ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.
The Caenorhabditis elegans elt-2 gene encodes a single-finger GATA factor, previously cloned by virtue of its binding to a tandem pair of GATA sites that control the gut-specific ges-1 esterase gene. In the present paper, we show that elt-2 expression is completely gut specific, beginning when the embryonic gut has only two cells (one cell cycle prior to ges-1 expression) and continuing in every cell of the gut throughout the life of the worm. When elt-2 is expressed ectopically using a transgenic heat-shock construct, the endogenous ges-1 gene is now expressed in most if not all cells of the embryo; several other gut markers (including a transgenic elt-2-promoter::lacZ reporter construct designed to test for elt-2 autoregulation) are also expressed ectopically in the same experiment. These effects are specific in that two other C. elegans GATA factors (elt-1 and elt-3) do not cause ectopic gut gene expression. An imprecise transposon excision was identified that removes the entire elt-2 coding region. Homozygous elt-2 null mutants die at the L1 larval stage with an apparent malformation or degeneration of gut cells. Although the loss of elt-2 function has major consequences for later gut morphogenesis and function, mutant embryos still express ges-1. We suggest that elt-2 is part of a redundant network of genes that controls embryonic gut development; other factors may be able to compensate for elt-2 loss in the earlier stages of gut development but not in later stages. We discuss whether elements of this regulatory network may be conserved in all metazoa.
The actin severing and capping protein gelsolin contains three distinct actin binding sites. The smallest actin binding domain of approximately 15,000 Mr was originally obtained by limited proteolysis and it corresponds to the first of six repeating segments contained in the gelsolin sequence. We have expressed this domain (here termed segment 1 or N150 to define its amino acid length) in Escherichia coli, together with a series of smaller mutants truncated at either N‐ or C‐terminal ends, in an attempt to localize residues critical of actin binding. Limited truncation of segment 1 by 11 residues at its N‐terminal end has no observable effect on actin binding, but on removal of a further eight residues, actin binding is totally eliminated. Although this loss of actin binding may reflect ablation of critical residues, we cannot rule out the possibility that removal of these residues adversely affects the folding of the polypeptide chain during renaturation. Truncation at the C‐terminus of segment 1 has a progressive effect on actin binding. Unlike intact segment 1, which shows no calcium sensitivity of actin binding within the resolution of our assays, a mutant with 19 residues deleted from its C‐terminus shows unchanged affinity for actin in the presence of calcium, but approximately 100‐fold weaker binding in its absence. Removal of an additional five residues from the C‐terminus produces a mutant that binds actin only in calcium. Further limited truncation results in progressively weaker calcium dependent binding and all binding is eliminated when a total of 29 residues has been removed. Although none of the expressed proteins on their own binds calcium, 45Ca is trapped in the complexes, including the complex between actin and segment 1 itself. These results highlight a region close to the C‐terminus of segment 1 that is essential for actin binding and demonstrate that calcium plays an important role in the high affinity actin binding by this domain of gelsolin.
We have previously shown that a tandem pair of (A/T)GATA(A/G) sequences in the promoter region of the Caenorhabditis elegans gut esterase gene (ges-1) controls the tissue specificity of ges-1 expression in vivo. The ges-1 GATA region was used as a probe to screen a C. elegans cDNA expression library, and a gene for a new C. elegans GATA-factor (named elt-2) was isolated. The longest open reading frame in the elt-2 cDNA codes for a protein of M(r) 47,000 with a single zinc finger domain, similar (approximately 75% amino acid identity) to the C-terminal fingers of all other two-fingered GATA factors isolated to date. A similar degree of relatedness is found with the single-finger DNA binding domains of GATA factors identified in invertebrates. An upstream region in the ELT-2 protein with the sequence C-X2-C-X16-C-X2-C has some of the characteristics of a zinc finger domain but is highly diverged from the zinc finger domains of other GATA factors. The elt-2 gene is expressed as an SL1 trans-spliced message, which can be detected at all stages of development except oocytes; however, elt-2 message levels are 5-10-fold higher in embryos than in other stages. The genomic clone for elt-2 has been characterized and mapped near the center of the C. elegans X chromosome, ELT-2 protein, produced by in vitro transcription-translation, binds to ges-1 GATA-containing oligonucleotides similar to a factor previously identified in C. elegans embryo extracts, both as assayed by electrophoretic migration and by competition with wild type and mutant oligonucleotides. However, there is as yet no direct evidence that elt-2 does or does not control ges-1.
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