The complete nucleotide sequence of a genomic clone encoding the mouse skeletal a-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal a-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal a-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein.Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle a-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal (-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal a-actin genes.The actins represent a multigene family of highly conserved proteins found in all eucaryotes. Differences in amino acid sequence among the various actins have shown that at least six different isoforms are expressed in vertebrates (52, 53). Two striated muscle isoforms, skeletal a and cardiac a (52), and two smooth muscle isoforms (53) are found in the contractile apparatus of muscle fibers, whereas two cytoskeletal isoforms, p and y, are present in the cytoskeleton of all cells (51). These actin proteins are extremely conserved in amino acid sequence.Actin gene expression is tissue specific and developmentally regulated (27,29,32,35). By studying the structural organization of the actin gene family, one can begin to look for the controlling elements which modulate the expression of these genes during development. Here we present the complete nucleotide sequence of the single genomic copy of the mouse skeletal a-actin gene. The coding region of this gene is interrupted by five introns which are located in the same positions as introns previously identified in other vertebrate skeletal a-actin genes (13, 54). A comparison of the nucleotide sequences of several vertebrate skeletal aactin genes reveals several blocks of highly conserved sequences in the 5'-flanking region and in both the 5'-and 3'-untranslated regions. Interestingly, the conserved sequences in the 5'-flanking region and within the first untranslated exon can potentially form several hairpin loops by base pairing between adjacent inverted complementary sequences. These regions do not correspond to the potential hairpin structure in the corresponding portion of the rat cytoskeletal ,B-actin gene (33). Furthermore, it is possible to form long hairpin loops within the first intron and one stem loop in the 3'-untranslated region upstream from the putative...

Initiation factor 2 (eIF-2) is phosphorylated in vitro by two different cyclic nucleotide-independent protein kinases. As previously shown, a protein kinase activity that comigrates with the major casein kinase activity from rabbit reticulocytes phosphorylates eIF-2,B. In addition, a second protein kinase that specifically phosphorylates eIF-2a has been identified. Both protein kinase activities demonstrate cyclic nucleotide-independent activity and are not inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase activities. Phosphorylation of eIF-2a is almost completely inhibited by 20-35,uM hemin, whereas phosphorylation of eIF-2ft is only partially inhibited. Hemin acts by decreasing the rate of incorporation of phosphate into eIF-2a. The protein kinase activity that modifies eIF-2a has been shown to have inhibitory activity in the cell-free protein-synthesizing system, whereas the protein kinase for eIF-2Bt has no effect. The identity of the former enzyme with the hemin-controlled repressor and role of hemin in the control of initiation are discussed. Hemoglobin biosynthesis in both reticulocytes (1) and reticulocyte lysates (2-4) is under the direct control of hemin, which acts as a positive effector for globin synthesis. In the absence of added hemin, protein synthesis is inhibited in a reticulocyte lysate at chain initiation with a concomitant decrease in binding of Met-tRNAf to 40S ribosomal subunits (5-7). This inhibition is potentiated by addition of ATP (8,9). Inhibition produced by hemin deprivation is prevented in the reticulocyte lysate by high concentrations of GTP, cyclic AMP, and various purines (8-10). In addition, inhibition of globin synthesis coincides with the appearance of a repressor (11-13). The hemin-controlled repressor (HCR) is thought to be activated from a prorepressor at hemin concentrations suboptimal for translation (5-7, 12, 14-16). Supporting the concept that HCR inhibits initiation is the observation that the addition of initiation factor 2 (eIF-2), the factor that forms a ternary complex with Met-tRNAf and GTP and then binds to 40S subunits (17-23), will reverse inhibition due to hemin deprivation in the lysate system (24, 25).eIF-2 is composed of three subunits (25-31) designated a, p, and y according to a decreasing relative mobility in the gel electrophoresis system of Laemmli (32). Phosphorylation of eIF-2fl by a cyclic nucleotide-independent protein kinase has been described (26, 33). It has been reported that purified preparations of HCR possess a protein kinase activity that phosphorylates the small subunit of eIF-2 (29,(34)(35)(36). This modification may be correlated with the inhibition of protein synthesis resulting from hemin depletion (34,37,38). In these studies we have examined phosphorylation of the two subunits of eIF-2 by the two different protein kinases. 2-mercaptoethanol). Under these conditions, the protein kinase activity did not adhere to the resin. When the enzyme was rechromatographed in the presence of 0.25 M ...

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A summer program was created for undergraduates and graduate students that teaches bioinformatics concepts, offers skills in professional development, and provides research opportunities in academic and industrial institutions. We estimate that 34 of 38 graduates (89%) are in a career trajectory that will use bioinformatics. Evidence from open-ended research mentor and student survey responses, student exit interview responses, and research mentor exit interview/ survey responses identified skills and knowledge from the fields of computer science, biology, and mathematics that are critical for students considering bioinformatics research. Programming knowledge and general computer skills were essential to success on bioinformatics research projects. General mathematics skills obtained through current undergraduate natural sciences programs were adequate for the research projects, although knowledge of probability and statistics should be strengthened. Biology knowledge obtained through the didactic phase of the program and prior undergraduate education was adequate, but advanced or specific knowledge could help students progress on research projects. The curriculum and assessment instruments developed for this program are available for adoption by other bioinformatics programs at

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.

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