Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.     

Short 5'-flanking regions of the Amy gene of Drosophila kikkawai affect amylase gene expression and respond to food environments

Evolution of the duplicated genes and regulation in gene expression is of great interest, especially in terms of adaptation. Molecular population genetic and evolutionary studies on the duplicated amylase genes of Drosophila species have suggested that their 5'-flanking (cis-regulatory) regions play an important role in evolution of these genes. For better understanding of evolution of the duplicated amylase genes and gene expression, we studied functional significance of the Amy1 gene of Drosophila kikkawai using in vitro deletion mutagenesis followed by P-element-mediated germline transformation. We found that a 1.6-kb of the 5'-flanking region can produce strikingly higher level of larval amylase activity on starch food compared with that on glucose food. We found two cis-regulatory elements, which increase larval amylase activity on starch food. We also found a larval cis-regulatory element, which responds to the food difference. This food-response element is necessary for the function of the element increasing larval activity on starch food. A 5-bp deletion in a putative GRE caused high amylase activity, indicating a cis-regulatory element decreasing amylase activity. These cis-regulatory elements identified in the 5'-flanking region could be the targets of natural selection.     

Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences

We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'-and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements.     

Localization of the human UBC polyubiquitin gene to chromosome band 12q24.3

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An congruent to 1.4-kb sequence from the 5'-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5'-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5'-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.     

Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. Identification of a 250-base pair fragment which confers cardiac-specific expression

The present study characterized the structure, organization, and expression of the rat cardiac myosin light chain (MLC) -2 gene. The rat cardiac MLC-2 gene has seven exons which display complete conservation with the exon structure of the rat fast twitch skeletal MLC-2 gene. A 250-base pair (bp) sequence of the 5'-flanking region contains CArG motifs and additional cis elements, each greater than 10 bp in length, which were conserved in sequence and relative position with the chick cardiac MLC-2 gene. A series of MLC-2/luciferase fusion genes consisting of nested 5' deletions of the MLC-2 5'-flanking region were constructed and transfected into primary neonatal rat myocardial cells and a non-myocardial cell line (CV-1), demonstrating that this 250 bp of the MLC-2 5'-flanking region was sufficient to confer cardiac specific expression on a luciferase reporter gene. This study suggests the presence of important proximal regulatory sequences in the MLC-2 5'-flanking region which are capable of directing the cardiac specific expression of the rat cardiac myosin light chain-2 gene.     

Cloning and characterization of porcine insulin gene

The complete porcine preproinsulin cDNA and 1022 bp of its 5'-flanking region have been cloned by PCR-based technology and characterized. The porcine insulin gene has the same structure of three exons and two introns as that found in all insulin genes sequenced to date. Northern blot analysis of isolated adult porcine islets demonstrated an increase in steady-state insulin mRNA levels in response to high concentrations of glucose. Highly conserved cis-acting elements were found in the 5'-flanking region of the porcine insulin gene including multiple E and A elements as well as a cAMP responsive element (CRE). Tissue-specific activity of the proximal promoter was confirmed by transient transfection of the promoter/reporter gene constructs. This information now makes it possible for regulation and expression of the porcine insulin gene to be analyzed.     

Molecular evolution of the 5'-flanking regions of the duplicated Amy genes in Drosophila melanogaster species subgroup

The nucleotide sequences of the 5'-flanking regions of the duplicated Amy genes in eight sibling species belonging to the melanogaster species subgroup are analyzed. In Drosophila melanogaster, a region of about 450 bp immediately upstream of the translation initiation site of the two paralogous genes (the proximal and distal genes) has sequence similarities. However, we could not detect any significant sequence similarity in the region more upstream than -450. This result indicates that the coding regions of the ancestral Amy gene were duplicated together with 450 bp of the 5'-flanking region as one unit. Multiple alignment of these 450-bp sequences in the proximal and distal genes of all eight species revealed a mosaic pattern of highly conserved and divergent regions. The conserved regions included almost all the putative regulatory elements identified in previous analyses of the sequences. A phylogenetic analysis of the aligned sequences shows that these 450-bp sequences are clustered into the proximal and the distal groups. As a whole, the divergence between groups in this region is very large in contrast to that in the coding regions. Based on the divergence between groups, the 450-bp region is divided into two subregions. We found that the ratios of the divergence between groups to that within groups differ in the two subregions. From these observations, we discuss a possibility of positive selection acting on the subregion immediately upstream of the Amy coding region to cause divergence of regulatory elements of the paralogous genes.      

Brachyury regulatory region active in embryonal carcinoma P19 cells

Brachyury (T) is involved in mesoderm induction during early mouse development. We analyzed the region regulating expression of T in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro. Transfection of plasmids encoding reporter genes under the control of the 5'-flanking region showed positive regulatory elements between -298 and -129 bp are responsible for driving T expression in mesodermal cells.