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V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the alpha and beta subunits of the capping protein (CP, alternatively called CapZ or beta-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1.CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 x 10(-7) m and a molecular stoichiometry of approximately 1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the sessile adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actomyosin Mg2+-ATPase activity in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only

The far upstream region (−1.2-0.9 kilobase pairs) of the mouse glycophorin gene contains the locus control region (LCR)-like region, which acts as an erythroid-specific enhancer dependent on chromosomal integration in murine erythroleukemia (MEL) cells. In the present study, we demonstrated that this region binds six nuclear factors. The binding of GATA-1 to corresponding sites did not show any change before or after induction with dimethyl sulfoxide, but the binding of Spi-1/PU.l and an unidentified factor called glycophorin regulatory element binding factor (GRBF) showed a change during induction. While binding activity of Spi-l/PU.l dropped soon after induction, the GRBF activity increased after induction when expression of the glycophorin gene began. After identification of the consensus binding site of GRBF, we cloned cDNA for that factor by Southwestern method, and it was identified as a previously reported transcription factor, delta, a murine form of YY-l which is a versatile transcription factor. Mutation analysis in the delta/YY-1 binding site within the LCR-like region indicated that delta/YY-1 acts as a regulatory protein in combination with the E-box-binding protein that binds to the neighboring sequence.

Previously, three structurally related proteins, MyoD, myogenin, and Myf5, have been identified, and each of them was found to convert C3H10T1/2 fibroblasts to myoblasts when their respective cDNAs were expressed under the control of a viral promoter. Here, we describe the cloning and DNA sequencing of myogenin cDNAs from chicken and mouse. They encode polypeptides highly homologous to each other, but the polypeptide sequences we have obtained are not homologous in the carboxyl-terminal 70 amino acids with those previously reported for mouse, rat, and human because of a single base deletion in the previously reported cDNAs. Determination of genomic sequence coding for myogenin revealed that the mouse myogenin cDNA presented here corresponds to a correct transcript of the gene, and the nucleotide sequence of the previously reported cDNA is incorrect. A comparison of chicken and mouse myogenins with other myogenic regulatory factors, MyoD and Myf-5, identified a domain with an interesting feature located in the carboxyl terminus of these proteins in addition to the myc homology domain previously reported.

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.

We have isolated cDNA clones of the mRNA for chick embryonic myosin light chain (MLC), L23, by cross-hybridization with chicken skeletal muscle MLC1 cDNA. The identification of the isolated cDNAs was carried out by in vitro translation of hybrid-selected mRNA. Sequence analysis of the cloned cDNAs revealed that the cDNA insert contained 832 nucleotides and predicted a polypeptide of 185 amino acids with a calculated molecular weight of 20,687. The deduced amino acid sequence for L23 showed high sequence similarities to those of adult alkali type MLCs from various tissues, indicating that L23 belongs to the alkali MLC group. Using the cloned cDNA as a hybridization probe, we have revealed by RNA blot analysis that the expression of L23 mRNA was regulated in temporal and tissue-specific manners. The L23 mRNA of 1.1 kilobases is transiently expressed in embryonic skeletal, cardiac, and smooth muscles of chickens. It is also found in the brain of chickens during all stages of development so far investigated. Only a single gene for L23 was detected by Southern blot of chick genomic DNA. We therefore suggest that L23 is expressed from a single gene in both embryonic muscles and brain.