogenesis. (+info)
Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes.
The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323-331(A) and 350-354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes. (+info)
Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells.
Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu. (+info)