Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.     

Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters

DNA sequences in the 5'-flanking region of rat and bovine oxytocin genes were examined for their capacity to confer estrogen responsiveness to their homologous promoters. In contrast to the 5'-flanking region of the rat oxytocin gene, upstream promoter sequences up to 3200 bp of the bovine gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene which were transfected in estrogen receptor expressing MCF-7 cells did not respond to estrogen. Testing 5'-deletion mutants of the rat upstream region linked to the luciferase gene in P19 embryocarcinoma cells co-transfected with an estrogen receptor expression plasmid showed that two regions each associated with approximately 15-fold stimulation of promoter activity were located between nucleotides -172 and -149 and between -148 and +16 in the rat gene. The former region contains the imperfect palindrome GGTGACCTTGACC which differs in one nucleotide from the estrogen response element (ERE) consensus. It is concluded that the corresponding motive CATAACCTTGACC of the bovine gene is not a functional ERE. Thus, the estrogen responsiveness of oxytocin genes is species-dependent.     

Roles of the TGACT repeat sequence in the yeast TRP5 promoter

Yeast genes under general amino acid control contain multiple copies of a sequence known as the TGACT repeat in the 5'-flanking DNA. The yeast TRP5 gene contains two copies of the TGACT repeat sequence in its 5'-flanking region. The upstream TGACT repeat of TRP5 is required for normal basal expression as well as derepression by general control. Synthetic oligonucleotides containing a TGACT sequence were inserted into previously constructed TRP5 control region deletion mutants. A synthetic 17-base pairs (bp) oligonucleotide containing a TGACT copy along with flanking nucleotides from HIS3 was able to restore derepression in all deletion mutants tested. The 17-bp oligonucleotide also functioned bidirectionally. Replacements in the upstream control region by synthetic oligonucleotides indicated that sequences other than the TGACT repeat are required for high basal expression. Replacements of the downstream repeat sequence by the 17-bp oligonucleotide suggest its main role in this position is for derepressed expression. High level derepressed expression was found to correlate with the presence of two repeats.     

Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5'' end of the chicken alpha-skeletal actin gene.

alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression.