We have constructed a mammalian expression vector consisting of 3 kilobases of the human fi-actin gene 5' flanking sequence plus 5' untranslated region and intervening sequence I linked at the 3' splice site to a short DNA polylinker sequence containing unique Sal I, HindIII, and BamHI restriction endonuclease sites followed by a simian virus 40 (SV40) polyadenylylation signal. Two derivatives, containing the selection markers obtained from pSV2gpt or pSV2neo, were also generated. We find that the promoter activity of this vector is as great or greater than that of the SV40 early promoter in a variety of human and rodent cells. The vector was used to generate y-actin and 8-tubulin antisense transcripts in human fibroblast cell lines. The antisense transcripts accumulate to levels comparable with that of the highly abundant y-actin and .-tubulin mRNAs.

We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one-or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from Nterminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from oneand two-dimensional polyacrylamide gels.Protein sequence analysis is central to modern biological research. Amino acid sequence data can aid in gene isolation either through the use of synthetic oligonucleotides (1) or through the use of antisera of predetermined specificity (2). Sequence data can also be used to correlate the gene structure with the expressed polypeptide chain, to map active sites and domain boundaries, to define posttranslational modifications, or to establish evolutionary relationships between proteins (3).Currently, 20 pmol amounts of protein can give limited N-terminal sequence information using optimized isolation methods (4) and a commercially available gas-phase sequenator (5). It is desirable to develop a method of comparable sensitivity for internal amino acid sequence analysis of proteins for the following reasons:(i) Many proteins are not susceptible to the Edman degradation. This is assumed to be due to artefactual or biosynthetic blocking of the a-amino groups of the proteins. The nature of the modified N terminus is not easily determined and only a few of the modifications can be chemically or enzymatically reversed (6). Therefore, the most general way to obtain sequence information from a protein with a blocked a-amino group is to sequence peptide fragments generated by cleavage of the polypeptide chain.(ii) In the molecular cloning of genes through the use of synthetic oligodeoxynucleotides reverse translated from the protein sequence, it is advantageous to screen appropriate cDNA libraries with either a set of multiple probes or a single probe with minimum degeneracy to reduce the incidence of "false positives." Furthermore, the use of an oligodeoxynucleotide probe derived from the N...

We have assigned six members of the human beta-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned beta-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome. Using intervening sequence probes, we showed that the functional gene is single copy and that all of the other beta-actin related sequences are recently generated in evolution and are probably processed pseudogenes. The entire nucleotide sequence of the functional gene has been determined and is identical to cDNA clones in the coding and 5' untranslated regions. We have previously reported that the 3' untranslated region is well conserved between humans and rats (Ponte et al., Nucleic Acids Res. 12:1687-1696, 1984). Now we report that four additional noncoding regions are evolutionarily conserved, including segments of the 5' flanking region, 5' untranslated region, and, surprisingly, intervening sequences I and III. These conserved sequences, especially those found in the introns, suggest a role for internal sequences in the regulation of beta-actin gene expression.

Abstract. Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors.

Heteroploid mouse NIH 3T3 fibroblasts and several rat fibroblast strains (Rat-1, Rat-2 and REF-52) are cell lines of special interest in the field of carcinogenesis because of their extensive use as normal cells in transformation assays for putative cancer-causing genes. Exposure of these cells to carcinogenic chemicals or oncogenic DNA produces anchorage-independent cells with retracted cytoplasms that lack actin cables. All human fibroblast strains, normal and transformed, synthesize two electrophoretic forms of actin (beta- and gamma-actin). In contrast, we discovered that early-passage mouse and rat strains synthesize abundant amounts of each of the three electrophoretic forms of actin (alpha-, beta- and gamma-actin) but mouse and rat cancer cells express only beta- and gamma-actins. We now show that in NIH 3T3 and Rat-2 fibroblasts a third actin, the smooth muscle alpha isoform, is abundantly co-expressed with beta- and gamma-actin. In every instance tested following transformation to tumorigenicity, the accumulation of alpha-actin messenger RNA and alpha-actin synthesis was greatly inhibited. Shutdown of alpha-actin expression thus appears to be a reproducible transformation-sensitive marker in rodent fibroblasts.

We identified six tropomyosin (Tm) isoforms in diploid human fibroblasts. We used computerized microdensitometry of 2-dimensional protein profiles to measure the relative rates of synthesis and abundance of the individual Tm isoforms and actin, the two major structural constituents of microfilaments. In carcinogen-transformed human fibroblasts (HuT-14), the rates of synthesis of three Tm isoforms (Tml, Tm2, and Tm6) were greatly decreased relative to normal diploid parental fibroblasts and to actin. In contrast, related nontumorigenic HuT fibroblasts which are "immortalized" and anchorage independent exhibited both slight down-regulation of Tml and Tm6 and 3.5-fold up-regulation of Tm3. Thus, Tm isoform switching from the predominance of the larger more avid Tm isoforms (Tml, Tm2, Tm3, and Tm6) to the smaller, less avid Tm isoforms (Tm4 and TmS) in microfilaments was a transformation-induced change correlated with tumorigenicity in human fibroblasts.

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