Three Sea Urchin Actin Genes Show Different Patterns of Expression: Muscle Specific, Embryo Specific, and Constitutive

Actin coding sequence cDNA probes were used to quantitate the number of transcripts in RNA from eggs, embryos, and tube feet of the sea star Pisaster ochraceus. Transcript concentrations were measured in both total RNA and in poly(A)+ RNA by titration and hybridization kinetic methods. Surprisingly, the actin transcript number in sea star eggs is two orders of magnitude greater than in sea urchin eggs. There are at least 2.9 X 10(5) actin transcripts per sea star egg, 1.2 X 10(5) per 48-h gastrula and 1.9 X 10(5) per 72-h gastrula. The number of actin transcripts per unit mass of extracted tube foot RNA is lower than in developmental stages. The relative abundance and size of actin transcripts was determined by Northern and dot blot analyses using probes containing actin coding DNA or 3'-untranslated-region sequences. The actin transcript in eggs and embryos is 2,300 nucleotides (nt) long and originates from the Cy (cytoplasmic) gene class. In tube feet, the most abundant actin transcript is 2,200 nt long and originates from the M (muscle) gene class. Tube feet also contain, at lower abundance, 2,300-nt transcripts of the Cy gene type expressed in eggs and embryos.

Section: Discussionmentioning

confidence: 99%

Quantitative Assessment of Actin Transcript Number in Eggs, Embryos, and Tube Feet of the Sea Star Pisaster ochraceus

Kovesdi

Smith

1985

Delimitation and Characterization of cis-Acting DNA Sequences Required for the Regulated Expression and Transcriptional Control of the Chicken Skeletal α-Actin Gene

Bergsma

Grichnik

Gossett

et al. 1986

We have previously observed that DNA sequences within the 5'-flanking region of the chicken skeletal alpha-actin gene harbor a cis-acting regulatory element that influences cell type and developmental stage-specific expression (J. M. Grichnik, D. J. Bergsma, and R. J. Schwartz, Nucleic Acids Res 14:1683-1701, 1986). In this report we have constructed unidirectional 5'-deletion and region-specific deletion-insertion mutations of the chicken skeletal alpha-actin upstream region and inserted these into the chloramphenicol acetyltransferase expression vector pSV0CAT. These constructions were used to locate DNA sequences that are required for developmental modulation of expression when transfected into differentiating myoblasts. With this assay we have delimited the 5' boundary of a cis-acting regulatory element to ca. 200 base pairs upstream of the mRNA cap site. In addition, we have preliminarily identified DNA sequences that may be important subcomponents within this element. A second major focus of this study was to identify those DNA signals within the regulatory element that control transcription. Toward this end, the expression phenotypes of progressive 5'-deletion and deletion-insertion mutants of the 5'-flanking region of the chicken skeletal alpha-actin gene were assayed in microinjected Xenopus laevis oocytes. These experiments defined a cis-acting transcriptional control region having a 5' border 107 base pairs preceding the alpha-actin RNA cap site. Proximal and distal functionally important regions of DNA were identified within this element. These DNA signals included within their DNA sequences the "CCAAT" and "TATA" box homologies.

A 29-Nucleotide DNA Segment Containing an Evolutionarily Conserved Motif is Required in cis for Cell-Type-Restricted Repression of the Chicken α-Smooth Muscle Actin Gene Core Promoter

Carroll

Bergsma

Schwartz

1988

A series of 5' deletion mutations of the upstream flanking sequences of the chicken alpha-smooth muscle (aortic) actin gene was prepared and inserted into the chloramphenicol acetyltransferase expression vector pSV0CAT. Deletion recombinants were transfected into fibroblasts, which actively express the alpha-smooth muscle actin gene, and primary myoblast cultures, which accumulate much lower quantities of alpha-smooth muscle actin mRNAs. The first 122 nucleotides of 5'-flanking DNA were found to contain a "core" promoter capable of accurately directing high levels of transcription in both fibroblasts and myotubes. The activity of this core promoter is modulated in fibroblasts by a "governor" element(s) located at least in part between nucleotides -257 and -123. This region contains sequences potentially conserved between mammalian and avian alpha-smooth muscle actin genes as well as one of a pair of 16-base-pair inverted CCAAT box-associated repeats which are conserved among all chordate muscle actin genes examined to date. A smaller DNA segment (-151 to -123) containing these upstream CCAAT box-associated repeats was sufficient to suppress expression of the core promoter in muscle cultures, suggesting that the upstream CCAAT box-associated repeats play a negative role in the alpha-smooth muscle actin gene promoter.