Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.     

The effect of regulatory sequences of alphaS1-casein gene on the expression of the lacZ-gene in loach Misgurnus fossilis L. transgenic embryos

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine alphaS1-casein gene tissue-specific promoter-enhancer region or highly homologous goat alphaS1-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1-3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the alphaS1-casein gene of goat was higher than with the promoter-enhancer region of the bovine alphaS1-casein gene. Thus, regulatory sequences of the bovine or goat alphaS1-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.     

The third and fourth tropomyosin isoforms of Caenorhabditis elegans are expressed in the pharynx and intestines and are essential for development and morphology.

The tropomyosin gene tmy-1/lev-11 of Caenorhabditis elegans spans 14.5 kb and encodes three isoforms by alternative splicing. To identify, characterize and compare the genome and tissue expression of a fourth isoform, the technique of rapid amplification of cDNA ends and microinjection with lacZ and gfp fusion plasmids were employed. We elucidated CeTMIV, a fourth isoform of tmy-1, which encoded a 256 residue polypeptide. CeTMIV isoform had a similar promoter region to CeTMIII isoform, but was alternatively spliced to generate a cDNA that differed in two exons. The tmy-1::lacZ and tmy-1::gfp fusion genes, with 3.2 kb promoter sequence and 1.1 kb of CeTMIV isoform specific exons, were expressed in the pharyngeal and intestinal cells. Further unidirectional deletion of the sequence located the primary promoter region 853 bp upstream from the initial codon. We show within the upstream region, the presence of B and C subelement-like sequences of myo-2, which may be used to stimulate pharyngeal expression. Despite the presence of a ges-1 like sequence, we were unable to locate the two GATA sites required for intestinal expression. Reassessing tissue expression for CeTMIII isoform with newly constructed fusion plasmids, we showed further expression in germ-line tissue and intestinal cells in addition to pharyngeal expression. Finally, to demonstrate that tropomyosin is essential for development, we inactivated the body wall and pharynx-specific isoforms by RNA-mediated interference. In addition to 50-75 % embryonic lethality in both cases, the worms that survived body wall interference had abnormal body morphology and uncoordinated movements, and those that survived pharynx interference had deformed pharynges and gut regions. These results show the function of tropomyosin in normal muscle filament assembly and embryonic development, and illustrate the different expression patterns characteristic of tropomyosin isoforms in C. elegans.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region.     

Nucleotide sequence of the 5' flanking region responsible for the enhancement of the expression of yeast enolase 1 gene

Several plasmids carrying different length of the 5' flanking region of yeast (Saccharomyces cerevisiae) enolase 1 gene (ENO1) which is fused in frame to the Escherichia coli lacZ gene were constructed by recombination in vitro. Promoter activity of ENO1 was assayed by measuring beta-galactosidase activity of the fused gene product. Comparison of the promoter activity of these plasmids suggests that the sequences required for a strong promoter activity lie within the DNA segment -724 to -353 base pairs (bp) upstream from the start of ENO1 coding sequence. The nucleotide sequence of this region was determined.